Vector NTI® Express Software

USER GUIDE
Vector NTI Express Software

Publication Part Number MAN0006009
Revision Date 15 January 2012
For Research Use Only. Not for use in diagnostic procedures.

For research use only. Not intended for any animal or human therapeutic or diagnostic use.
Copyright
2012 Life Technologies Corporation. All rights reserved. No part of this publication may be reproduced, transmitted, transcribed,
stored in retrieval systems, or translated into any language or computer language, in any form or by any means: electronic,
mechanical, magnetic, optical, chemical, manual, or otherwise, without prior written permission from Life Technologies Corporation
(hereinafter, Life Technologies). The information in this guide is subject to change without notice. Life Technologies and/or its
affiliates reserve the right to change products and services at any time to incorporate the latest technological developments.
Although this guide has been prepared with every precaution to ensure accuracy, Life Technologies and/or its affiliates assume no
liability for any errors or omissions, nor for any damages resulting from the application or use of this information. Life Technologies
welcomes customer input on corrections and suggestions for improvement.
Trademarks
BioBrick is a trademark of the BioBricks Foundation, Inc. The BioBrick trademark is used herein merely for the purpose of fair use.
The BioBricks Foundation, Inc. is not affiliated with Life Technologies Corporation. The BioBricks Foundation, Inc has not authorized,
sponsored or otherwise endorsed this product or our use of the BioBrick trademark herein. Information about BioBrick and the
BioBricks Foundation, Inc. can be found at www.biobricks.org.
TaqMan is a registered trademark of Roche Molecular Systems, Inc.
GeneArt is a registered trademark of GENEART AG.
Windows and Microsoft are registered trademarks of Microsoft Corporation.
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Limited Use Label License: For Research Use Only

The purchase of this product conveys to the purchaser the limited, non-transferable right to use the product only to perform internal
research for the sole benefit of the purchaser. No right to resell this product or any of its components is conveyed expressly, by
implication, or by estoppel. This product is for internal research purposes only and is not for use in commercial applications of any
kind, including, without limitation, quality control and commercial services such as reporting the results of purchaser's activities
for a fee or other form of consideration. For information on obtaining additional rights, please contact outlicensing@lifetech.com or
Out Licensing, Life Technologies, 5791 Van Allen Way, Carlsbad, California 92008.
Technical Resources
Additional technical resources for Vector NTI Express Software are available online at: http://lifetechnologies/vectornti
To obtain personalized technical support by telephone or email, you must have a paid annual software maintenance and support
contract. To purchase a contract, email bioinfosales@lifetech.com or contact your local Life Technologies office.
If you have a paid annual support contract:
Email your question to bioinfosales@lifetech.com
Or phone
800 955 6288 (North America)
+44 (0) 141 814 6318 (Europe, Middle East, Africa)
For additional technical support, visit www.lifetechnologies.com/support.
Contents
About This Guide . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 11

Note on screen captures . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 11
CHAPTER 1 Database Explorer . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 13

Overview . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 13
About this chapter . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 13
Archives . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 14
Open the Database Explorer . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 14
Components of the Database Explorer . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 15
Types of databases . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 15
Subsets . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 16
Open files . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 16
Database Explorer operations . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 17
Create DNA/RNA and protein molecules . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 17
Create a DNA/RNA molecule . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 18
Create a Protein molecule . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 19
Create gel markers, oligos, and enzymes . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 19
Create a gel marker . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 19
Create an oligo . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 19
Create an enzyme . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 20
Import data . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 21
Import molecules . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 21
File formats . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 21
Import files . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 22
Manage data . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 23
Manage Database, Projects, and Results databases . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 23
Manage Database, Projects, and Results subsets . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 23
Manage database objects . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 25
Edit data . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 28
Edit enzyme properties . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 28
Edit oligo properties . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 29
Edit gel marker properties . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 29
Open other applications . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 30
Copy, save, and print molecules . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 30
Copy a molecule . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 30
Save a molecule . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 32
Vector NTI ExpressSoftware User Guide 3

Contents
Print molecule data . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 33

Manage the Projects database . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 33
Create a project . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 33
Open a project . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 33
Manage Projects subsets . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 33
Delete Projects subsets . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 34
Manage the Results database . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 34
View results . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 34
Manage Results subsets . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 34
Workgroup Shared Database . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 34
About workgroup shared databases . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 35
Connect to a workgroup shared database . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 35
Add users to a workgroup shared database . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 36
A user name should be one word without spaces.Edit users in a workgroup shared database
37
Delete users from a workgroup shared database . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 37
Upload data to a workgroup shared database . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 37
Download data from a workgroup shared database . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 37
Disconnect from a workgroup shared database . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 37
Set preferences . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 37
Set display preferences for molecules . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 37
Set display preferences for sequences . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 38
Register with the NCBI . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 38
CHAPTER 2 Molecule Editor . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 39

Create or open a molecule . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 39
Create a new molecule . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 39
Open an existing molecule . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 39
Molecule Editor window . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 40
Molecule display tools . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 41
Enter or edit a sequence . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 41
Enter a sequence . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 41
Select a sequence . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 42
Cut or copy a sequence . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 42
Paste a sequence . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 42
Replace a sequence . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 42
Delete a sequence . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 42
Reverse a sequence . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 42
Molecule features . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 42
Create a molecule feature . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 42
Select a feature . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 43
Hide or display a feature . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 43
Edit or delete a feature . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 43
Restriction Analysis . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 43
4 Vector NTI ExpressSoftware User Guide

Contents
Selecting enzymes for analysis . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 44

Configuring the enzyme list . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 44
Filter the analysis results . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 45
Perform the analysis . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 45
ORF Finder . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 45
Define start and stop codons . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 46
ORF Types . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 46
Perform the analysis . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 46
Translation tool . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 47
Translation results . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 47
Oligo Duplex Analysis . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 48
Entering or selecting oligos . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 48
Analysis parameters . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 49
Run the analysis . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 49
Silent Mutation Analysis . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 49
Web analyses . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 50
Back Translation . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 51
Protein Domain and Motif Finder Analyses . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 52
Protein Domain AnalysisPROSITE and PRINTS databases . . . . . . . . . . . . . . . . . . . . . . . . . . . 52
Motif FinderInterProScan sequence search . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 54
CHAPTER 3 Primer Design . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 57

Open the primer/probe design tools . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 57
Save and load settings . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 58
Run the design tool . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 58
Primer/probe design results . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 58
Save primer/probe designs . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 59
Find PCR Primers Inside Selection settings . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 60
Amplify Selection settings . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 61
PCR Using Existing Oligos settings . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 63
Sequencing Primers settings . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 64
Hybridization Probes . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 65
Shared Advanced settings . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 66
CHAPTER 4 BioAnnotator . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 71
Selecting an analysis . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 71
Graph and Sequence panes . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 72
Highlight sequence region in the graph . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 72
Magnify the graph . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 72
Determine the value at a point in the graph . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 72
Analysis parameters . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 72
Window size . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 73

Contents
Analyses descriptions and parameters . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 73
CHAPTER 5 Regenerator . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 75
Regenerator Workflow . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 75
Open Regenerator . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 75
Regenerator tool features . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 76
Create mutations in the input sequence . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 76
Clear mutations . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 77
View the mutated sequence . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 77
Refresh the in silico DNA sequence . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 77
Optimize the expression system and genetic code . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 77
Add attachments . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 77
Clear attachments . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 78
Generate a new sequence and send for synthesis . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 78
CHAPTER 6 BLAST and Entrez Searches . . . . . . . . . . . . . . . . . . . . . . . . . 81

BLAST search . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 81
Open the BLAST search tool . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 81
BLAST search settings . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 81
Perform the BLAST search . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 84
BLAST Result Viewer . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 85
Entrez Search . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 86
Open Entrez search tool . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 86
Entrez search settings . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 87
Entrez search results . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 87
Editing and deleting queries . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 88
CHAPTER 7 GenomeBench . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 89
Download data from public DAS servers . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 89
Data Loading Monitor . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 91
Local GenomeBench Projects . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 91
GenomeBench Project Viewer . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 92
Map Overview . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 92
Feature Map . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 92
Genome Features and Genome Sequence . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 93
Editing features . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 93
CHAPTER 8 Align Multiple Sequences . . . . . . . . . . . . . . . . . . . . . . . . . . . 95

Open AlignX . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 95
AlignX window . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 95
Manage AlignX projects . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 96
Save and rename a project . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 96

Contents
Open a project . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 96
Close a project . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 97
Export a project . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 97
Save consensus sequence . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 97
Open .dnd file . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 97
Select fragments to align . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 97
Add fragments . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 98
Remove fragments . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 98
Select fragments to align . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 98
Alignment settings . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 99
Program . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 99
Molecule type . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 99
Pairwise alignment . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 99
Multiple sequence options . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 100
Perform the alignment . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 101
Guide Tree pane . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 101
Graphs pane . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 101
Sequence similarity graph . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 101
Sequence complexity graph . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 101
Magnify the graphs . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 102
Select a region . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 102
Alignment pane . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 102
Select a region . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 102
Dot Plot . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 102
Display the Dot Plot . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 103
CHAPTER 9 3D Molecule Viewer . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 105

Download 3D Structure Files . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 105
Open a molecule in 3D Molecule Viewer . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 105
Elements of the 3D Molecule Viewer window . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 106
Magnify and rotate the molecule . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 106
Highlight an amino acid or a chain . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 107
Additional menu operations . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 107
CHAPTER 10 Sim4 and Spidey Analysis . . . . . . . . . . . . . . . . . . . . . . . . . 109

Sim4 . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 109
Launch Sim4 analysis tool . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 109
Analysis Jobs settings . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 110
Submit the job . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 110
View analysis results . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 111
Spidey . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 111
Launch Spidey analysis tool . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 111
Analysis Jobs settings . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 112

Contents
Spidey Parameters . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 112

Submit the job . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 112
View analysis results . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 113
CHAPTER 11 Clone2Seq . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 115

Launch Clone2Seq . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 115
Clone2Seq window . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 115
Select molecules . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 115
Molecule requirements . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 116
Display restriction sites in molecules . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 116
Generate molecule fragments . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 116
Edit the Fragment List . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 117
Modify fragment ends . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 118
Assemble the molecule . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 118
Multiple fragment cloning . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 119
CHAPTER 12 Gateway Cloning . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 121

For more information . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 121
Gateway Cloning Workflow . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 121
Gateway Cloning Tool . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 122
Open the Gateway Cloning Tool . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 122
Gateway Project pane . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 122
Task List pane . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 123
Current Task pane . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 124
Create, save, and load projects . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 124
Gateway Cloning workflow . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 125
. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 125
Amplify fragments to Use in a BP reaction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 125
Recombine Entry Clones by BP . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 127
Preview Entry Clones . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 129
Add Entry Clones to Use in LR Reaction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 129
Create Expression Clones by LR . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 130
Preview Expression Clones . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 131
CHAPTER 13 TOPO Cloning . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 133

TOPO Cloning in Vector NTI Express . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 133
TOPO Cloning Tool . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 133
Open the TOPO Cloning Tool . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 133
TOPO Project pane . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 134
Task List pane . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 135
Current Task pane . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 135
Create, save, and load projects . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 136
TOPO Cloning workflow . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 136

Contents
. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 136
Amplify fragments to Use in TOPO reaction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 136
Create TOPO Clones . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 139
CHAPTER 14 GeneArt Cloning . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 141

Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 141
GeneArt Seamless Cloning Overview . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 141
GeneArt High Order Assembly Overview . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 141
How GeneArt Assembly Works . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 142
Open the GeneArt Assembly Tool . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 143
From the molecule editor . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 143
From the main toolbar with no molecule selected . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 143
GeneArt Assembly Wizard . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 144
Assembly settings . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 144
Add and organize fragments . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 144
Design PCR primers to create end homology . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 145
Design stitching oligos (High Order Assembly only) . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 146
Create the assembled molecule . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 146
CHAPTER 15 Parts Assembler . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 149

Additional Information about Parts and Standards . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 149
Using the Parts Assembler . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 149
Selecting Parts . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 149
Restrictions on Parts . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 150
Assembly Settings . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 150
Completing and previewing the assembly . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 151
Viewing the assembly in Molecule Editor . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 152
CHAPTER 16 Contig Assembly using ContigExpress . . . . . . . . . . . . . . 153

About this chapter . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 153
Open ContigExpress . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 153
ContigExpress window . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 154
Manage ContigExpress projects . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 155
Save and rename a project . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 155
Open a project . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 155
Close a project . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 155
Export a project . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 155
Navigate the Task List . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 155
Add fragments to assemble . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 156
Add fragments . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 156
Remove fragments . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 156
Rename fragments . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 156
Fragment trimming . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 156

Contents
Select fragments to trim . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 157

Trim ends . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 157
Trim vector contaminations . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 158
Assembly settings . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 160
Perform the assembly . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 164
Save, delete, and rename contigs . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 165
Save contig assemblies . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 165
Delete a contig assembly . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 165
Re-assemble contigs . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 165
Rename a contig . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 166
View the assembly . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 166
Contig Viewer Graph Pane . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 166
Contig Viewer Alignment pane . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 167
Translate the consensus sequence . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 168
Edit the contig sequence . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 169
Insertions . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 169
Deletions . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 169
Saving sequence changes . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 169
APPENDIX A Symbols and Formats: IUB (IUPAC) Ambiguity Codes and

ASCII Format . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 171
Format for ASCII Sequence Files . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 171
IUB Formats recognized by Vector NTI Express . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 171
APPENDIX B Primer Tm Calculations . . . . . . . . . . . . . . . . . . . . . . . . . . . 173

General Information . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 173
Usefulness of Thermodynamic Tm Versus %GC Tm . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 173
Effects of Primer (Probe) and Salt Concentration on Tm Calculations . . . . . . . . . . . . . . . . . . . . . . . 173
%GC Tm Calculation . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 173
Thermodynamic Tm Calculation . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 174
Example . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 175
Oligos Containing IUB Ambiguity Characters . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 176
RNA Oligos . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 176
Primer/Probe Tm, TaOpt and Similarity Calculations . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 177
Primer/Probe Tm Values . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 177
TaOpt Values . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 177
Primer/Probe Similarity Values . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 178
References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 178
Index . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 179

About This Guide
ThisuserguideprovidesreferenceinformationforVectorNTIExpressSoftware,a
Javabasedcrossplatformapplicationthatcanrunoncomputersusingthefollowing
operatingsystems:
MicrosoftWindowsXP(SP3)andWindows7
MacOSX
Linux
Forinstallationandlicensinginformation,seetheVectorNTIExpressReleaseNotes
andInstallation/LicensingGuide.
Thisguideusesconventionsandterminologythatassumeaworkingknowledgeofthe
youroperatingsystem,theInternet,andInternetbrowsers.
Note on screen ThisguideincludesscreencapturesfortheMicrosoftWindowsversionofthe

captures software.ForMacintoshandLinuxusers,thescreenswilllookslightlydifferentbut
thecommandsandfunctionswillbethesame.

About This Guide

1 Database Explorer
Overview
TheVectorNTIExpresslocaldatabaseisadatabasefileandassociatedfolderthatby
defaultareinstalledintherootdirectoryofyourlocalharddrive(e.g.,
C:\VntiExpress_Database).DatabaseExploreristhetoolinVectorNTIExpressfor
managingallthemolecules,oligos,projects,analysisresults,andotherdatainthe
localdatabase.
Eightdifferenttypesofobjectsarestoredandorganizedindatabasesandsubsetsin
theVectorNTIExpresslocaldatabase:
DNA/RNAmoleculesthesecontainanucleicacidsequenceaswellas
annotations,primers,restrictionsites,analysisresults,andothermoleculedata.
Uponimportfromothersources,nucleicaciddataareparsedandstoredinan
internalformat.Youcanaddmoleculestothedatabasebyimportingorcreating
basicorconstructedmolecules.
Proteinmoleculesthesecontainanaminoacidsequenceaswellasannotations
andotherfeatures.LikeDNAmolecules,uponimportfromothersources,protein
moleculedataareparsedandstoredinaninternalformat.Youcanaddmolecules
tothedatabasebyimportingorcreatingbasicmolecules.
ProjectsincludingAlignment,ContigAssembly,andCloningprojects.
Restrictionenzymes(RENs)importedfromtheREBASEdatabase.Datafor
restrictionenzymesareparsedandstoredinaninternalformat.Youcanadd
otherRENsfromtheREBASEfileincludedintheVectorNTIExpressSoftware.
Oligonucleotidesthesecanbecreatedbytheuserorgeneratedbyprimerdesign
toolsinVectorNTIExpress.Severalexampleoligosareincludedinthesoftware
fordemonstrationpurposes.
Gelmarkersthesecanbecreatedbytheuser.Commonlyusedgelmarkersare
includedwiththeVectorNTIExpressinstallation.
BLASTresultsthesearegeneratedbyBLASTsearchesrunVectorNTIExpress,
andcanbestoredasresultsfilesinthedatabase.
AnalysisresultssuchasPCRanalysisorPFAManalysisofmolecules,canbe
storedasresultsfilesinthedatabase.
About this chapter

Thischaptercovers:
Archivesonpage14
OpentheDatabaseExploreronpage14
Openfilesonpage16
DatabaseExploreroperationsonpage17

Database Explorer
1 Archives
CreateDNA/RNAandproteinmoleculesonpage17
Creategelmarkers,oligos,andenzymesonpage19
Importdataonpage21
Managedataonpage23
Editdataonpage28
Openotherapplicationsonpage30
Openotherapplicationsonpage30
Copy,save,andprintmoleculesonpage30
ManagetheProjectsdatabaseonpage33
ManagetheResultsdatabaseonpage34
WorkgroupSharedDatabaseonpage34
Setpreferencesonpage37
Archives
Parentdescendantrelationships(tokeeptrackofyourconstructs),userfields,
comments,andkeywordsarekeptforallmoleculesinthedatabase.
Alldatabasemoleculesandotherobjectscanbeplacedintoarchivesdatafilesof
specialformatthatcanbetransferredtoanothercomputer(MacintoshorWindows)
andreadbyVectorNTIExpressSoftware.Througharchives,youcansharemolecules,
constructs,orotherobjectswithyourcolleagues,orsimultaneouslyusethemon
severalcomputers(forinstance,atworkandathome).
InVectorNTIExpressSoftwarearchives:
AllDNA/RNAmoleculeinformationiswrittento,andreadfrom,anarchivefile.
Thisinformationincludesmoleculecomponentfragments(ifthemoleculeis
constructedfromothermolecules)andparentdescendantconnectionsbetween
molecules.
VectorNTIExpressSoftwareautomaticallycheckstheconsistencyofmolecule
archiveinformation,addingnecessaryparents(includingDNAparentsof
translatedproteinmolecules)ordisconnectingthemifyouneglectedtotransfer
themtothearchives.
Whenthearchiveisloadedintoanewdatabase,VectorNTIExpressSoftware
checkstheinformationconsistencyonanyofdatabasemoleculesandrecalculates
themifnecessary.
Open the Database Explorer

TheDatabaseExplorercanbeopenedtwoways:
InWindows,clickStartPrograms(orAllPrograms)LifeTechnologies
VectorNTIExpress VectorNTIExpress.Bydefault,VectorNTIExpress
openswiththeDatabaseExplorerscreen.

Open the Database Explorer 1
Inthemaintoolbar,clicktheDatabaseExplorerbutton:
Components of the TheDatabaseExplorerscreenconsistsofthefollowingcomponents:

Database Explorer Threedatabases:Database,Projects,andResults.
AnExplorerViewerfornavigatingamongthreetypesofdatabases.Theviewer
contentschangewhenyouclicktheExplorerbuttonsbelowtheviewer.
ExplorerbarsforopeningandnavigatingtheDatabase,Projects,andResults
databases.
ARecordsViewerpaneforviewingthedataandrecordsassociatedwiththe
Database,Projects,andResults.
ASummaryViewerforviewingasnapshotofaselectedrecord.
Note: Youcanwiden,narrow,lengthen,andshortenthesizeofviewersbyclicking
anddraggingtheviewerframes.
Main Menu
Toolbar
Subset
Records
Viewer
Explorer
Viewer
Summary
Explorer Bars Viewer
Drag frames to resize viewers
Types of databases TherearethreetypesofdatabasesintheDatabaseExplorer:

DatabaseincludesdataassociatedwithDNAandRNArecords;proteinrecords;
andenzymes,oligos,andgelmarkers.
Projectsincludedataandresultsassociatedwithalignmentprojects,contig
assemblyprojects,andcloningprojects.
ResultsincludeBLASTandanalysisresults.

Database Explorer
1 Open files
Subsets Asubsetisagroupofobjectsorganizedbyaspecifiedcriteria,suchascommon
features.Forexample,youmighthaveonesubsetforeachofyourmoleculefamilies,
andoneforeachtaxonomicgroup.Subsetsareshownasfolders intheDatabase,
Projects,andResultspanes.Youcansearchforrecordsbynameandcreateandstore
datainsubsets.
Open files
Youcanopenassemblyprojects,molecules,andBLASTresults.Thefollowingfile
formatsarerecognizedbyVectorNTIExpressSoftware:
File Format
DNA/RNA All nucleotide files (*.gb, *.gbwithparts, *.fasta, *.fas, *.fa, *.mpfa,
molecules *.fna, *.fsa, *.seq, *.embl, *.gcg, *.ma4, *.ddbj)
Genbank (*.gb, *.gbwithparts)
DDBJ (*.ddbj)
Fasta (*.fasta, *.fas, *.mpfa, *.fna, *.fsa, *.seq)
EMBL (*.embl)
GCG (*.gcg)
Vector NTI Archive (*ma4)
Protein molecules All protein files (*.fasta, *.fas, *.fa, *.mpfa, *.fna, *.fra, *.fsa, *.seq,
*.gp, *.gpwithparts, *.swp, *.pa4, *.trembl)
Fasta (*.fasta, *.fas, *.mpfa, *.fna, *.fsa, *.seq)
GenPept (*.gp, *.gpwithparts)
Swiss-Prot (*.swp)
TrEMBL/EMBL (*.trembl,*.embl)
Vector NTI Archive (*pa4)
3D molecules *.pdb
Assembly projects Contig Project (*.cepx, *.cep)
MSA projects Alignment Project (*.apr, *.aprx)
BLAST results Vector NTI Archive (*.ba6)
Toopenafile:
1. ClickFileOpen.
2. Inthedropdownlist,selectasubset.

Database Explorer operations 1
3. IntheOpenFilewindow,navigatetothefile,selectit,thenclickOpen.
Database Explorer operations

InDatabaseExplorer,youcan:
CreateDNA/RNAandproteinmolecules(thispage),gelmarkers,oligos,and
enzymes(page 19),andprojects(page 33).
Importdata(page 21)andexportdata(page 27)(notavailableinthe
demonstrationversion).
Manageyourdatabyorganizingyourdataintoconvenientgroups(subsets),and
sorting,duplicating,anddeletingdata(page 23).
Editdata(page 28).
Searchthedatabasefortextsequence,motifs,featuretypes,keywords,andother
information(page 28).
Openotherapplications(page 30).
Copyamolecule(page 30)saveamolecule(page 32),andprintmoleculedata
(page 33).
Create DNA/RNA and protein molecules

TherearefivedifferentwaystocreateDNA/RNAandproteinmolecules:
ImportamoleculefromDDBJ,EMBL,FASTA,GCG,GenBank,GenPept,
SWISSPROT,TrEMBL,andVectorNTIArchiveformats,orfromanASCIIfile
offlexibleformat.Thesequenceandfeaturemapareconvertedfromthefile,and
thenewmoleculebecomespartoftheVectorNTIExpressSoftwaredatabase.
Createmoleculesfromnucleotideoraminoacidsequencesthatyoudefine.You
canmanuallyenterthesesequencesoryoucanpastefromaclipboardandenter
thesequenceasanewmolecule.
TranslateacodingregionofanexistingDNAorRNAmoleculetocreateprotein
molecules.
ConstructnewDNA/RNAmoleculesfromuserdefinedcompatiblecomponent
fragmentsfromothermolecules.
DesignDNA/RNAmoleculesfromcomponentsinauserdefinedfragmentlist,
usingthebuiltinbiologicalknowledgeofVectorNTIExpressSoftwaretodesign
therecombinationprocess.Allnewmoleculesareintegratedintothedatabase
andcanbeusedinallfutureoperationsandanalyses.

Database Explorer
1 Create DNA/RNA and protein molecules
Create a DNA/RNA TocreateaDNA/RNAmolecule:

molecule 1. Inthemainmenu,clickFileNew.
2. Inthedropdownlist,selectDNAtoopentheNewDNAMoleculewindow.
3. Enteraname,selecttheform,andenteradescription,thenclickOK.

Create gel markers, oligos, and enzymes 1
Create a Protein Tocreateaproteinmolecule:

molecule 1. Inthemainmenu,clickFileNew.
2. Inthedropdownlist,selectProteinintheNewProteinMoleculewindow.
3. Enteranameanddescription,thenclickOK.
Create gel markers, oligos, and enzymes

Gelmarker,oligonucleotide,andenzymeobjectscanbecreatedfromscratchusing
editorsinVectorNTIExpressSoftwareorbyimportingthemfromFASTAfiles,Vector
NTIArchivefiles,anoligolist,andaREBASEdatabase.
Create a gel Tocreateagelmarker:

marker 1. Inthemainmenu,clickFileNew.
2. Inthedropdownlist,selectGelMarkertoopentheGelMarkerwindow.
3. Enteraname.Clickthebrowsebutton(...)toopentheChooseDatabaseGel
Markerwindow,thenclickOK.
4. Selectagelmarkertype.
5. ClicktheGelMarkertab,enterafragmentlength,thenclickOK.
Create an oligo Tocreateanoligo:
1. Inthemainmenu,clickFileNew.
2. Inthedropdownlist,selectOligotoopentheOligowindow.
3. IntheGeneraltab,enteraname,thenclicktheOligotab.
4. Enterthenucleotidesequence,thenselecttheoligotype.
Toreplacetheoligowithitscomplementarysequence,checktheReverse
Complementarybox.
Enteradescription,thenclicktheUserFieldstab.
5. Clickacellinthetable,clickChangeValuetoopenadataentrywindow,then
enteravalue.
Toremoveavalue,clickRemoveValue.
Afteryouhavefinshedenteringvalues,clicktheCommentstab.
6. Entercommentsabouttheoligo,thenclicktheKeywordstab.
7. Toaddakeywordfortheoligo,enteranewwordorselectaniteminthelistof
existingkeywords.
Tomovethekeywordintothekeywordlist,click<Add.Toremoveanitemfrom
thekeywordlist,selectit,thenclick>Remove.
8. Tosaveyourdata,clickOK.ToclosetheOligowindowwithoutsavingyourdata,
clickCancel.

Database Explorer
1 Create gel markers, oligos, and enzymes
Create an enzyme Tocreateanenzyme:
1. Inthemainmenu,clickFileNew.
2. Inthedropdownlist,selectEnzymetoopentheEnzymewindow.
3. IntheGeneraltab,enteraname,thenclicktheEnzyme/Methylasetab.
4. Entertherecognitionstringoftheenzyme.
5. IntheCleavagePoint/MethylationBasefield,enterthenumberofthenucleotide
immediatelyafterthedirectstrandcleavagepoint.Thefollowingexample
demonstrateshowcleavagepointsofpalindromicsitesaredefined.
Cleavage Point = 2
A A T A T T
1 2 3 4 5 6
T T A T A A
IntheCleavagePointonComplementaryStrandfield,enterthenumberofthe
nucleotideimmediatelyafterthecomplementarystrandcleavagepoint.The
followingexampledemonstrateshowcleavagepointsaredefinedfor
nonpalindromicsitesonbothdirectandcomplementarystrands.
Cleavage Point on Complementary Strand = 10
Cleavage Point = 8
A A G T N N N N N N
1 2 3 4 5 6 7 8 9 10
T T C A N N N N N N
Cleavage Point on Complementary Strand = -4
Cleavage Point = -2
N N N N N N N A A G T
-7 -6 -5 -4 -3 -2 -1 1 2 3 4
N N N N N N N T T C A
6. Selecttheenzymetype,regularrestrictionenzymeormethylase,thenclickthe
UserFieldstab.
7. Clickacellinthetable,clickChangeValuetoopenadataentrywindow,then
enteravalue.
Toremoveavalue,clickRemoveValue.
Afteryouhavefinshedenteringvalues,clicktheCommentstab.
8. Entercommentsabouttheenzyme,thenclicktheKeywordstab.

Import data 1
9. Toaddakeywordfortheenzyme,enteranewwordorselectaniteminthelistof
existingkeywords.
Tomovethekeywordintothekeywordlist,click<Add.Toremoveanitemfrom
thekeywordlist,selectit,thenclick>Remove.
10. Tosaveyourdata,clickOK.ToclosetheEnzymewindowwithoutsavingyour
data,clickCancel.
Import data
YoucanimportDNA/RNAandproteinmolecules,enzymes,oligos,andgelmarkers,
assemblyprojects,andBLASTresultsintheVectorNTIExpressSoftwareLocal
Database.
Import molecules Youcanimportmolecules(includingtheirfeaturetables)fromDDBJ,EMBL,FASTA,

GCG,GenBank,GenPept,SWISSPROT,TrEMBL,andVectorNTIAdvanceArchive
files.TwotypesofDNA/RNAmoleculescanexistintheVectorNTIExpressLocal
Database:
CircularDNAMolecules
LinearDNA/RNAMolecules
MoleculesareimportedintheDNA/RNAMoleculessubsetandProteinMolecules
subsetwithintheLocalDatabase.TheDNA/RNAMoleculesandProteinMolecules
subsetscontaindescriptionsofamoleculesfeatures.
YoucanalsoimportnucleotideoraminoacidsequencesfromanASCIIfileofflexible
format,andVectorNTIExpressSoftwarewillautomaticallycreatethenewdatabase
moleculeandassignthesequencetothemolecule.
File formats ThefollowingfileformatscanbeimportedintoVectorNTIExpress:
File Format
DNA/RNA All nucleotide files (*.gb, *.gbwithparts, *.fasta, *.fas, *.fa, *.mpfa, *.fna,
molecule *.fsa, *.seq, *.embl, *.gcg, *.ma4, *.ddbj)
Genbank (*.gb, *.gbwithparts)
DDBJ (*.ddbj)
Fasta (*.fasta, *.fas, *.fa, *.mpfa, *.fna, *.fsa, *.seq)
EMBL (*.embl)
GCG (*.gcg)
Vector NTI Archive (*ma4)

Database Explorer
1 Import data
File Format
Protein molecule All protein files (*fasta, *fas, *fa, *mpfa, *.fna, *fsa, *seq, *.gp,
*.gpwithparts, *.swp, *.pa4, *.trembl)
Swiss-Prot (*.swp)
TrEMBL/EMBL (*.trembl,*.embl)
Vector NTI Archive (*pa4)
Enzyme REBASE (*.rebase), Vector NTI Archive (*.ga4)
Oligo Vector NTI Archive (*oa4), Oligo List (*.txt), Fasta (*.fasta, *.fas, *.fa,
*.mpfa, *.fna, *.fsa, *.seq)
Gel marker Vector NTI Archive (*.ga4)
Assembly Contig Project (*.cepx, *.cep)
Project
MSA projects Alignment Project (*.apr, *.aprx)
BLAST results Vector NTI Archive (*.ba6)
Import files Therearetwowaystoimportfiles:bydragginganddroppingfilesinWindows

ExplorerorbyusingthemainmenuintheVectorNTIExpressSoftware.
Drag and drop files into a subset

Todraganddropfilesintoasubset:
HighlightthefilenamesinWindowsExplorer.Selectthefiles,thendraganddrop
themontoasubsetintheLocalDatabasefolderintheExplorerViewer.
Import files via the main menu

Toimportobjectsviathemainmenu:
1. ClickFile>Import.
2. Inthedropdownlist,selectthetypeoffileyouwanttoimport.
3. IntheImportintoDatabasewindow,navigatetothefile,selectit,thenclickOpen.

Manage data 1
Manage data
Thissectiondescribeshowto:
ManageDatabase,Projects,andResultsdatabases(page 23)
ManageDatabase,Projects,andResultssubsets(page 23)
ManagedataintheDatabase,Projects,andResultsdatabasesbycreatingsubsetsof
subsets(subfoldersoffolders),groupingdatawithinsubsets,andorganizingsubsets.
Subsetscanbehierarchicallyorganizeddowntosixlevels.
Youcan:
CreateDNAandproteinmolecules,gelmarkers,oligos,enzymes,andprojects.
Openanassemblyproject;DNA,protein,and3Dmolecules;andBLASTresults.
ImportDNAandproteinmolecules,gelmarkers,enzymes,oligos,BLASTresults,
andassemblyprojects.
Editmoleculesbyinserting,deleting,andreplacingsequencefragmentsand
features.Youcanalsomodifythedisplayformatandgeneraldataofmolecules.
Manage Database, IntheDatabaseExplorer,youcancreatesubsetsinadatabaseandsearchdatabases.

Projects, and
Results databases Create a Local Database subset
Tocreateasubset:
1. IntheExplorerViewer,clickLocalDatabase,rightclickasubset(forexample,
DNA/RNAMolecules),thenselectAddsubsettoopentheCreateSubsetwindow.
2. Enterasubsetname(forexample,Hexokinase),descriptionofthesubset(for
example,Acollectionofhexokinasegenes),thenclickOK.
Search a Local Database subset

TosearchaLocalDatabasesubset:
1. IntheExplorerViewer,clickLocalDatabase,thenexpandadatabase.
2. Rightclickadatabase,thenselectSearchtoopentheSearchwindow.
3. Entertext.Tosearchfortheexacttext,checkExactmatchonly.ClickOKtobegin
thesearch.
Manage Database, ManageDatabase,Projects,andResultssubsetsintheExplorerViewer.Youcan:

Projects, and Addasubset(page 24)
Results subsets Searchalocaldatabaseorsubset(page 24)
Editthepropertiesofalocaldatabasesubset(page 24)
Renamealocaldatabasesubset(page 24
Clearalocaldatabasesubset(page 24)
Dismisslocaldatabasesubsets(page 25)
Deletethecontentsofalocaldatabasesubset(page 25)
Viewasummaryofalocaldatabasesubset(page 25)

Database Explorer
1 Manage data
TomanageLocalDatabasesubsets:
2. Rightclickasubset,thenselectamanagerialoperation.
Add a subset
ToaddasubsettoaLocalDatabase:
1. IntheExplorerViewer,clickLocalDatabase.
2. Rightclickadatabase,thenselectAddsubsettoopentheCreateSubsetwindow.
3. Enterasubsetnameanddescription,thenclickOK.
Search a local database or subset

Tosearchalocaldatabaseorsubsetinalocaldatabase:
1. IntheExplorerViewer,clickLocalDatabase.
2. Rightclickadatabase,thenselectSearchtoopentheSearchwindow.
3. Entertext.Tosearchfortheexacttext,checkExactmatchonly.ClickOKtobegin
thesearch.
Edit the properties of a local database subset

ToeditthepropertiesofaLocalDatabasesubset:
2. Rightclickadatabase,thenselectEditpropertiestoopentheEditSubset
window.
3. Enteranewnamefor,and/ordescriptionof,thesubset,thenclickOK.
Rename a local database subset

TorenameaLocalDatabasesubset:
1. IntheExplorerViewer,clickLocalDatabase,thenselectadatabase.
2. IntheRecordsViewer,rightclickanobject,thenselectRenametoopenthe
Renamewindow.
3. Enteranewname,thenclickOK.
Clear a local database subset

Note: ClearingaLocalDatabasesubsetofitscontentsdoesnotpermanentlydeletethe
contentsfromalocaldatabase.
Toclearasubsetofitscontents:
2. Rightclickasubset,selectClearsubsettoopentheSubsetManagementwindow,
thenclickOK.

Manage data 1
Dismiss local database subsets

Dismissingasubsetdeletesthesubsetfromalocaldatabase.
TodismissLocalDatabasesubsets:
2. Rightclickasubset,thenselectDismisssubsetstoopentheSubsetManagment
window,thenclickOK.
Delete the contents of a local database subset

Note: DeletingthecontentsofasubsetpermanentlydeletesthemfromtheLocal
Database.
TodeletethecontentsofaLocalDatabasesubset:
2. Rightclickasubset,selectDeleteSubsetContentstoopentheVectorNTI
Expresswindow,thenclickOK.
View a summary of a local database subset

ToviewasummaryofaLocalDatabasesubset:
2. Rightclickasubset,thenselectSubsetSummarytoopentheSubsetSummary
window.
Manage database ManagedatabaseobjectsintheRecordsViewer.Youcan:

objects Groupdatabaseobjects(page 25)
Sortdatabaseobjects(page 26)
Addanobjecttoasubset(page 26)
Renameanobject(page 26)
Excludeanobjectfromasubset(page 26)
Deleteanobject(page 27)
Duplicateanobject(page 27)
Viewobjectproperties(page 27)
Exportdata(page 27)
Showorhidecolumns(page 28)
Tomanagedatabaseobjects:
2. IntheRecordsViewer,rightclickadatabaseobject,thenselectamanagerial
operation.
Group database objects

Youcangroupdatabaseobjectsinsubsets.

Database Explorer
1 Manage data
Togroupdatabaseobjects:
2. IntheRecordsViewer,selectseveralobjects.
Note: Toselectspecificobjects,presstheCtrlkeyandclickobjects.Toselecta
blockofobjects,clickthefirstobjectintheblock,presstheShiftkey,thenclickthe
lastobject.
3. Addtheobjectstoasubset:
Rightclicktheselectedobjects,thenselectAddtosubsettoopentheSubset
Managementwindow.Selectasubset,thenclickOK,or
DragtheselectedobjectsintothesubsetintheExplorerViewer.
Note: Theobjectsyougroupedwerenotmovedoutoftheparentsubset.
Sort database objects

Tosortdatabaseobjectsinascendingordescendingorder:
2. IntheRecordsViewer,clickacolumnheader.
Add an object to a subset

Toaddanobjecttoasubset:
2. IntheRecordsViewer,rightclickanobject,thenselectAddtosubsettoopenthe
SubsetManagementwindow.
3. Selectthesubsetyouwanttoaddtheobjectto,thenclickOK.
Rename an object
Torenameanobject:
2. IntheRecordsViewer,rightclickanobject,thenselectRenametoopenthe
Renamewindow.
3. Enteranewname,thenclickOK.
Exclude an object from a subset

Note: Excludinganobjectfromasubsetdoesnotpermanentlydeleteitfromthe
database.
Toexcludeanobjectfromasubset:
2. IntheRecordsViewer,rightclickanobject,selectExcludefromsubsettoopen
theVectorNTIExpresswindow,thenclickOK.

Manage data 1
Delete an object
Topermanentlydeleteanobject:
2. IntheRecordsViewer,rightclickanobject,selectDeletefromDatabase,then
clickOK.
Duplicate an object
Toduplicateanobject
2. IntheRecordsViewer,rightclickanobjectormultipleobjects,thenselect
Duplicate.
Duplicatesofobjectsarecreatedinthedatabaseandincludedinthecurrentsubset.
TheduplicateofanobjectisnamedCopyof,forexample,CopyofEPAC.Multiple
duplicatesarenumbered,forexample,Copy#2ofEPAC.
Note: Duplicatesofobjectsarenotrelatedtotheoriginalobjects.Therefore,changes
thatyoumaketotheoriginalobjectarenotmadeinduplicates.
View object properties

Toviewobjectproperties:
2. IntheRecordsViewer,rightclickanobject,thenselectPropertiestoopenthe
propertieswindow.
Note: Ifoneobjectisselected,allthenamedobjectfieldswiththeirvaluesare
displayed.Someobjectdata(likesequenceandcomments)arenotstoredinnamed
fieldsandarenotdisplayedinthepropertieswindow.
Export data
ToexportDNA/RNAandproteinmolecules,enzymes,oligos,ContigAssembly
projects,andBLASTresults:
1. IntheExplorerViewer,clickLocalDatabase,thenselectadatabase.
2. IntheRecordsViewer,opentheExportDatawindow:
Selectanobject,thenclickFileExport,or
Rightclickanobject,thenselectExport.
3. Navigatetotheexportlocation.
4. (Optional)Renamethefileand/orselectanewfiletype,thenclickOK.

Database Explorer
1 Edit data
Youcanexportdatainthefollowingfileformats:
File Format
DNA/RNA Genbank (*.gb, *.gbwithparts)
molecule
DDBJ (*.ddbj)
EMBL (*.embl)
GCG (*.gcg)
ASCII (*.txt)
Protein molecule Fasta (*.fasta, *.fas, *.fa, *.mpfa, *.fna, *.fsa, *.seq)
SWISS-PROT (*.swp)
TrEMBL (*.trembl)
ASCII (*txt)
Enzyme REBASE (*.rebase)
Oligo CSV (*.csv)
BLAST results Tab-delimited (*.txt)
Contig Assembly Contig Project (*.cepx)
Project
Show or hide columns

ForalldatabasesintheLocalDatabase,youcanshoworhidecolumnsintheRecords
Viewer.
Toshoworhidecolumns:
2. IntheRecordsViewer,rightclickanywhereintherecordstable,thenselect
Column.
3. Inthedropdownlist,checkoruncheckthecolumnsthatyouwantshownor
hidden.
Edit data
TheoperationsdescribedinthissectionapplytotheEnzymes,Oligos,andGel
MarkersdatabasesintheLocalDatabase.Thissectiondescribeshowto:
Editenzymeproperties(page 28)
Editoligoproperties(page 29)
Editgelmarkerproperties(page 29)
Edit enzyme Toeditanenzyme:

properties 1. IntheExplorerViewer,clickLocalDatabase,thenselectEnzymes.

Edit data 1
2. IntheRecordsViewer,rightclickanenzyme,thenselectEdit.
3. Intheenzymewindow,clicktheEnzyme/Methylasetabtoeditthefollowing
properties:
:
Recognition String Enter a nucleotide string that can be recognized as

the enzyme.
Cleavage Point/Methylation Base Enter the number of the nucleotide immediately
after the direct-strand cleavage point.
Description Enter or edit the enzymes description.
4. ClicktheUserFieldstabtoeditthevaluesofthefollowingfields:
CommercialSources Organism
FreezerPosition WWWSource
5. ClicktheCommentstabtoentercommentsabouttheenzyme.
6. ClicktheKeywordstabtoaddorremovekeywordsthatwillimprovedatabase
searches.
Edit oligo Toeditanoligo:

properties 1. IntheExplorerViewer,clickLocalDatabase,thenselectOligos.
2. IntheRecordsViewer,rightclickanoligo,thenselectEdit.
3. Intheoligowindow,clicktheOligotabtoeditthefollowingproperties:
Nucelotide Sequence Enter or edit the oligos nucleotide
sequence. Valid characters: ATUCG
DNA/RNA Order Select a DNA or RNA sequence, then click
Order to open your browser and order
your primer sequence from Life
Technologies Corporation.
Reverse Complementary Check the box to replace the oligo
sequence with the complementary
sequence.
Description Enter or edit the oligos description.
4. ClicktheUserFieldstabtoeditthevaluesofthefollowingfields:
CommercialSources Organism
FreezerPosition WWWSource
5. ClicktheCommentstabtoentercommentsabouttheenzyme.
6. ClicktheKeywordstabtoaddorremovekeywordsthatwillimprovedatabase
searches.
Edit gel marker Toeditagelmarker:

properties 1. IntheExplorerViewer,clickLocalDatabase,thenselectGelMarkers.
2. IntheRecordsViewer,rightclickagelmarker,thenselectEdit.

Database Explorer
1 Open other applications
3. IntheGeneraltabofthegelmarkerwindow,clickthe...buttontoselectaname
forthegelmarker.
4. ClicktheGelMarkertabtoeditthefollowingproperties:
Fragment Lists by length all the fragments making
up the marker.
Click Add or Delete to add a fragment to
the marker, or remove a fragment from
the marker.
Fragment (length in b.p.) Enter the length of the fragment in base
pair (bp), then click Add.
Description Enter or edit the gel markers description.
Open other applications

YoucanrightclickonDNA/RNAandproteinmoleculesinthedatabaseandopenthe
followingapplications:
WebAnalyses(seepage 50)
AnalysisMonitor(seeChapter10onpage109)
BLASTSearch(seeChapter6onpage81
BioAnnotator(seeChapter4onpage71)
AlignX(seeChapter8onpage95)
Toopenotherapplications:
1. IntheExplorerViewer,clickLocalDatabase,thenselectDNA/RNAMolecules
orProteinMolecules.
2. IntheRecordsViewer,rightclickamolecule,thenselectanapplication.
Copy, save, and print molecules

CopyamoleculebytakingascreenshotwiththeCameratool.Savethescreenshotas
textorasanimageinanapplicationsuchasMicrosoftWordorMicrosoftExcel.Print
thetextorimage.
Copy a molecule UsetheMoleculeEditortocopymolecules.IntheDatabaseExplorer,openthe

MoleculeEditorbyopeningamolecule:
1. IntheExplorerViewer,clicktheDNA/RNAMoleculesfolderortheProtein
MoleculesfolderintheLocalDatabasefoldertodisplaythemoleculesinthe
RecordsViewer.
2. IntheRecordsViewer,doubleclickamoleculetodisplayagraphicalmapofa
moleculeanditssequence.
TheExplorerViewerisreplacedbythePropertiesViewer.TheRecordsVieweris
replacedbytheMoleculeEditor.TheSummaryViewerisreplacedbythe
SequenceViewer.

Copy, save, and print molecules 1
Note: Youcanwiden,narrow,lengthen,andshortenthesizeoftheviewersand
MoleculeEditorbyclickinganddraggingtheviewerframes.
FormoreinformationabouttheMoleculeEditor,seeChapter2onpage39.
Molecule
Properties Editor
Viewer
Sequence Viewer
Copy molecule properties, feature map, and analysis results

Tocopymoleculeproperties,featuremap,andanalysisresults:
orProteinMolecules.
2. IntheRecordsViewer,doubleclickamoleculetoopenit.
3. RightclickanywhereintheProperties,FeatureMap,orAnalysisResultsviewers,
thenselectCameratotakeascreenshotofthedata.
Thedataiscopiedtotheclipboardandcanbepastedinotherapplications.
Copy molecule image

TocopyanimageofDNA/RNAmoleculesandproteinmolecules:
orProteinMolecules.
2. IntheRecordsViewer,doubleclickamoleculetoopentheMoleculeEditor.
3. Rightclickthegraphicaldepiction,thenselectCamera.
4. Clickanywhereinthegraphictotakeascreenshot.Theimageiscopiedtothe
clipboardandcanbepastedinotherapplications.
Copy a sequence
Tocopyasequence:

Database Explorer
1 Copy, save, and print molecules
orProteinMolecules.
2. IntheRecordsViewer,doubleclickamoleculetoopentheMoleculeEditor.
3. Selectasequence:
Clickanddragacrossasequenceregioninthemoleculeimage,or
ClickanddragacrossthetextintheSequenceViewer.
Rightclick,thenselectCopy
Thedataiscopiedtotheclipboardandcanbepastedinotherapplications.
Save a molecule MoleculesyoucreatecanbesavedtoonlytotheLocalDatabase.Tosavedatabase

molecules:
1. ClickFileSaveinthemainmenu,or
ClickFileSaveAstosavethemoleculewithadifferentnameand/ortoa
differentlocation:
a. Enteranewnameforthemolecule.
b. Selectthedestinationfolderforthemolecule.
Optionallysavetheobjectasafile.Selectthefiletype:
ForDNA/RNAmolecules:Genbank,GenbankOldFormat,EMBL,
Fasta,DDBJ,orASCIItext
Forproteinmolecules:ASCIItext,GenPept,GenPeptOldFormat,
SwissProt,Fasta,orTrEMBL
2. ClickOK.

Manage the Projects database 1
Print molecule Toprintmoleculedata:

data 1. IntheExplorerViewer,clickLocalDatabase,thenselectDNA/RNAMolecules
orProteinMolecules.
2. IntheRecordsViewer,doubleclickamoleculetoopenit.
3. RightclickinthePropertiesViewer,MoleculeEditor,orSequenceViewer,then
selectCameratotakeascreenshot.
4. OpenanapplicationsuchasMicrosoftWord,Excel,Paint,orNotepadandpaste
thescreenshot.
5. Printthedatafromtheapplication.
Manage the Projects database

YoucanperformthefollowingoperationstomanagetheProjectsdatabase:
Createaproject(page 33)
Openaproject(page 33)
ManageProjectssubsets(page 33)
DeleteProjectssubsets(page 34)
Create a project Tocreateaproject:
1. Inthemainmenu,clickFileNew,thenselectthetypeofprojectyouwantto
create.
Alignment GatewayCloning
ContigExpress Clone2Seq
TOPOCloning PartsAssembler
2. Intheprojectwindow,enteraprojectnameanddescription,thenclickNew.
Open a project Toopenaproject:
1. ClicktheProjectsbar.
2. IntheProjectsViewer,expandtheLocalDatabase.
3. Selectthefoldercontainingthetypeofprojectyouwanttoopen.
4. IntheRecordsViewer,doubleclickaprojecttoopentheprojectapplication
(for example,Alignment)andviewitsproperties.
Manage Projects TomanagesubsetsintheProjectsdatabase:

subsets 1. ClicktheProjectsbar.
2. IntheProjectsViewer,expandtheLocalDatabase,thenexpandaproject.
3. Selectthesubsetyouwanttomanage.
4. Rightclickasubset,thenselectamanagerialoperation.Formoreinformation,see
ManageDatabase,Projects,andResultssubsetsonpage23.

Database Explorer
1 Manage the Results database
Delete Projects YoucandeletesubsetsofAlignment,ContigAssembly,orCloningprojects.Youcannot

subsets deletetheAlignment,ContigAssembly,andCloningfoldersintheLocalDatabase.
TopermanentlydeletethesubsetofanAlignment,ContigAssembly,orCloningproject
fromthedatabase:
1. ClicktheProjectsbar.
3. Selectthefoldercontainingthetypeofprojectyouwanttodelete.
4. Rightclick,thenselectDismisssubsets.
Manage the Results database

YoucanviewBLASTresultsandanalysisresultsandmanagetheBLASTResultsand
AnalysisResultsdatabases.
View results ToviewBLASTresultsandanalysisresults:
1. ClicktheResultsbar.
2. IntheResultsViewer,expandtheLocalDatabase,thenselectBLASTResultsor
AnalysisResults.
3. IntheRecordsViewer,doubleclickaresulttoopentheBLASTVieweror
MoleculeEditor.
Manage Results TomanagesubsetsintheResultsdatabasse:

subsets 1. ClicktheResultsbar.
3. Rightclickasubset,thenselectamanagerialoperation.Formoreinformation,see
ManageDatabase,Projects,andResultssubsetsonpage23
Workgroup Shared Database

Thissectiondescribeshowto:
Connecttoaworkgroupshareddatabase(page 35)
Adduserstoaworkgroupshareddatabase(page 36)
Ausernameshouldbeonewordwithoutspaces.Editusersinaworkgroup
shareddatabase(page 37)
Deleteusersfromaworkgroupshareddatabase(37)
Uploaddatatoaworkgroupshareddatabase(page 37)
Downloaddatafromaworkgroupshareddatabase(page 37)
Disconnectfromaworkgroupshareddatabase(page 37)

Workgroup Shared Database 1
About workgroup Youcancreatespecialdatabases,repositoriesofDNA/RNAorproteinmolecules,

shared databases enzymes,oligonucleotides,andgelmarkersandsharethemamongseveralVectorNTI
ExpressSoftwareusersonanetwork.Workgroupshareddatabasesarenota
replacementforlocaldatabases;eachVectorNTIExpressSoftwareapplicationstill
musthaveitsownlocaldatabase.Thelocaldatabaseisusedinalloperations,for
example,theconstruction,design,andcreationofviewers.Thelocaldatabaseisalso
theplaceforstoringprivateandtemporarydata.Themainpurposeoftheworkgroup
shareddatabaseistostorecommondata.
Theonlyoperationsyoucanperformonworkgroupshareddatabasesare:
Copyingdatatoandfromthelocaldatabase.Forinstance,youcancopysomeof
yourmoleculesandenzymesfromyourlocaldatabaseintotheworkgroupshared
database.Inordertousetheminthedesignprocess,yourcolleaguemustfirst
copythemtohisorherlocaldatabase.
Performingvariousdatabasemanagementoperationssuchascreatingand
deletingsubsets.
Runningdatabasesearches.
Inadditiontobiologicaldata,eachdatabasecontainsinformationaboutitscreatorand
registeredusers.Onlythecreatorandregistereduserscanhaveaccesstodatabase
data.Thedatabasecreatorcanalsochangedatabaseproperties,removeregistered
users,anddefinethepasswordrequiredtobecomearegisteruser.
Note: VectorNTIExpressworkgroupshareddatabasesuseportabledataformat
(PDF)andfilenamingconventionstoensurethatbothMacintoshandWindowsusers
ofVectorNTIExpressSoftwarecanaccesscommondatabases.Workgroupshared
databasescanbelocatedonawiderangeoffileservers.VectorNTIExpressSoftware
canusenotonlyservicesnativetoeachsystem(MicrosoftNetworkorAppleTalk),but
alsovariousUnix(NFSorSamba)andNetWareservices.
TheworkgroupshareddatabasecapabilityisapurchasedadditiontoVectorNTI
ExpressSoftware.Whenyoupurchasetheworkgroupshareddatabasecapability,you
areissuedaVectorNTIExpressSoftwareworkgroupshareddatabaselicensethat
enablesyoutocreateworkgroupshareddatabases.Aworkgroupshareddatabase
licenseisaspecialtypeofstaticlicensethatenablesyoutocreatenumerous
workgroupshareddatabases.Butthelicensealsolimitsthenumberofusersforeach
databaseyoucreate.Youdonotneedaworkgroupshareddatabaselicensetoaccess
workgroupshareddatabases.
Note: WorkgroupshareddatabasescanbeaccessedfromVectorNTIExpressSoftware
usingalicensethatissharedthroughanetworkserver(DynamicLicense).
Connect to a Youcanconnecttoaworkgroupshareddatabasethroughonlyahostnameand
workgroup shared IP address,notviatheWorldWideWeb.
database Whenthenetworkdirectoryforanewworkgroupshareddatabaseisarranged,
connecttoaworkgroupshareddatabase.Beforeyoucanconnecttoaworkgroup
shareddatabase,theworkgroupshareddatabaseservermustbestarted.
Start the workgroup shared database server

InC:\ProgramFiles\LifeTechnologies\VectorNTIWorkgroupSharedDatabase
Admin\h2_setup,doubleclickstartSharedServerandopenthecmd.exewindow:

Database Explorer
1 Workgroup Shared Database
Note: Thecmd.exewindowautomaticallyopensaftertheinstallationofShared
DatabaseAdmin.
Connect to the workgroup share database server

1. OpenVectorNTIExpressSoftware,then:
Inthemainmenu,clickToolsConnecttoworkgroupshareddatabase,or
OpentheDatabaseExplorer.IntheExplorerViewer,clickWorkgroup
SharedDatabase.
2. IntheConnecttoWorkgroupSharedDatabasewindow,logon:
HostName:localhost
UserName:Yourusername
PasswordYouruserpassword
Note: (HostNamefield)IftheWorkgroupSharedDatabaseAdminapplication
andVectorNTIExpressSoftwarearenotinstalledonthesamecomputer,enter
theIPaddressofthecomputeronwhichtheSharedDatabaseAdminapplication
isinstalled.TheIPaddressisshowninthecmd.exewindow:
3. ClickConnect.
4. ClosetheConnecttoWorkgroupSharedDatabasewindow.Yourconnectionwill
notbebroken.
Add users to a Youmustbeanadministratortoadduserstoaworkgroupshareddatabase.

workgroup shared Toadduserstoaworkgroupshareddatabase:
database
1. InC:\ProgramFiles\LifeTechnologies\VectorNTIWorkgroupSharedDatabase
Admin\h2_setup,doubleclickstartSharedServertostarttheserver.
2. DoubleclicktheSharedDatabaseAdministrationicononyourdesktoptoopen
theLoginwindow.
3. Login:
DatabaseHostname/IP:localhost
Administratorusername:admin
Password:admin

Set preferences 1
4. ClickLogintoopentheSharedDatabaseAdministratorwindow.
5. ClickAdd,thenenterinformationintheAddUserwindow.
A user nameshould Toeditusersinaworkgroupshareddatabase:

beonewordwithout 1. OpentheWorkgroupSharedDatabaseAdminapplicationandloginasan
spaces.Edit users in administrator.
a workgroup
2. IntheWorkgroupSharedDatabaseAdministratorwindow,doubleclickauserto
shared database
opentheEditUserwindow.
3. Edittheuserinformation,thenclickOKtosaveyouredits.
Delete users from Todeleteusersfromaworkgroupshareddatabase:

a workgroup 1. OpentheWorkgroupSharedDatabaseAdminapplicationandloginasan
shared database administrator.
2. IntheWorkgroupSharedDatabaseAdministratorwindow,selectauser,click
Delete,thenOK.
Upload data to a TouploaddatafromtheLocalDatabasetoaworkgroupshareddatabase:

workgroup shared 1. Connecttoaworkgroupshareddatabase.
database
2. IntheExplorerViewer,clickLocalDatabase,thenselectasubset.
3. IntheRecordsViewer,rightclickanobject,thenselectUploadtoworkgroup
shareddatabase.
Download data TodownloaddatafromaworkgroupshareddatabasetotheLocalDatabase:

from a workgroup 1. Connecttoaworkgroupshareddatabase.
shared database
2. IntheExplorerViewer,clickWorkgroupSharedDatabase,thenselectasubset.
3. IntheRecordsViewer,rightclickanobject,thenselectUploadtolocaldatabase.
Disconnect from a Todisconnectfromaworkgroupshareddatabase:

workgroup shared 1. 1.ClickToolsinthemainmenu,thenConnecttoworkgroupshareddatabase.
database
2. 2.IntheConnecttoWorkgroupSharedDatabasewindow,clickDisconnect.
Set preferences
Youcansetpreferencesfordisplayingmoleculesandsequencesandregisteryour
emailaddresswiththeNationalCenterforBiotechnologyInformation(NCBI).
Set display Tosetdisplaypreferencesformolecules:

preferences for 1. Inthemainmenu,clickToolsPreferencestoopenthePreferenceswindow.
molecules

Database Explorer
1 Set preferences
2. IntheDisplayPreferenceview,clickthebackgroundcolorandhighlightcolor
buttonstoopentheColorpalette.
3. Selectbackgroundandhighlightcolors,thenclickOK.
4. Tosaveyourpreferences,clickApply,orclickRestoreDefaultstokeepthe
originalsettings.
Set display Tosetdisplaypreferencesforsequences:

preferences for 1. ExpandDisplayPreference,thenclickSequencePreference.
sequences
2. ClickSequencefillcolorandSequencelinecolortoopentheColorpaletteand
selectcolors.
3. Entervaluesforthesequencewidthandsequencelinewidthparameters.
4. Tochangethetitlefont,clicktheChangebutton,thenselectanddefineafont.
5. Tochangethetitlefontcolor,clickthebuttontoopentheColorpaletteandselect
acolor.
6. ClickApplytosaveyourpreferences.
Register with the ToregisteryouremailaddresswiththeNCBI:

NCBI 1. ClickNCBIConfiguration.
2. Enteryouremailaddress,thenclickRegistertoopenyouremailapplication.
3. ClickOKtoclosethePreferenceswindow.

2 Molecule Editor
TheMoleculeEditorinVectorNTIExpressdisplaysagraphicalrepresentationofa
DNAorproteinmolecule,themoleculesequence,informationaboutthemolecule,and
theresultsofanyanalysisperformedonthemolecule.
UsingtheMoleculeEditor,youcan:
Createandeditamoleculesequence
Annotatethemolecule
Analyzethemolecule
ForDNA/RNAmolecules,youcanperformrestrictionanalysis,design
primersfromthesequence,findORFs,translatethesequenceintoamino
acids,andotherfunctions.
Forproteinmolecules,youcanscanformotifs,performwebsearchesonthe
sequence,performbacktranslation,andotherfunctions.
Moleculesequencesmayalsobeanalyzedusingavarietyofwebtoolsand
publiclyavailableanalysisengines
Displaytheanalysisresults
Create or open a molecule

Create a new 1. Tocreateanewmolecule,clickonFile>NewandselectDNAorProteinasthe
molecule moleculetype.
Note: ForDNAorRNAmolecules,specifyLinearorCircular.
2. TheblankmoleculewillbedisplayedintheMoleculeEditorbutwillnotyetbe
savedinthedatabase.
Open an existing Toopenanexistingmolecule:

molecule Formoleculesinthedatabase,doubleclickonthemoleculenameintheDatabase
Explorerwindow
Formoleculesthatarenotinthedatabase,selectFile>Openandselecta
moleculeoftheappropriatefiletype(.gb,.fasta,etc.)
ThemoleculewillbedisplayedintheMoleculeEditor.

Molecule Editor
2 Molecule Editor window
Molecule Editor window

TheMoleculeEditorwindowconsistsoffivemainpanesaswellasvarioustoolsand
analysisfunctions:
Display tools Graphics pane
Analysis tools
Properties pane
Feature Map
Analysis Results
Sequence pane
TheGraphicspanedisplaysagraphicalrepresentationofthemolecule,including
features.
TheSequencepanedisplaysthemoleculesequence,aswellastheresult
ThePropertiespanedisplaysinformationaboutthemolecule.
TheFeatureMapdisplaysalistofthedefinedfeaturesinthemolecule.

Enter or edit a sequence 2
TheAnalysisResultsdisplaysanalysesthathavebeenperformedonthe
molecule.
TheAnalysistoolstoolbarcontainsanalysisfunctionsspecifictotheMoleculeEditor.
TheDisplaytoolstoolbarcontainstoolstomagnifythemolecule,ordisplayaDNA
sequenceaslinearorcircular.
Molecule display Usethetoolsatthetopofthewindowtozoominandoutonthemolecule,fitthe

tools moleculewithinthewindow,ordisplayDNA/RNAmoleculesaslinearorcircular.
Enter or edit a sequence

ThemoleculesequenceisdisplayedintheSequencepane,andagraphical
representationofthesequenceisdisplayedintheGraphicspane.
Enter a sequence 1. Toenteranewsequence,clickonaninsertpointintheSequencepaneandbegin

typing.
2. TheEditSequencedialogwillopen,displayingyourtypedsequence.
3. ContinuetypingandclickonOKtoinsertthesequence.

Molecule Editor
2 Molecule features
Select a sequence Toselectpartorallofasequence:

DragyourcursoroverthedesiredportionsequenceintheSequencepaneor
Graphicspane.
ClickonafeatureintheGraphicspaneorFeatureMapthatencompassesthat
sequence.
ClickCtrl+aorrightclickandselectSelectalltoselecttheentiresequence.
Cut or copy a Tocutasequence,selectitintheSequenceorGraphicspaneandclickCtrl+x,or

sequence rightclickandselectCut,orselectthecommandfromtheEditmenu.Youwillbe
promptedtoremovethesequence.
Tocopyasequence,selectitandclickCtrl+c,orrightclickintheSequenceor
GraphicspaneandselectCopy,orselectthecommandfromtheEditmenu.
Paste a sequence 1. Topasteasequencefromtheclipboard,clickonaparticularinsertionpointinthe

SequenceorGraphicspaneandclickCtrl+vorselectEdit>Paste.
2. TheEditSequencedialogwillopen,displayingyourpastedsequence.Clickon
OKtocompletetheaction.
Replace a Toreplacepartorallofasequence,selectitintheSequencePaneandtypeorpasteas
sequence describedabove.
Delete a sequence Todeleteallorpartofasequence,selectitintheSequenceorGraphicspaneandclick

theDeletekeyorrightclickandselectDelete.
Reverse a Youcanreverseallorpartofasequence:
sequence 1. SelectallorpartofthesequenceintheSequenceorGraphicspane.
2. RightclickandselectReverseSelectiontoComplementary.
Molecule features
MoleculefeaturesareregionsofaDNAorproteinsequencethatyoudefineand
annotate.ThefeatureisthendisplayedintheGraphicspaneandlistedintheFeature
Map.
TypicalDNAmoleculefeaturesmightincludecodingregions,specificORFs,primer
bindingsites,etc.Proteinmoleculefeaturesmightincludestructuralfeatures,binding
sites,functionaldomains,etc.
Create a molecule Tocreateamoleculefeature:

feature 1. SelectaportionofthemoleculesequencebydraggingintheGraphicspaneorin
theSequencepane.
2. RightclickandselectCreatefeaturefromselection.
3. IntheFeaturedialog,selectacategoryfromtheFeatureTypelist.Various
miscellaneouscategoriesareavailableunderMisc.

Restriction Analysis 2
Feature dialogs for DNA (left) and protein (right)
4. EnteraFeatureNameandoptionalDescription.
5. TheLocationrangeisautopopulatedbasedonyourselection.Enteradifferent
rangeinthefieldsifdesired.
6. ForaDNAfeature,selecttheComplementarycheckboxifthefeatureislocated
onthereversestrand.
7. ClickonAddorDeletetoaddorremoveAnnotationkeys.Whenaddingakey,a
blankkeyiscreatedinthetable;typetoreplacethetext.
8. Whenyouarefinished,clickonOK.ThefeaturewilldisplayedintheGraphics
paneandintheFeatureMap.
Select a feature Toselectamoleculefeature,clickonitintheGraphicspaneorintheFeatureMap.
Hide or display a Tohideafeatureorgroupofsimilarfeatures,deselecttheappropriatecheckboxinthe

feature FeatureMap.Toredisplayit,reselectthecheckbox.
Edit or delete a Toeditafeature,rightclickonitintheGraphicspaneandselectEditfeature.The

feature Featuredialogwillopen.
Todeselectafeature,rightclickonitintheGraphicspaneandselectDeletefeature.
Youwillbepromptedtoconfirmthedeletion.
Restriction Analysis
YoucanidentifytherestrictionsitesinaDNA/RNAsequenceforhundredsof
restrictionenzymes.
Tobegin,withaDNA/RNAmoleculeopen,clickontheRestrictionAnalysis
buttonontheAnalysistoolbar.

Molecule Editor
2 Restriction Analysis
TheRestrictionAnalysistoolincludescommandsforselectingandconfiguringalistof
restrictionenzymes,andrunningtheanalysis.
Selecting enzymes Adefaultlistofrestrictionenzymestobeusedintheanalysiswillbeselected.

for analysis Clickonenzymesinthelisttoselectordeselectthem.
Toclearthelist,clickonClearSelection.
Toselectalltheenzymesinthelist,clickSelectAll.
Configuring the 1. ToconfiguretheenzymesintheRestrictionAnalysistool,clickonConfigureList.

enzyme list 2. UsethecommandsintheSelectedRestrictionEnzymesdialogtoaddorremove
theselectedenzymes,thenclickonApply.

ORF Finder 2
Filter the analysis Tofiltertheanalysisresultsbytheterminustypeoftherestrictioncutsite,selector

results deselectthecheckboxesunderPermittedTerminusTypes.
Note: 5and3refertotheoverhangoftheresultingcutsite,e.g.,with5deselected,
restrictionsiteswith5overhangswillberemovedfromtheanalysis.
IgnoreRENsHavingLessThan/MoreThanSiteshidesrestrictionsitesthatdonot
fallwithintherangeofthespecifiednumberofcutsites.SuchRENswillbelistedbut
deselectedintheRestrictionMapintheAnalysisResults.Theywillnotbedisplayedat
allintheGraphicsandSequencepanes.
Perform the Whenyouhavemadeyourselections,clickonRun.Thesiteswillbedisplayedinthe

analysis GraphicsandSequencepanesandlistedintheAnalysisResults.
TheresultsofRestrictionAnalysisaresavedwiththemolecule.
ORF Finder
To identify ORFs in a DNA or RNA sequence, click on the ORF Finder button
in the Analysis toolbar of the Molecule Editor.
The ORF Finder tool contains settings for identifying ORFs within the sequence.

Molecule Editor
2 ORF Finder
Define start and IntheStartandStopCodonfields,enterstartandstopcodonsforthenewORFs.

stop codons ClicktheDefaultStart&Stopbuttontosetthestartandstopcodonstothe
followingconventionalvalues:StartcodonsATG,GTG;StopcodonsTAA,
TGA,TAG.
ChecktheIncludeStopCodoninORFboxifyouwantthestopcodontobe
consideredasapartoftheORF.Otherwise,thestopcodonisnotconsideredas
partoftheORFandisnotincluded.
ORF Types Complete:TheCompletecheckboxisselectedbydefaultandintheMinimum

Sizefieldtheminimumsizeinbasepairsisspecifiedas150.Thiscanbechanged
accordingtoyourrequirement.ChecktheNestedcheckboxtolookfornested
ORFs.TheseareORFsthatoccurwithinthemainORF.
Incomplete:ChecktheIncompletecheckboxandintheMinimumSizefield
specifytheminimumsizeinbasepairsfortheincompleteORFtobedisplayed.
ChecktheUndefinedStart/StopcheckboxtosearchforORFswithundefined
starts,stopsorboth.ChecktheNestedcheckboxtolookfornestedORFs.
Note:
AnORFwithanundefinedstartisonethathasastopcodonattheendbutno
correspondingstartcodoninthesameframe
AnORFwithanundefinedstopisonethathasastartcodonbutnostopcodonin
thesameframe.
IncompleteORFsaredisplayedasadashedarrowinboththeGraphicsandthe
SequencePane.CompleteORFsaredisplayedassolidarrows.
Perform the Click on Run to perform the ORF analysis. The ORFs will be marked with directional
analysis arrows in the Graphics and Sequence panes and listed in the Analysis Results.
TheresultsofORFanalysisaresavedwiththemolecule
By default, ORFs are displayed for the direct and complementary strands. If single-
stranded sequence is displayed, only the ORFs for that strand are shown.

Translation tool 2
Translation tool
To translate a RNA or RNA sequence into amino acids, click on the Translate
button in the Analysis toolbar of the Molecule Editor.
TheTranslationtoolcontainssettingsfortranslatingthesequence.
1. Selectthegeneticcodeforthedesiredorganismtouseasthebasisforthe
translationfromtheGeneticCodedropdownlist.Formoreinformationon
geneticcodes,visitwww.ncbi.nih.gov/Taxonomy/Utils/wprintgc.cgi?mode=c.
2. SelecttheTranslationSourcefromamongthefollowingoptions:
DirectStranddisplaysatranslationofthedirectstrandsequenceinthe
currentframe
ComplementaryStranddisplaysatranslationofthecomplementary
strandsequenceinthecurrentframe
6FrameTranslationdisplaysupto6translationsofthesequence
dependingontheselectionsintheTranslationFramecheckboxes
TranslateORFsifyouhaveusedtheORFFindertoidentifyORFsinthe
sequence,thisselectionwilltranslatetheORFs
3. Ifyouselected6FrameTranslation,selectnumberofdirectandcomplementary
TranslationFramesusedtotranslatethesequence.Uptothreedirectstrand(+1,
+2,+3)andthreecomplementarystrand(1,2,3)translationsmaybeselected.
4. Tocreateanewproteinmoleculefromthetranslation,selecttheTranslateinto
newprotein.OtherwisethetranslationwillbedisplayedwithintheDNA/RNA
sequence.
5. ClickonRunbuttontoperformthetranslation.
Translation results IfyouselectedTranslateintonewprotein,youwillbepromptedtonamethenew

proteinandtheproteinmoleculewillbecreated.
Otherwise,translationsofthedirectstrandwillappearabovetheDNAsequenceand
translationsofthecomplementarystrandwillappearbelowthesequence.

Molecule Editor
2 Oligo Duplex Analysis
Translated sequence using 6 Frame Translation with +2 Frames selected
Oligo Duplex Analysis

OligoDuplexAnalysisenablestheanalysisofoneormoreoligosforpotentialcross
reactivityanddimerization.
Toopenthetool,clickontheOligoDuplexAnalysisbuttonintheMolecule
Editor.
Entering or Usingthetool,youcananalyzeoligosequencesthatyouenterinthefieldsatthetop,
selecting oligos oryoucananalyzesavedoligosinthedatabase.
EnteranoligonameandsequenceunderUserDefinedPrimersattopofthe
windowandclickonAddtoListtoaddittothelistofoligosequencesbelow,
and/orSavetoDatabasetosaveitasanoligointhedatabase.
ClickonSelectOligosinDatabasetoselectoneormoresavedoligosinthe
databasetoanalyze.UseCtrl+clickandShift+clicktoselectmultipleoligosfrom
theOligosinDatabasedialog.

Silent Mutation Analysis 2
TheselectedoligoswillappearintheSelectedOligos/Primerslist.
Analysis Selectfromthefollowingadditionalanalysisparameters:
parameters dGTemperature:enterthetemperatureindegreesCelsiustobeusedfor
calculatingfreeenergyvalues.
StemLength:entertheminimumacceptablenumberofbasepairsinahairpinor
dimerstem.
Run the analysis ClickonAnalyzetoruntheanalysis.Analysisresultsaredisplayedatthebottomof

thewindow.
ClickonSaveResultstosavetheanalysisasaseparatetextfile.
Note: Inthegraphicaldepictionofduplexes,verticallinesindicatetheprimary
interaction,basedonthestemlengthset,andplussymbolsindicatesecondary
interactions.ThegreaterthedGvalue,theweakertheinteraction;secondary
interactionsarenotconsideredinthedGcalculation.
Silent Mutation Analysis

IntheMoleculeEditor,youcansearchforsilentmutationsinaDNA/RNAsequence
orselectedregionofasequencethatdonotaffectaminoacidtranslationbutresultin
thecreationordisappearanceofoneormorerestrictionsites.
Note: YoudonotneedtoperformrestrictionanalysisbeforerunningSilentMutation
Analysis.
1. Selectaregionofthesequenceormakenoselectiontoanalyzetheentire
sequence.
2. ClickontheSilentMutationsAnalysisbuttontoopenthetool.
3. ClickonAddEnzymefromDatabasetoselectfromalistofrestriction
enzymesinthedatabase.UseCtrl+clickandShift+clickcommandstoselect
multipleenzymesintheEnzymeinDatabasedialog.
4. ClickonOKtoaddtheselectionstotheAvailableEnzymeslist.
5. Usethe>>,All>>,<<,and<<AllbuttonstomoveenzymesfromtheAvailableto
theUselist.

Molecule Editor
2 Web analyses
6. ClickonRuntoinitiatethemutagenesissearch.VectorNTIExpressanalyzesthe
sequenceorselectedregionandattemptstogeneratesuitablesilentmutations.
Thereadingframeforaminoacidsisdefinedbythestartoftheselectedregionso
thatthefirstthreenucleotidesoftheselectedregionformthefirstcodon.
Thefoldercontainsalistofmutationoptionsthatresultintheappearingand/or
disappearingofatleastonerestrictionsite.Theoptionsaresortedbythepositionof
thefirstalterednucleotide.Ifyouselectedthecomplementarystrandoption,mutation
coordinatesonbothcomplementaryanddirectstrandsarelisted.
Note: Theprogramisabletofindbothsingle(justonenucleotidealtered)and
multiple(severalneighbornucleotidesaltered)mutationsforanyelementaryevent
(appearingand/ordisappearingofatleastonesite)significantlywideningthesetof
possiblesolutionscomparedtojustsinglemutationanalysis.
Web analyses
ADNAorproteinmoleculesequenceorpartofasequencecanbeanalyzedusinga
varietyofonlinedatabases,searchengines,andanalysistools.
Note: TheseanalysesrequireanactiveInternetconnection.
1. IntheGraphicsorSequencepane,selecttheregionofthesequencetoanalyze,or
selectCtrl+Atoselecttheentiresequence.
2. RightclickandselectWebAnalyses>[searchtype]>[searchdatabase].
Note: YoucanalsorightclickonamoleculeintheDatabaseExplorertoanalyzethe
entiresequence.
3. Thesequencewillbeautomaticallytransferredtotheanalysisengineontheweb:

Back Translation 2
DNA Molecule Web Analyses.
Protein Molecule Web Analyses
Back Translation
BackTranslationallowsyoutoobtainaDNAsequencefromaproteinsequenceby
reversingthetranslationprocess.Youcanselectfromalistoftranslationandcodon
usagetableoptions

Molecule Editor
2 Protein Domain and Motif Finder Analyses
1. Withaproteinmoleculeopen,clickontheBackTranslationbuttoninthe
Analysistoolbar.
2. IntheBackTranslationTool,selectthetranslationtabletype:UseTranslation
TableorUseCodonUsageTable.
3. Selectthespecifictranslationtablefromthedropdownlist.
Note: Formoreinformationabouttranslationandcodonusagetables,visit
www.ncbi.nih.gov/Taxonomy/Utils/wprintgc.cgi?mode=c.
4. SelecttheTranslateintonewnucleotidecheckboxtocreateaDNA/RNA
moleculefromtheresultinganalysis.
5. ClickontheSubmitbutton.
Ifyouselectedthecheckboxinstep3,youwillbepromptedtoenteranameforthe
moleculeandsaveittothedatabase.
TheresultswillbedisplayedintheBackTranslationResultfield.
Protein Domain and Motif Finder Analyses

YoucananalyzeaproteinsequenceinVectorNTIExpressagainstdatabasesofknown
proteindomains,families,functionalsites,andmotiffingerprints.
Protein Domain PROSITEandPRINTSaredatabasesofbiologicallysignificantproteinpatternsand

AnalysisPROSITE profilesthatcanbeusedtoidentifythefunctionofuncharacterizedproteins.
ComparinganunknownpolypeptidesequenceinVectorNTIExpresstothemotifsin
and PRINTS
thesedatabasescanhelpdetermineanyknownproteinfamiliesanddomainstowhich
databases thesequencemaybelong.

Protein Domain and Motif Finder Analyses 2
FormoreinformationaboutthePROSITEdatabase,visithttp://
prosite.expasy.org/.
FormoreinformationaboutthePRINTSdatabase,visitwww.bioinf.man.ac.uk/
dbbrowser/PRINTS/index.php.
LocalversionsofthePROSITEandPRINTSmotifdatabasesareprovidedaspartofthe
VectorNTIExpressinstallation.
1. Tobegintheanalysis,withaproteinmoleculeopen,clickontheProteinDomain
buttonintheAnalysistoolbaroftheMoleculeEditorwindow.
2. SelectthelocalversionofthePROSITEorPRINTSdatabase,installedwithVector
NTIExpress.
3. IntheAnalysisJobswindow,thenameofthejobwillappearlistedintheleft
handpaneandthestatuswillbeNewsubmit...
4. Selecttheparametersthatwillbeincludedwiththeresults,thenclickonSubmit.
5. Whentheanalysisiscomplete,itwillbelistedinthelefthandpaneas
Complete.
6. Doubleclickonthecompletejobtoreturntothemoleculewiththeresults
displayedintheSequencepaneandtheAnalysisResults.
ClickonafeatureintheAnalysisResultspanetoshowexpandeddetailsforthat
result.
ClickonacheckboxintheAnalysisResultspanetoshoworhidethatfeaturein
theSequencepane.
RightclickintheAnalysisResultspaneandselectSaveAllAnalysisResultsto
DatabasetosavethesetoAnalysisResultsinyourlocaldatabase.

Molecule Editor
AnalysisResultssavedtothedatabasewilllistedintheDatabaseExplorer,under
ResultsintheAnalysisResultsfolder.
Motif Finder MotifFinderusesalocalversionoftheInterProScansequencesearchenginetoscana

InterProScan proteinsequenceforproteindomainsandfunctionalsites.Thissearchtoolintegrates
analysisenginesfrommultipleproteinsignaturedatabases.Usingthistool,youcan
sequence search
analyzeasequenceusingallInterProanalysisenginesorasubset.
FormoreinformationaboutInterProScanandthedatabases/analyticaltoolsit
includes,visitwww.ebi.ac.uk/Tools/pfa/iprscan/.
AlocalversionoftheInterProScansearchengineisprovidedaspartoftheVector
NTIExpressinstallation.
1. Tobegintheanalysis,withaproteinmoleculeopen,clickontheMotifFinder
buttonintheAnalysistoolbaroftheMoleculeEditorwindow.
2. IntheAnalysisJobswindow,thenameofthejobwillappearlistedintheleft
handpaneandthestatuswillbeNewsubmit...
3. Selecttheanalyticalapplication(s)tousefromtheavailablelistofInterProScan
tools,thenclickonSubmit.
Note: Themoreapplicationsyouselect,thelongertheanalysiswilltake.
4. Whentheanalysisiscomplete,itwillbelistedinthelefthandpaneas
Complete.

Protein Domain and Motif Finder Analyses 2
5. Doubleclickonthecompletejobtoreturntothemoleculewiththeresults
displayedintheSequencepaneandtheAnalysisResults.
ClickonafeatureintheAnalysisResultspanetoshowexpandeddetailsforthat
result.
ClickonacheckboxintheAnalysisResultspanetoshoworhidethatfeaturein
theSequencepane.
RightclickintheAnalysisResultspaneandselectSaveAllAnalysisResultsto
DatabasetosavethesetoAnalysisResultsinyourlocaldatabase.
AnalysisResultssavedtothedatabasewilllistedintheDatabaseExplorer,under
ResultsintheAnalysisResultsfolder.

Molecule Editor

3 Primer Design
ThischapterdescribesthefunctionsfordesigningprimersandprobesinVectorNTI
Express,includingsettingsfordesigningPCRprimers,sequencingprimers,and
hybridizationprobes.VectorNTIExpresscandesignprimersforanentireDNA
moleculesequenceorpartofasequenceselectedintheMoleculeEditorwindow.After
selectingthetargetsequence,themaximumandminimumproductlengthand
parametersaredetermined,andthesoftwareevaluates,ratesandsortsseveraldesign
options.Youcanfurtherfinetunetheoligosandannealingparametersifyouwish,
savetheprimersorprobesasseparatemoleculesinthedatabaseortotheOligoList,
ordercustomoligosfromLifeTechnologies,orusetheprimersinrecombinantcloning
strategies.
Thefollowingtablesummarizesthevariousprimer/probedesignoptionsin Vector
NTIExpress:
Design Tool Purpose

Find PCR Primers Inside Specify limits for PCR primer search such as length of target
Selection sequence, output options, attach restriction sites, etc.
Amplify Selection Similar to Find PCR Primers except that primer hybridization
domains upstream and downstream from the target sequence
can be specified. Primers will be generated anywhere within
the designated upstream and downstream domains.
Sequencing primers Set parameters for sequencing and primer regions and
primer; analyze primers.
Hybridization Probes Set parameters for target region, output options; analyze
probes.
PCR Using Existing Oligos Similar to Find PCR Primers, but, for the selected
amplification region, allows you to search for suitable PCR
primers from among those selected from the Vector NTI
Express oligo list.
Open the primer/probe design tools

ThedesigntoolsarelocatedontheMoleculeEditortoolbar.
1. WithamoleculeloadedintheMoleculeEditor,youcandesignoligosforthe
entiremoleculesequenceoraselectedregion:
Todesignprimers/probesfortheentiremolecule,makenosequence
selectionbeforeyouopenthedesigntool.
Todesignprimers/probesforaspecificregionofthesequence,selectthe
region(e.g.,bydraggingorclickingonafeatureintheGraphicsorSequence
pane)andthenopenthetool.

Primer Design
3 Save and load settings
2. ClickonPrimerDesigninthetoolbarandselectfromthedropdownlistofdesign
tools:
3. EachprimertoolopensinaseparatepaneintheMoleculeEditorwindow,with
thebasicsettingsaccessibleinthepane.Foradvancedsettings,clickonthe
Advancedbuttoninthepanetoopenaseparatedialogbox.
Primer Design pane
Advanced settings
Save and load settings

TheSaveandLoadbuttonsatthebottomofeachpane(oratthebottomofeachtabin
theAdvancedsettingsdialog)allowyoutosaveyourprimer/probedesignsettingstoa
fileandloadthesettingsfileforsubsequentanalyses.Designsettingsaresavedas
*.pcrfiles.
Thisisusefulforsavingfrequentlyusedsettings.
Run the design tool

Afteryouhaveselectedthedesiredsettings(seethefollowingpagesformoredetails
aboutindividualsettings),clickonRunintheprimerdesigntool,orclickonOKinthe
Advancedsettingsdialogofthetool.
Primer/probe Theprimerorprobedesignswillbeaddedasfeaturesinthemolecule,andlistedinthe
design results FeatureMapanddisplayedintheGraphicspaneoftheMoleculeEditor(see
Moleculefeaturesonpage42).
PrimerandprobedesignsaresortedindescendingorderintheFeatureMapaccording
totheirratingvaluescalculatedbasedontheimportancefactorsassignedinthe
Qualitiestab(seepage69).Themoleculeregionofeachdesignislistedinparentheses.

Run the design tool 3
TheresultsarealsolistedunderAnalysisResultsintheMoleculeEditorwindow,
alongwithspecificinformationabouteacholigosequence.
PrimerandprobedesignsarealsobelistedintheOrderingdialog,underPrimers.
Save primer/probe Thereareanumberofwaystosaveprimer/probedesigns:

designs TosavethemasAnalysisResultsinthedatabase,rightclickonatopbranchinthe
MoleculeEditorAnalysisResultspaneandselectSaveAllAnalysisResultsto
Database.
TosavethemasoligosintheOligosDatabase,rightclickonaprimer/probe
sequenceintheMoleculeEditorAnalysisResultspaneandselectSavePrimersto
OligoDatabase.
ToaddthemtotheOligosList,rightclickonaprimer/probesequenceinthe
MoleculeEditorAnalysisResultspaneandselectAddPrimerstoOligoList.

Primer Design
3 Find PCR Primers Inside Selection settings
Find PCR Primers Inside Selection settings

SelectFindPCRPrimersInsideSelectiontodesignprimerswithinaselectedregion
ortheentiremoleculeforPCRamplification.WiththemoleculeopenintheMolecule
Editor,selecttheregiontobesequenced,thenselectPrimerDesign>FindPCR
PrimersInsideSelectionfromtheMoleculeEditortoolbar.:
ThefollowingaretheuniquesettingsforFindPCRPrimers.Thesearealsoavailable
underthePrimerstabifyouclickontheAdvancedbutton:
Find PCR Primers

Description
setting
Region of Analysis The start and end coordinates of the region to amplify. You can enter
new coordinates, or select a region of the sequence before opening
the tool to pre-populate the region coordinates.
Product Length Enter the maximum and minimum lengths of the molecule target
region. Note: Unless you specify differently here, the minimum
amplicon length may be less than the target sequence you selected.
Maximum Number Enter the number of sense-antisense primer pairs to be found. The
of Primers actual result may contain fewer than this number if there are not
enough possible primers.
Analysis Conditions settings

Click on the Analysis Conditions dropdown to access these settings:
Analysis Conditions
Description
setting
DNA/RNA radio button Select the type of target nucleotide sequence.
Salt Concentration Enter the PCR reaction salt concentration in mMol, if known.
Probe Concentration Enter the value of probe concentration in pMol, if known.
dG Temperature Enter the temperature in degrees Celsius to be used for
calculating free energy values.

Amplify Selection settings 3
Analysis Conditions
Description
setting
Tm Enter limits in degrees Celsius for primer melting temperature
(Tm) (temperature at which 50% of primer is a duplex) and the
difference between Tm for sense and antisense primers.
%GC Enter the limits of G/C percentage in the primer and the difference
between GC percentages for sense and antisense primers.
Length Enter primer length limits. Note: Nucleotide sequences such as
RENs attached to a primers 5 end are included when calculating
primer length.
Note: ThecalculationforTmisdependentonprimerandsaltconcentrations;varying
theseconcentrationscangreatlyaffecttheTmforanygivenprimer.Makesureto
adjusttheseparametersaccordingtoyourreactionconditionswhenperformingyour
PCRanalysistoensurethatyouobtainaccurateTmvalues.
Attach to 5 Terminus settings

Click on theAttachto5Terminusdropdown to access these settings
Attach to 5
Description
Terminus setting
Attach to 5 Terminus Enter a short (=/<18 bp) nucleotide sequence (if any) to be attached
of Sense Primer to the 5 end of either primer. To choose from recognition sites of
and/or Antisense database RENs, click the Browse button next to each field.
Primer
Note: This sequence, while considered in primer parameters, does
not affect the calculation of complementarity between primer and
molecule. A sequence can be attached to the primer whether or not
the primers are user-defined or designed by the software.
Advanced settings: Primers tab

ClickontheAdvancedbuttonbelowthemainsettingstoopentheAdvancedsettings
dialog.Under the Primers tab, the settings described above are listed, in addition to the
following:
Advanced Primers
Description
setting
User-Defined Enter user-defined primer sequences or a primer from the oligo
Primers database. The search engine checks the compatibility of the primers
according to primer parameters.
AdditionaladvancedsettingsaredescribedinSharedAdvancedsettingsonpage66
Amplify Selection settings

SelecttheAmplifySelectionprimerdesigntooltoamplifyanentireselectedregionof
amolecule.WiththemoleculeopenintheMoleculeEditor,selecttheregiontobe
sequenced,thenselectPrimerDesign>AmplifySelectionfromtheMoleculeEditor
toolbar.

Primer Design
3 Amplify Selection settings
ThesesettingsaresimilartotheFindPCRPrimerssettingsexceptthatyoucanspecify
primerhybridizationregionsupstreamanddownstreamofthetargetsequence.
AmplifySelectionpicksprimerstoamplifytheentireselection.Ifsuitableprimers
cannotbefoundinsidetheselectedregion,thesearchwillexpandwithinthespecified
upstreamanddownstreamflankingregions.
ThefollowingaretheuniquesettingsforAmplifySelection.Thesearealsoavailable
Amplify Selection
Description
setting
Amplicon Must Set the 5 and 3 positions for region of the molecule that must be
Include Region of included in the final amplified product.
Molecule
Max bp before Provides additional upstream region where the Primer may be
selection made.
Max bp after Provides additional downstream region where the Primer may be
selection made.

For information about the Analysis Conditions settings, see page 60.

For information about the Attach to 5 Terminus settings, see page 61.
Advanced settings: Primers tab

dialog.For information about the Advanced settings on the Primers tab, settings, see
page 61.
Additional Advanced settings

AdditionaladvancedsettingsaredescribedinSharedAdvancedsettingsonpage66

PCR Using Existing Oligos settings 3
PCR Using Existing Oligos settings

SelectPCRUsingExistingOligostosearchforsuitablePCRprimersfromamong
thoseintheVectorNTIExpressOligoDatabase.Withthemoleculeopeninthe
MoleculeEditor,selecttheregiontobesequenced,thenselectPrimerDesign>PCR
UsingExistingOligosfromtheMoleculeEditortoolbar.
ThesesettingsaresimilartotheFindPCRPrimerssettingswithafewexceptions.The
followingaretheuniquesettingsforPCRUsingExistingOligos.Thesearealso
availableunderthePrimerstabifyouclickontheAdvancedbutton:
PCR Using Existing

Description
Oligos setting
Sense Primers Click the Sense Primers button and select the desired primer(s)
from the Oligo Database.
Antisense Primers Click the Antisense Primers button and select the desired primer(s)
from the Oligo Database.
Note: Sinceyoucanchoosethenumberof3and5primers,thesesettingseffectively
enableyoutoanalyzeone3primeragainstanarrayof5primersorviceversa.

For information about the Analysis Conditions settings, see page 60.

For information about the Attach to 5 Terminus settings, see page 61.
Advanced settings
dialog.AdvancedaredescribedinSharedAdvancedsettingsonpage66

Primer Design
3 Sequencing Primers settings
Sequencing Primers settings

SelectSequencingPrimerstofindprimersforsequencingaDNAmoleculefragment.
WiththemoleculeopenintheMoleculeEditor,selecttheregiontobesequenced,then
selectPrimerDesign>SequencingPrimersfromtheMoleculeEditortoolbar.
PrimerswillbegeneratedanywherewithinthedesignatedPrimerHybridization
Domain(upstreamanddownstream).
Ifthesequencingregionislongenough,itisdividedbyVectorNTIExpressinto
smallersequencingdomains,areasinwhichasinglesequencingreactionwilltake
place.Thesizeoftheprimerhybridizingdomainmaythenbeset,aswellasother
primerparameters.Severalprimeroptionsareevaluatedandsortedfrombestto
worst.
Note: Sequencingprimersaredesignedforasequenceregion,notanentiremolecule.
ThefollowingaretheuniquesettingsfortheSequencingPrimerstool.Thesearealso
availableunderthePrimerstabifyouclickontheAdvancedbutton:
Sequencing
Description
Primers setting
Sequencing Region Region that you want to sequence. Enter the start and end
coordinates of the region to be sequenced.
Sequencing Domain Enter the number of bases to be sequenced in a single sequencing
reaction.
Primer Hybridization Enter the length of region where primers for each sequencing
Domain domain should be sought. Primers are generated within the set
domain.
Maximum Number Enter the number of primers to be found for each sequencing
of Primers domain. (The actual result may contain fewer primers than this
number if there are not enough possible primers.)
DNA/RNA Select the type of nucleotide sequence.
Complementary Select if you are sequencing the complementary strand.
Strand

Hybridization Probes 3
Sequencing
Description
Primers setting
User-Defined First Enter a user-defined nucleotide sequence to be evaluated as a
Primer primer for the FIRST sequencing domain, instead of leaving the
primer search to Vector NTI Express Software.

For information about additional Analysis Conditions settings, see page 60.
Advanced settings
dialog.AdvancedsettingsaredescribedinSharedAdvancedsettingsonpage66.
Hybridization Probes
SelecttheHybridizationProbesettingstofindoligonucleotidesthatwillhybridizeto
aselectedmoleculesequence.Vector NTIExpress can generate a set of oligos or use
user-defined or database-stored oligos to test for hybridization efficiency with a target
molecule.
WiththemoleculeopenintheMoleculeEditor,selectthehybridizationregion,then
selectPrimerDesign>HybridizationProbesfromtheMoleculeEditortoolbar.
ThefollowingaretheuniqueHybridizationProbessettings.Thesearealsoavailable
Hybridization
Description
Probes setting
Search Region Region for which you want to design the probe. Enter the start and
end coordinates.
Maximum Number Enter the number of probes to be found for the region. (The actual
of Output result may contain fewer probes than this number if there are not
enough possible designs.)
DNA/RNA Select the type of nucleotide sequence.

Primer Design
3 Shared Advanced settings
Hybridization
Description
Probes setting
Complementary Select if you are sequencing the complementary strand.
Strand
User-Defined Oligo Enter a user-defined nucleotide sequence to be evaluated as a probe
instead of leaving probe search to the software.

For information about additional Analysis Conditions settings, see page 60.
Advanced settings
dialog.AdditionaladvancedsettingsaredescribedinSharedAdvancedsettingson
page66.
Shared Advanced settings

dialog.The following Advanced settings are the same for all the primer/probe design
tools.
Amplicon tab
ClicktheAmplicontabtocustomizeparametersrelatingtotheresultingPCRproduct.
%GCcontentfortheproductoraportionoftheproductandallowedbasesadjacentto
theprimerannealingsitecanbespecified.:
Amplicon setting Description

Amplicon %GC Enter the minimum and maximum for the desired %GC content in
the PCR product.
Next to Primer Choose accepted bases for the four successive bases adjacent to the
Annealing Site primer annealing site. Set minimum and maximum %GC range for
a specified length of the amplicon adjacent to the primer annealing
site.
Structure tab
ClicktheStructuretabtosetacceptablelimitsfornucleotiderepeats,palindromesand
hairpinloopsfortheprimers.Youcanalsocheckyourprimers/productforaselected
groupofrestrictionsitesfromthistab.
Structure setting Description

Nucleotide Repeats Enter the maximum permitted length of nucleotide repeats in
primers.
Palindromes Enter the maximum permitted length of palindromes in primers.

Shared Advanced settings 3
Structure setting Description

Hairpin Loops Stem Length: Enter the minimum number of base pairs in a
hairpin stem. (This value is also used as a minimum stacking
length for primer-primer complementarity and primer-primer 3
end complementarity.) Permitted with dG: Check the Permitted
box for hairpin loops; enter the minimum permitted value for free
energy of hairpin loops. Primers with hairpin loops which have
free energy values > /= to this number will be accepted.
Check Hairpin Loops, Check this box and enter the length of a 3 region if all of a
Palindromes, primers features (repeats, palindromes, hairpin loops, dimers)
Nucleotide Repeats should be checked only within that 3 region.(If this box is empty,
and Dimers Only Within the whole primer will be evaluated.)
3 Region of ...
Check Primers For Check to find possible cloning sites inside primers and attached
Restriction Sites From nucleotide sequences (if any). In the drop-down menu, specify the
REN subset. Enzymes will be checked for the presence of their
sites in the primers and attached sequences, and within the PCR
product.
Pairs tab
ClickthePairstabtospecifyhowcloselyparameterssuchasTmand%GC,etc.must
matchbetweentwoprimersinageneratedprimerset.
Pairs setting Description

Tm Difference Enter difference in degrees Celsius between Tm for sense and
antisense primers.
%GC Enter the difference between GC percentages for sense and
antisense primers.
Primer-Primer Check the Permitted box for primer-primer complementarity;
Complementarity enter the minimum permitted value for duplex free energy.
Primer-Primer 3 End Check the Permitted box for primer-primer 3 end
Complementarity complementarity; enter the minimum permitted value for duplex
free energy.
Similarity tab
ClicktheSimilaritytabtodeterminethesimilarityrelationshipbetweentheprimers
andthetargetsequence.
Similarity setting Description

Best Fit Check this button to specify the search for site(s) with maximum
similarity with no set threshold.
With Similarities > = Check this button to indicate similarity site search above the
Similarity Threshold specified similarity threshold.
Similarity Threshold Enter the percentage of minimally acceptable similarity.
Last ... Nucleotides Check and specify the number of nucleotides necessary to have
Must Have 100% 100% complementarity with the target sequence at the 3 end.
Similarity

Primer Design
Similarity setting Description

Similarity Between Specifies acceptable similarity between ambiguous nucleotides (if
Ambiguous any). The Average, Minimum, and Maximum buttons indicate that
Nucleotides the average, minimum, and maximum possible similarity will be
calculated respectively for any nucleotide pair. For instance, if you
are calculating similarity between N and A, then the average
similarity is 25%, the minimum similarity is 0%, and the maximum
similarity is 100%. In case of R and A they are 50%, 0%, and 100%;
in case of R and T0%, 0%, and 0%.
ThesimilaritytableusedbyVectorNTIExpressisasfollows:
N-N N-R N-A R-W R-A R-T

Maximum 100 100 100 100 100 0
Average 25 25 25 25 50 0
Minimum 0 0 0 0 0 0
3 End tab
Clickthe3Endtabtosetspecificationsforthe3endoftheprimersgeneratedby
VectorNTIExpress.ParameterssuchasdGandspecificnucleotidecontentforthe3
endofbothsenseandantisenseprimerscanbesethere.
3 End setting Description

dG <= Specify the maximum permitted value of 3 end free energy.
Length for Analysis Enter the length of the primers 3 region that should be analyzed.
Sense Primer 3 Check the nucleotide boxes to specify permitted last primer
Nucleotides nucleotides for the sense primer.
Antisense Primer 3 Check the nucleotide boxes to specify permitted last primer
Nucleotides nucleotides for the antisense primer.
Uniqueness Tab
ClicktheUniquenesstabtoselectsettingstodeterminetheuniquenessofthe
generatedprimers.Theseparameterscanbeusedtohelpensurethatgenerated
primersbindtothedesiredtemplateareawithgreaterspecificitythantotherestofthe
PCRproduct.
Uniqueness tab
Description
setting
Uniqueness Checks for Choose the area of the molecule to check for primer uniqueness.
Either the entire molecule or the Amplicon only can be selected
for the uniqueness check.
Max Allowed Similarity Check this box and enter the similarity threshold to check primer
uniqueness on the molecule. Primers which have parasitic
hybridization with similarity > /= this threshold will be rejected.
Note: this similarity threshold must be </= the minimum similarity
required for hybridization of user-defined primers (if any).
Max Consecutive Match Check this box and enter the maximum acceptable match of
for Entire Primer consecutive bases for the entire primer and the Amplicon.

Shared Advanced settings 3
Uniqueness tab
Description
setting
Primer 3 End Check the first box and enter the number of consecutive 3 bases
that must match the amplicon with 100% similarity. Check the
second box and specify the maximum acceptable % match
between the Amplicon and the designated number of bases on the
3 end of the primer.
Qualities tab
ClicktheQualitiestabtosetparametersthatgovernprimerqualitybydetermining
howmuchweightshouldbeassignedtotheparametersonothertabs.Thesevalues
affectscoringfunctionsthatevaluatethequalityratingoftheprimersetsgenerated.
Theimportancefactorsareintegersbetween1and10usedincalculatingthescore
evaluatingprimer/oligoquality.Thelowerthefactor,thelessweightgiveninthe
calculation.Forexample,forminimalimportance,enter1intheappropriatebox.For
maximumimportance,enter10.

Primer Design

4 BioAnnotator
BioAnnotatorisasequenceanalyzerthatperformscertaintypesofDNA/RNA
sequenceanalysesanddisplaystheresultsaslineargraphics.
Launching BioAnnotator
ToopenBioAnnotator:
RightclickonaDNA/RNAmoleculeintheDatabaseExplorerandselect
Bioannotator,or
WithamoleculeopenintheMoleculeEditor,clickontheBioannotator
buttonontherightsidetoolbar.
TheBioAnnotatorwindowcontainsthreepanes:aSequenceInfopane,Graph
pane,andSequencePane.
Selecting an analysis
IntheSequenceInfopane:
1. SelectthegraphtodisplayfromtheSelectGraphdropdownlist.
2. Selectanyadditionalsettingsforthespecificgraph(seeAnalysisparameterson
page 72).
3. ClickonRefreshGraphtodisplaythegraph.

BioAnnotator
4 Graph and Sequence panes
Graph and Sequence panes

ThegraphsinBioAnnotatordisplayphysiochemicalpropertiesoftheDNAmolecule.
TheGraphpaneconsistsofthegraphicalanalysesregion:avertical(Y)axis,showing
minimalandmaximalvaluesofanalysisresults,andanindividualhorizontal(X)axis
displayingnumericalpositionsinthesequence,scrollbars,andthelegendthat
displaysthenameofeachanalysis.
Atanypointalongthesequence(Xaxis),thevalue(Yaxis)forthepropertyisderived
notjustfromthespecificbaseatthatpoint,butfromadjacentbasesaswell.Foreach
property,thealgorithmdeterminestheoptimumwindowofadjacentbasestobe
consideredwhencalculatingthevalueforapoint.Forinstructionsinmodifyingthe
WindowSizeparameter,seeWindowsizeonpage 73.
Highlight sequence Tohighlightaparticulardataregion,dragyourcursorintheGraphorSequencepane

region in the graph andthecorrespondingregionwillbehighlightedinbothpanes.
Magnify the graph InWindows,clickintheGraphpaneandusethemousewheeltochangethe

horizontalscaleofthegraphtozoominonparticularfeatures.Ascrollbarwillappear
belowthegraphasyoumagnify.
Determine the Todeterminethevalueataparticularpointonthegraph,holdyourcursoroverthe

value at a point in barinthegraphatthatpoint.Apopupnumberwillbedisplayed,reflectingthevalue
atthatpoint.
the graph
Ifyouhavemagnifiedthegraphandhighlightedaregioninthesequence,youcansee
thevaluecalculatedforthatspecificregion.
Note: TheWindowSizeparameterdescribedbelowmeansthatthereisnotnecessarily
a1:1correlationbetweenaparticularbaseorsmallgroupofbasesinthesequenceand
avalueinthegraph.Duetophysiochemicalinteractionswithinasequence,each
analysisalgorithmnecessarilycalculatesgraphvaluesfromawindowof
surroundingdatapoints.
Analysis parameters
ThefollowingparametersandsettingareavailableintheSequenceInfopane.

Analysis parameters 4
Window size AllthegraphshaveaWindowSizeparameter.Thisisthenumberofadjacentdata

pointsusedinaveragingvaluesforeachdisplayeddatapoint,andtheoptimalvalueis
analysisdependent.Thedefaultwindowsizehasbeenoptimizedbasedonthe
selectedanalysis,butthisvaluecanbechanged.Youcanexperimentwithdifferent
windowsizesandtheireffectontheresultinggraphbyenteringadifferentnumberin
thisfield.
Afteranychange,clickonRefreshGraph.
Analyses Meltingtemperatureandfreeenergyarecalculatedusingthenearestneighbors
descriptions and method.Forconstantsandalgorithmsusedtocalculatethermodynamicparameters,
seeAppendixB.
parameters
TheavailableDNA/RNAanalysesandtheirspecificparametersare:
FreeEnergy(dG)(kcal/mol)
Thiscanberecalculatedforadifferenttemperaturebyenteringanewvalue
intheTemperaturefield( C).
MeltingTemperature(GCContent)( C)
ThiscanberecalculatedforadifferentSaltConcentration(mMol)and%
Formamideofthesolutionbyenteringnewvaluesintheappropriatefields.
SequenceComplexity
GCContent(%)
NucleicAcidDistribution(%)
Selectthebasesforwhichyouwanttocalculatethepercentdistribution
usingthecheckboxesunderSequenceincludes.
MeltingTemperature(Thermodynamic)( C)
ThiscalculationisdependentontheSaltConcentration(mMol)and%
Formamideofthesolution,aswellastheconcentrationofanyprobeinthe
solution(ProbeConcentrationinpMol).
Entropy(dS)(cal/K/mol)
Enthalpy(dS)(kcal/mol)
Afteranychange,clickonRefreshGraph.

BioAnnotator
4 Analysis parameters

5 Regenerator
TheRegeneratorapplicationinVectorNTIExpressSoftwareenablesyoutoback
translateaDNAsequencefromaproteinsequence,ormodifyanexistingDNA
sequenceforagivenexpressionsystem.Youcanintroducemutationssuchas
insertions,deletions,andsubstitutions,ormodificationslikerestrictionorGateway
sites,inthebacktranslatedDNAsequence,whichcanthenbeoptimizedforaspecific
expressionsystem.
Regenerator Workflow
1. LoadaproteinorDNAsequenceintoRegeneratorfromtheMoleculeEditor.
2. Ifdesired,introduceinsertions,deletions,orsubstitutionsintothesequence.
3. Selectthedesiredexpressionsystem.
4. Ifdesired,addattachmentsrelevanttodownstreamcloningtothebacktranslated
DNAsequence.
5. SavethenewlycreatedDNAsequenceasamoleculeinthedatabaseand/orsend
itforsynthesis.
Open Regenerator
TolaunchRegenerator:
1. LoadaproteinorDNAmoleculeintotheMoleculeEditor.
2. Selectpartofthesequenceormakenoselectiontoselecttheentiresequence.
3. RightclickintheGraphicspaneandselectRegeneratororclickonthe
RegeneratorbuttonintheMoleculeEditortoolbar.

Regenerator
5 Regenerator tool features
TheRegeneratortoolwillopen.
Regenerator tool features

TheRegeneratortoolhastwomainsequencepanes,aswellasvariouscontrolsfor
editingthesequence:
ThetoppanecontainstheinputaminoacidsequenceorDNAsequenceandis
editable,allowingyoutoaddordeleteaminoacidsorbasesdirectlyinthe
sequence.
ThemiddlepanecontainstheinsilicoDNAsequencegeneratedfromthe
mutationsselectedorenteredinthetool.
Create mutations in the input sequence

Youcaninsert,delete,orsubstituteaminoacids(forproteins)orbases(forDNA)inthe
inputsequencedirectly.
Toinsertaminoacidsorbases,placeyourcursorupstreamoftheinsertionpoint
andtypeinyourdesiredchanges.

Optimize the expression system and genetic code 5
Tosubstituteoneormoreaminoacidsorbases,highlightthedesiredpartofthe
inputsequenceandtypeinyourdesiredchanges.
Todeletepartoftheinputsequence,highlightthesequenceandusetheDeleteor
Backspacekey.
Clear mutations ClicktheClearallMutationsandAttachmentstoremovemutationsandrestorethe

originalsequence.
View the mutated TheViewMutantProtein/DNAbuttonisenabledonceanymutationisintroduced

sequence intotheinputsequencepane.ClickthisbuttontoviewthemutatedproteinorDNA
sequenceintheMoleculeEditor.
TheproteinisgivenanameusingthefollowingconventionVNTI_<Protein
Name>_mutated.
Refresh the in silico ToupdatetheInSilicoDNASequencepanewithanymutations,clickontheRefresh

DNA sequence DNASequencebutton.
Optimize the expression system and genetic code

SelectthedesiredExpressionsystemfromthedropdownlist.TheinsilicoDNA
sequencewillautomaticallyupdatebasedonaninternalcodonusagetableforthe
expressionsysteminVectorNTIExpress.
SelectthedesiredGeneticcodefromthedropdownlist.TheinsilicoDNA
sequencewillautomaticallyupdatebasedontheselection.
Add attachments
Youcanaddthefollowingattachmentstothe5and3endsoftheinsilicoDNA
sequence:

Regenerator
5 Generate a new sequence and send for synthesis
Note: The3endattachmentwillalwaysbetheReverseComplementofthesequence
displayedintheattachmentsdialogbox.
SelecttheRestrictionSitescheckboxandthenselectfromthedropdownlistto
addarestrictionsitetotheselectedend(s)oftheinsilicoDNAsequence.The
selectedRestrictionSiteappearsintheDNAsequencepaneatthe5and3end,
basedonyourselection.
SelecttheGatewaySitescheckboxtoaddattBsitesforGatewaycloningtothe
backtranslatedmolecule.IntheChooseattBExtensiondialogboxthatappears
basedontheterminusyouhaveselected(5or3),selectafragmentfromthelist
andclickOK.TheselectedGatewaysiteappearsintheDNAsequencepaneat
the5or3end,basedonyourselection.
SelecttheUserDefinedcheckboxtoaddspecialsequences(promoter,tags,etc.)
tothebacktranslatedmolecule.Enterthesequenceinthefield;theThesequence
willappearintheDNAsequencepaneatthe5or3end,basedonyourselection.
Note: TheattachmentsmadetotheDNAsequencewillnotappearintheprotein
sequencepane.
Clear attachments ClickonClearAllMutationsandAttachmentstoclearallattachmentsandrestorethe

originalsequence.
Generate a new sequence and send for synthesis

ClickonViewInSilicoDNAtoopenthenewlycreatedDNAmoleculeinthe
MoleculeEditorYoucanthensavethemolecule,andcopyandsubmitthe
sequenceforsynthesisasdescribedbelow.

Generate a new sequence and send for synthesis 5
ClickontheGotoGeneSynthesisbuttontogototheLifeTechnologies
GeneArtgenesynthesisservicewebsite.Thereyoucancreateanaccountand
submittheinsilicoDNAsequenceforsynthesis.

Regenerator
5 Generate a new sequence and send for synthesis

6 BLAST and Entrez Searches
VectorNTIExpressSoftwareincludesthesearchenginesBLASTandEntrezQueryfor
queryingsequences.
BLAST search
BLAST(BasicLocalAlignmentSearchTool)isasearchengineforexploringavailable
publicsequencedatabasesforDNAorproteinsequencesimilaritiestoaquery
sequence.BLASTprogramshavebeendesignedforspeed,withaminimalsacrificeof
sensitivitytodistantsequencerelationships.BLASTscoreshaveawelldefined
statisticalinterpretation,makingrealmatcheseasytodistinguishfromrandom
backgroundhits.BLASTusesaheuristicalgorithmthatseekslocalasopposedto
globalalignmentsandisthereforeabletodetectrelationshipsamongsequencesthat
shareonlyisolatedregionsofsimilarity.
FordetailedinformationonBLASTsearchtypes,settings,parameters,search
databases,etc.,visithttp://blast.ncbi.nlm.nih.gov/Blast.cgi.
Open the BLAST ToBLASTsearchanentiremoleculeinthedatabase:

search tool SelectthemoleculeinDatabaseExplorer,rightclick,andselectBLAST
Search.
OpenthemoleculeintheMoleculeEditorandclickontheBLASTbuttonon
themaintoolbar.
ToBLASTsearchpartofasequence,openthesequenceintheMoleculeEditor,
selectthedesiredpartofthesequenceintheGraphicsorSequencepane,and
rightclicktoselectBLASTsequence.
ToopenablankBLASTsearchwindowandtypeandpasteasequencedirectly
intothetool,clickontheBLASTbuttononthemaintoolbarwithoutselectinga
moleculefirst.
BLAST search TheBLASTsearchtoolsettingsaredisplayedintherighthandpaneofthetool;the

settings resultsofasearcharedisplayedinthelefthandpane.
Selectthefollowingsettingstodefineyoursearch:
Program
IntheProgramdropdownmenu,specifythetypeofdatabasesearchtobeperformed:
blastn Compares a nucleotide query sequence against a nucleotide sequence

database.

BLAST and Entrez Searches
6 BLAST search
blastp Compares an amino acid query sequence against a protein sequence

database.
blastx Compares a nucleotide query sequence translated into all reading frames
against a protein sequence database.
tblastn Compares a protein query sequence against a nucleotide sequence
database dynamically translated in all six reading frames (both strands).
tblastx Compares the six-frame translations of a nucleotide query sequence
against the six-frame translations of a nucleotide sequence database. This
program cannot be used with the nr database.
Algorithm
IntheAlgorithmdropdownmenu,specifythetypeofalgorithmsearchtobe
performed.Inadditiontoblastnandblastp,algorithmsearchesinclude:
megablast Concatenates many queries to save time spent scanning the database. It
is optimized for aligning sequences that highly similar and is up to 10
times faster than more common sequence similarity programs. It can be
used to quickly compare two large sets of sequences against each other.
MEGABLAST permits searching with batches of ESTs or with large cDNA
or genomic sequences.
PHI blast Pattern Hit Initiated BLAST. A program for searching a protein database
using a protein query; it seeks only alignments that preserve a specified
pattern contained within the query.
PSI blast Position Specific Iterated BLAST. A program for searching protein
databases using protein queries to find other members of the same
protein family.
Database
Inthedropdownlist,selecttheGenBankdatabasetoquery:
Menu item Description

nr/nt Peptide Sequence Database: All non-redundant GenBank
sequences and CDS translations.
Nucleotide Sequence Database: All GenBank+EMBL+PDB
sequences (no EST, STS, GSS or phase 0, 1 or 2 HTGS sequences).
No longer non-redundant.
Refseq RNA Human Genome BLAST databases: human RefSeq mrna with
NM_#### or XM_#### accessions
Refseq Genomic Human Genome BLAST databases: human genomic contig
sequences with NT_#### accessions
Chromosome Nucleotide Sequence Database: complete chromosomes
EST Nucleotide Sequence Database: EST (Expressed Sequence Tags)
mouse and human.
EST-others Nucleotide Sequence Database: EST (Expressed Sequence Tags)

other than mouse and human.

BLAST search 6
Menu item Description

GSS Nucleotide Sequence Database: Genome Survey Sequence,
includes single-pass genomic data, exon-trapped sequences, and
Alu PCR sequences.
HTGS Nucleotide Sequence Database: Unfinished High Throughput
Genomic Sequences.
PAT Protein sequences from the Patent division of GenBank.
PDB Peptide Sequence Database: Saccharomyces cerevisiae protein
sequencesgenomic CDS translations.
Nucleotide Sequence Database: Saccharomyces cerevisiae
genomic nucleotide sequences.
Alu repeats Peptide Sequence Database: Translations of select Alu repeats
from REPBASE.
Nucleotide Sequence Database: Select Alu repeats from REPBASE.
DBSTS Nucleotide Sequence Database: Database of
GenBank+EMBL+DDBJ sequences from STS Divisions.
ENV-NT
Limit by Entrez Query

ThischeckboxletsyoulimittheBLASTsearchtotheresultsofanEntrezqueryagainst
thedatabasechosen.ThiscanbeusedtolimitsearchestosubsetsoftheBLAST
databases.
Selectthecheckboxandinthefieldbelowentertermsthatwouldnormallybeallowed
inanEntrezsearchsession.Forexample:
proteaseNOThiv1[Organism]thisformofasearchwilllimitthesearchtoall
proteases,exceptthoseinHIV1.
biomol_mrna[PROP]ANDbrainthisformofasearchcanbeusedtolimitsearches
toaparticularmoleculetype
Musmusculus[Organism]thisformofasearchwilllimitthesearchtoaspecific
organism.EnterthenameoftheorganismintheEntrezQueryfieldwiththe
[Organism]qualifier.
General Parameters
TheparametersherearealmostidenticaltoparametersforthevariousBLASTsearches
attheNCBIwebsite.Formoreinformationregardingtheseparameters,visithttp://
www.ncbi.nlm.nih.gov/blast/html/blastcgihelp.html.
MaxtargetsequencesThemaximumnumberofalignedsequencestodisplay.
ExpectthresholdThestatisticalsignificancethresholdforreportingmatchesagainst
databasesequences.Thedefaultvalueof10meansthatinadatabaseofthecurrent
size,10matcheswouldbeexpectedmerelybychance(stochasticmodelofKarlinand
Altschul,1990.)HitsshowingastatisticalsignificancegreaterthantheExpect
thresholdarenotreported.IncreasingtheEvalueabove10producesalargerlistwith
morelowscoringhits(chancematches).Lowerexpectationvaluethresholdsaremore
stringent,leadingtofewerchancematchesbeingreported.

6 BLAST search
Ifyourquerypeptideornucleotidesequenceisshort,youmightwanttoincreasethe
Expectvalue.Becauseashortqueryismorelikelytooccurbychanceinthedatabase,
evenaperfectmatchcanhavelowstatisticalsignificanceandmaynotbereported.
IncreasingtheEvalueletsyoulookfartherdownthehitlistandseematchesthat
wouldnormallybediscardedbecauseoflowstatisticalsignificance.
WordsizeWordsizeisroughlytheminimallengthofanidenticalmatchan
alignmentmustcontainifitistobefoundbythealgorithm.MegaBLASTismost
efficientwithwordsizes16andlarger,althoughwordsizeaslowas8canbeused.If
thevalueWofthewordsizeisdivisibleby4,itguaranteesthatallperfectmatchesof
lengthW+3willbefoundandextendedbyMegaBLASTsearch,howeverperfect
matchesoflengthaslowasWmightalsobefound,althoughthelatterisnot
guaranteed.AnyvalueofWnotdivisibleby4isequivalenttothenearestvalue
divisibleby4(with4i+2equivalentto4i).
Scoring Parameters
Match/MismatchScoresRewardandpenaltyformatchingandmismatching
bases.Manynucleotidesearchesuseasimplescoringsystemthatconsistsofa
rewardforamatchandapenaltyforamismatch.The(absolute)reward/
penaltyratioshouldbeincreasedasonelooksatmoredivergentsequences.A
ratioof0.33(1/3)isappropriateforsequencesthatareabout99%conserved;a
ratioof0.5(1/2)isbestforsequencesthatare95%conserved;aratioofaboutone
(1/1)isbestforsequencesthatare75%conserved.
GapcostsThepenaltytoopenaGapandpenaltytoextendaGap.Increasing
theGapCostsdecreasesthenumberofGapsintroducedinthealignment.
Filters
LowcomplexityThisfiltermasksoffsegmentsofthequerysequencethathave
lowcompositionalcomplexity(asdeterminedbytheSEGprogramofWootton&
Federhen,ComputationalChemistry,1993).Regionswithlowcomplexity
sequencecancreateproblemsinsequencesimilaritysearchingbyproducing
artificialhits,sequencesthatarenottrulyrelated.Suchhitscanproducehigh
scoresbecauseofthepresenceoflowcomplexityregions.
HumanRepeatsThisoptionmasksHumanrepeatsandisespeciallyusefulfor
humansequencesthatmaycontaintheserepeats.
MaskforLookupThisoptionmasksonlyforpurposesofconstructingthe
lookuptableusedbyBLAST.TheBLASTextensionsareperformedwithout
masking.
MasklowercasecharactersWiththisoptionselectedyoucancutandpastea
FASTAsequenceinuppercasecharactersanddenoteareasyouwouldlike
filteredwithlowercase.Thisallowsyoutocustomizewhatisfilteredfromthe
sequenceduringthecomparisontotheBLASTdatabases.
Perform the BLAST 1. Toperformthesearch,clickonSubmit.

search 2. ThesearchprogresswillbedisplayedlefthandpaneoftheBLASTsearchviewer.
Whenthesearchiscomplete,thesearchwillbeflaggedasComplete.
3. Rightclickonthesearchresultandselectfromthefollowingoptions:
SavetoLocalDatabasesavesthesearchinthelocaldatabase.
Saveastabdelimitedfilesaveasatextfile.

BLAST search 6
EditsearchchangethesearchparametersandreSubmit.
DeleteBLASTjobclearstheresultsfromthelist.
4. DoubleclickonthesearchresulttoopentheresultintheBLASTResultViewer.
Note: BLASTresultsthathavebeensavedtothedatabasemaybeopenedinDatabase
Explorer;clickonResultsandopentheBLASTResultsfolder.
BLAST Result DoubleclickonaBLASTsearchresultinDatabaseExplorerortheBLASTtooltoopen

Viewer theresultintheBLASTResultViewer.
Table pane
Graphics pane
Sequence pane
TheViewercontainsthreemainpanes:
TheTablepanedisplaysatextuallistofqueryhitmoleculesforthesequence
TheGraphicspanedisplaysthecorrespondingsequenceofeachhitingraphical
form
TheSequencepanedisplaystheQuerysequence,hitsequence(withAccession
number),andConsensussequence
ClickonarowintheTablepaneorafeatureintheGraphicspanetodisplaythe
correspondingsequenceintheSequencepane.
Table pane columns

EValue:Thisvaluereflectsthelikelihoodthatthesimilaritybetweenthe
sequenceswouldoccurbychancewhensearchingadatabaseofaparticularsize.
Azeroorextremelylownumbersuggeststhatthematchissoperfectthatitis
extremelyunlikelythatthesimilaritywouldoccurrandomly.
Name:TheNCBIsequenceidentifierofthequeryhit.
Accession:TheGenBankAccessionnumberofthehit.
BitScore:Ameasureofhowclosetheidentityofthematchistothequery
sequence.
Identity:Theratio(andpercentage)ofmatchingresiduesinthehitelements.The
numbersn/nrefertothenumberofidenticalresiduesoutofthenumberof
matchesinthehitelement.Thisisimportanttoconsiderwhendeterminingthe
significanceofthisstatistic.Ahighidentitypercentagemaymeannothingifalow
numberofnucleotidesisbeingcompared.

6 Entrez Search
Positives:theratio(andpercentage)ofsimilarresiduesinthehitelements
QueryFrom/To/Orientation:Thestartandendpositionnumbersinthequery
sequencematchingthatofthehitelement,andthestrandthatcorrespondstothe
hitelement.
HitFrom/To/Orientation:Thestartandendpositionnumbersinthehitsequence
matchingthatofthequeryelement,andthestrandthatcorrespondstothequery
element.
Download sequence features and save them as molecules

IntheTablepane,clickonthelinkintheNamecolumntodownloadthesequenceand
databaseinformationforthatquerymoleculefromtheNCBIdatabaseandsaveitasa
separatemoleculeinVectorNTIExpressSoftware.Thedatabaseinformationforthe
sequencewillbepopulatedinthePropertiesofthecreatedmolecule.
Note: Youwillbepromptedtosubmityouremailaddresswhenconnectingtothe
NCBIdatabase.
Entrez Search
YoucanusetheNCBIEntrezcrossdatabasesearchenginedirectlyfromVectorNTI
ExpressSoftware.Formoreinformationaboutthissearchengine,visit
www.ncbi.nlm.nih.gov/sites/gquery.
Note: AnInternetconnectionisrequiredtoperformthissearch.
Open Entrez search ToperformanEntrezsearch,clickonthePublicDatabaseSearchbuttononthemain

tool toolbar.
TheEntrezsearchtoolwillopen.
TheEntrezsearchtoolincludesthefollowingpanes:
QuerypaneContainssettingsforformulatingaquery.
QueryListpaneListsthestatusofeachqueryrequest.
ResultspaneDisplaystheresultsofaquery.

Entrez Search 6
Entrez search TheEntrezsearchengineallowsyoutosearchacrossawidevarietyofNCBI

settings databases.
1. Tobegin,selectthedesiredNCBIdatabasefromtheDatabasedropdownlist:
PubMed:Adatabaseofbiomedicalliteraturecitationsandabstractsfrom
MEDLINE,lifesciencejournals,andonlinebooks
Gene:Widerangingdatabasecontainingnomenclature,Reference
Sequences(RefSeqs),maps,pathways,variations,phenotypes,andlinksto
genome,phenotype,andlocusspecificresourcesworldwide.
OMIM:Databaseofhumangenesandgeneticphenotypes.
Protein:DatabaseoftranslationsfromannotatedcodingregionsinGenBank,
RefSeqandTPA,aswellasrecordsfromSwissProt,PIR,PRF,andPDB.
Structure:Databaseofthreedimensionalgeneticandproteinstructures.
Popset:DatabaseofDNAsequencesthathavebeencollectedtoanalyzethe
evolutionaryrelatednessofapopulation.
Nucleotide:Databaseofgenome,geneandtranscriptsequencesfrom
severalsources,includingGenBank,RefSeq,TPAandPDB.
SNP:Databaseofsinglenucleotidepolymorphisms(SNPs).
2. Searchtermswillvarybydatabase.Youcanenterknownsearchtermsdirectlyin
theSearchTermfield,oryoucanusetheSearchBuilderfunctiontoconstruct
yoursearchfromknowndatabaseelementsandBooleanoperators.Fora
descriptionofsearchtermsandoperators,visitwww.ncbi.nlm.nih.gov/books/
NBK3837/.
3. SelectthedesiredPagesize.
4. Whenyouhavemadeyourselections,clickonSubmit.
5. ThesearchprogresswillbedisplayedQueryListpane.Whenthesearchis
complete,thesearchwillbeflaggedasCompleteandthesearchresultswillbe
displayedintheResultspane.
Entrez search EntrezsearchresultsaredisplayedintabularformintheResultspane.

results
Thetableofresultsincludescolumnsappropriateforthedatabaseyouarequerying.
ClickonthelinkintheResultspanetoopentheonlinedatabaserecordforeachresult.
Note: EntrezqueriesarenotsavedintheVectorNTIExpressSoftwaredatabase.

6 Entrez Search
Editing and TodeleteanEntrezquery,rightclickonitintheQueryListpaneandselect

deleting queries Deletesearchjob.
Toeditaquery,rightclickonitintheQueryListpaneandselectEditsearch.

7 GenomeBench
GenomeBenchcanbeusedtodownload,view,analyze,annotate,andsavelocalcopies
ofreferencegenomicDNAsequencesfromseveralprincipalDistributedAnnotation
System(DAS)servers.
GenomeBenchwasdesignedtosupportthefollowingworkflows:
RetrievalofgenomicdatafrompublicDASservers.
Annotatingandcreatingalocalcopyofagenomicregion.
Insupportoftheseworkflows,itacceptsmegabasesizedgenomicsequencesandall
attendanttrackannotations.Allannotatedfeaturesarelinkedtotheircorresponding
regionofthegenomicsequencebackbone;additionalinformationforanysequence
basedannotation(forexample,mRNAs,ESTs,andSTSs),includingotherdefined
featuresfromtheGenBankrecord,iseasilydownloadable.Annotatedsequencescan
bealignedwiththegenomicbackbonesimplybydragginganddroppingintoan
alignmentwindow.
ProprietaryDNAsequencesstoredintheVectorNTIExpresslocaldatabasecanalso
bepositionedalonggenomicbackbonesusingSpideyorSim4alignmentalgorithmsto
determinetheintronexonstructureofgenesofinterest.Genomicsequencesin
GenomeBenchcanbeexportedtoVectorNTIExpressforfurthersequenceanalysis,
suchasPCRprimerdesignorthecreationofsplicedtranscripts.
GenomeBenchalsoprovidesthecapabilitytoquerypublicdatabasesandperform
sequenceandfeaturesearches.
Download data from public DAS servers

VectorNTIExpresshaspreconfiguredDASservers
TolaunchGenomeBenchanddownloaddata:
1. ClickontheGenomeBenchbuttononthemaintoolbar.
2. Whenyoufirstopentheapplication,anemptyprojectwillbedisplayed.
3. IntheGenomeBenchtoolbar(ontherightsideofthewindow),clickon
theLoadfromDASServerbuttontodownloadgenomicdatafromone
ofthepreconfiguredpublicDASservers:
GenomeReferenceConsortium:human(GRCh)
NationalCenterforBiotechnologyInformationmousegenomeassembly
(NCBIM)
RatGenomeSequencingConsortium(RGSC)

GenomeBench
7 Download data from public DAS servers
NCBIs32KBACarray,BACendsequencepairs,andFosmidcloneend
sequencepairs
4. IntheDASServerdialog,selecttheserverfromtheDASAuthoritylist.
5. SelectthegenomicsourcefromtheSourceslist.
6. SelectthedesiredchromosomesfromtheChromosomeslist,thenclickonOK.
7. IntheLoadFragmentdialog,specifythebaserangeofthesequencetodownload
usingtheStartandLengthfields
8. SelectthefeatureinformationtodownloadfromtheFeatureTypelist,which
displaysaselectedlistoffeaturesavailableintheDASdatabases.
UseCtrl+clickandShift+clicktomakemultipleselections.
ClickonRemovetoremoveselectedfeaturesfromthedownload.

Local GenomeBench Projects 7
ClickonAddtoopentheFeatureTypesFilterandselectfromallavailable
featuretypestoaddthemtotheFeatureTypelist.
9. ClickontheOKintheLoadFragmentdialogtobeginthedownload.
Note: Itcantakesometimeforallthefeaturestoload,butyoucanstartworkingon
featuresthathaveloadedbeforetheremainingfeaturesareloaded.
10. Thedownloadtimecanvaryconsiderablydependingontheserverconnection
anddatavolume.
11. Whenthedownloadisfinished,theGenomeBenchProjectviewerwilldisplaythe
project.
Data Loading YoucanmonitorthedownloadprogressbyclickingontheDownload

Monitor ProgressbuttonontheGenomeBenchtoolbar.
IntheDataLoadingMonitor,featuresarelistedasLoadingorInQueue.To
deleteanInQueuefeatureinthelist,selectitandclickonAbort.
Local GenomeBench Projects

GenomeBenchProjectsthathavebeendownloadedcanbesavedinthelocalVector
NTIExpressdatabaseforfutureaccess:
Tosaveaprojecttothelocaldatabase(i.e.,afterdownloadingthedatafromthe
DASserver),selectFile>SaveAsandspecifyanamefortheprojectinthedialog
box.
Toopenasavedproject,openGenomeBenchandclickontheLoadfrom
LocalDatabasebutton.ThenselecttheprojectnamefromtheExisting
GenomeProjectsdialog.

GenomeBench
7 GenomeBench Project Viewer
GenomeBench Project Viewer

TheGenomeBenchProjectViewercontainsthefollowingpanes:MapOverview,
FeatureMap,GenomeFeatures,andGenomeSequenceView.
Map Overview
Feature Map
Genome Features/
Sequence View
Map Overview TheMapOverviewpanepresentsagraphicaldepictionoftheentirechromosomeand

hasthefollowingfeatures:
Arulermarksthelengthofthechromosome.
Therodshapedbanddepictsthesequence/chromosome.
Narrowedareasontherodindicatecentromericregions.
Cytobandpatternsareindicatedbytheverticaldarklinesthatrunthelengthof
therod.Pausethecursoroveraparticularcytobandtodisplayatooltipwith
informationaboutthecytoband.
CentromericregionsandcytobandpatternsareshownonlyforthoseDASserversthat
providetheinformationinthecorrectformat.
Feature Map TheFeatureMapPaneshowsagraphicaldepictionofthefeaturesforthecurrently

loadedfragment.
LiketheOverviewPane,theFeatureMapPanehasaruleratthetoptohelpyou
visualizetheorientationoffeaturesalongthelengthofthefragment.

Editing features 7
FeaturetypesortracksdisplayalongtheleftsideoftheFeatureMapPane.Different
featuresaredepictedwithvariousgraphicalrepresentationsandarelabeledwiththe
featurename.Shownfeaturesarelistedalongwithrelevantfeatureinformationinthe
GenomeFeaturespane.
TheassociatedGenBankaccessionnumberisderivedfromtheTargetAccession
numberofthefeature.
Magnifying tools
UsethemagnifyingtoolstotherightoftheFeatureMaptomagnifyfeaturesinthe
panehorizontally.
Genome Features TheGenomeFeaturespaneliststhefeaturesinthemapbyname,andindicatestheir

and Genome type,location,orientation,andfeaturecategory.
Sequence
TheGenomeSequencepanedisplaystheentiredownloadedsequence
Editing features
IntheFeatureMapandGenomeFeatureslist,therightclickmenuallowsyouto
create,edit,ordeletefeatures.
Rightclickonafeatureinthemaporlistandselect:
Edittheselectedfeature:Editbasicsettingsandinformationforthefeature,
includingfeaturename,type,orientation,andpositioninthesegmentormultiple
segments.
CreateNewFeature:Createanewfeature,usingthesamesettingsandfieldsasin
theEditFeaturedialog.

GenomeBench
7 Editing features
Deletetheselectedfeature:Deletestheselectedfeature.

8 Align Multiple Sequences
Thesimultaneousalignmentofmultiplenucleotideoraminoacidsequencesisan
essentialtoolinmolecularbiology.AlignmentenablesyoutodesignPCRprimersfor
amplifyingaregionofalignedDNA/RNAmolecules.Multiplealignmentsareusedto
finddiagnosticpatterns,characterizeproteinfamilies,aswellasdetectordemonstrate
asimilaritybetweennewsequencesandexistingfamiliesofsequences.Theyarealso
usefulinpredictingsecondaryandtertiarystructuresofnewsequences,suggesting
oligonucleotideprimersforPCRandservingasanessentialpreludetomolecular
evolutionaryanalysis.
AlignXisthemultiplesequencealignmenttoolofVectorNTIExpressSoftware.In
additiontoaligningsequences,itcanbeusedtocreateandmanagesequence
alignmentprojects.ItusesamodifiedClustalWalgorithmandincorporatesthe
followingfeatures:
Profilealignment
Guidetreeconstruction,displayedingraphicalrepresentation
Useofresiduesubstitutionmatrices
Secondarystructureconsideration
Multicoloredalignmentpresentation
Automaticconsensuscalculation
Fullalignmenteditingcapabilities
DotPlotcomparisonofanytwosequences
Open AlignX
ThereareseveralwaystoopentheAlignXtool:
ClickontheAlignXbuttononthemaintoolbar.
InDatabaseExplorer,rightclickonamoleculeorCtrl+clickonmultiple
moleculesandselectAlignmenttoloadthosemoleculesintothetool.
LoadanexistingAlignXprojectasdescribedinManageAlignXprojectson
page96.
AlignX window TheAlignXwindowconsistsofthefollowingpanes:

ProjectPropertiespane:ContainstheProjectDescriptionandtheFragmentslist.
AsmoleculesareaddedtoanAlignXproject,theyarelistedintheFragmentslist.
AlignmentSettingspane:Containsthesettingsusedtoperformthealignment.
Graphspane:Displaysagraphicalrepresentationofthealignedmolecules,
showingbothsimilarityandsequencecomplexity.

Align Multiple Sequences
8 Manage AlignX projects
Alignmentpane:Displaysthealignedsequences,withalignedregions
highlighted
GuideTreepane:Agraphicalrepresentationofthedegreeofsimilarityamong
sequences(forthreemoresequences).
Project description
Guide tree
Graphics pane
Fragments list
Alignment pane
Alignment settings
Manage AlignX projects

Thetoolsforsaving,editing,andloadingprojectsarelocatedintheProjectDescription
regionoftheProjectPropertiespane:
Save and rename a TosaveanewAlignXproject,clickonSaveProjectinthepane.Inthedialog,

project enteranameandanydescriptionfortheproject.
Tosavechangestoanexistingproject,clickontheSaveProjectbutton.
Tochangeaprojectnameordescription,clickonEditProject.
Open a project Toloadanexistingprojectinthedatabase,inDatabaseExplorer,gotothe

Projectslist,doubleclickontheProjectsfolder,selecttheAlignmentProjects
folderfromtheLocalDatabase,anddoubleclickontheAlignXprojectinthelist
toopenit.

Select fragments to align 8
Toloadanexistingprojectthathasbeensavedasa.apr,.aprx,or.alnfile,clickon
theLoadProjectsbuttonintheProjectPropertiespaneandselecttheprojectfrom
theOpendialog.
Close a project Tocloseaproject,clickontheCloseProjectbutton.Ifthereareunsavedchanges,you

willbepromptedtosavetheprojectbeforeclosing.
Export a project ToexportanAlignXprojectasa.aprxfile,clickontheExportProjectbuttonandenter

aprojectnameintheExportMSAProjectdialog.
Save consensus Afteryouperformanalignment,youcansavetheconsensussequenceasaseparate

sequence moleculeinthedatabase.ClickonSaveConsensusandspecifyasequencefilename.
Open .dnd file Toopenaexternal.dndfilegeneratedfromaClustalWanalysis,clickonOpenDND.
Select fragments to align

ThetoolsforselectingthefragmentstoalignarelocatedintheFragmentsListregion
oftheProjectPropertiespane:

8 Select fragments to align
Add fragments Toaddfragments:

ClickAddfromFiletoselect.gb,.gp,orfastasequencefiles.
ClickAddfromDatabasetoselectDNAorproteinmoleculesfromthelocal
database.
Note: PresstheCtrlorShiftkeystoselectmultiplefiles/molecules.
Remove fragments ToremoveafragmentfromtheAlignXproject,selectthecheckboxnexttothe

fragmentnameintheFragmentListandclickontheDeletebutton.Thiswillremove
thefragmentfromtheproject,notdeleteitfromthedatabase.
Select fragments Toselectfragmentsforalignment,selectthecheckboxesnexttothefragmentnamesin

to align theFragmentsList.

Alignment settings 8
Alignment settings
ClickontheAlignmentSettingsbuttonbelowtheProjectPropertiespanetoviewthe
settingsforperforminganalignment.
Program Clustal W (local)

ThelocalClustalWalgorithmcalculatesacrudesimilaritybetweenallpairsof
sequences,calledaParitiesalignment.Thesescoresarethenusedtocalculatea
guidetreeordendrogram,whichdeterminestheorderinwhichthesequencesare
aligned.Thesequencesarethenalignedinlargerandlargergroupsuntiltheentire
sequencesareincorporatedinthefinalalignment.
Usingtheadditionalsettings,youcandesignatethescoringmatrixusedbythe
algorithmratherthanthealgorithmmakingthechoiceasisdoneinatraditional
ClustalWalignment.
Clustal W (EBI)
ThisselectionusestheClustalWalgorithmdevelopedbytheEuropean
BioinformaticsInstitute(EBI).
Molecule type Select DNA or Protein from this dropdown list.
Pairwise alignment ThesesettingscontrolpairwisedistancesbasedontheClustalWalgorithmselected.

TheAlignmentTypeoptionscontrolthespeed/sensitivityoftheinitialalignments:
Fast(approximate)method
Slow(moreaccurate)methodusestwogappenalties(foropeningorextending
gaps)andafullaminoacidweightmatrix.Bydefault,thisslowermethodisused.

8 Alignment settings
Therearetwogroupsofparameters,dependinguponwhichmethodaboveischosen:
Slow options Description

DNA Weight Matrix IUB (default) or Clustal W
Gap Open penalty The penalty for the first residue in a gap
Gap Extension penalty The penalty for additional residues in a gap
Fast options Description

KTUP (Word size) Change the K-tuple value to limit the word-length the
search should use. A word-length of 2 is sensitive
enough for most protein database searches. The
general rule is that the larger the word length, the less
sensitive, but faster the search will be.
Window Length The number of diagonals around each of the best
diagonals used
Score Type Percent or absolute
TOPDIAG Number of the k-tuple matches on each diagonal used
in the alignment
PAIRGAP Penalty for the existence of a gap
Multiple sequence These parameters control the final multiple alignment.

options
Option Description
DNA/Protein Weight Matrix All algorithms designed to evaluate pairwise sequence
alignment are based on systems which rank aligned
residues. Nucleotides or amino acids that are identical
or similar in alignment score higher than those less
similar. Matrices generated with these assigned scores
are used to detect similarities between differing
sequences. The most common of many different scoring
systems are based on substitutions of amino acids in
related proteins.
Gap Open The penalty for the first residue in a gap
Gap Extension The penalty for additional residues in a gap
Gap Distance Tries to decrease the distances between gaps
No End Gaps Does not penalize for gaps introduced at the end of a
sequence
Iteration None (default), tree, or alignment
Num of Iteration 1(default) to 10
Clustering Method used to create the Guide Tree: Neighbor Joining
(NJ) or Unweighted Pair Group Method with Arithmetic
Mean (UPGMA) (see description under Guide Tree pane
below)

Guide Tree pane 8
Perform the Whenyouhaveselectedthealignmentsettings,clickonSubmit.

alignment
Guide Tree pane

TheGuideTree,whichresemblesaPhylogenetictree,isdisplayedwhenthreeormore
sequencesarealigned.TheGuideTreecanbebuiltusingtwodifferentmethods:
TheNeighborJoiningmethod(NJ)ofSaitouandNeiworksonamatrixof
distancesbetweenallpairsofsequencetobeanalyzed.Thesedistancesarerelated
tothedegreeofdivergencebetweenthesequences.
TheUnweightedPairGroupMethodwithArithmeticMean(UPGMA)isasimple
agglomerativeorhierarchicalclusteringmethodinwhich,ateachstep,the
nearesttwoclustersarecombinedintoahigherlevelcluster.
TheGuideTreeiscalculatedafterthesequencesarealigned.AlignXdisplaysthe
calculateddistancevaluesinparenthesisfollowingthemoleculenameintheGuide
Treepane.
Graphs pane
TheGraphspaneprovidesagraphicalrepresentationofsequencesimilarityand
complexityamongthealignedsequences.
ThegraphsinthispaneinteractwiththesequencesaslistedintheAlignmentpane.
Note: Allgraphsdisplaythevaluesaveragedinawindowofaspecificlength(defined
bywindowparameter)thatslidesalongthealignment.
Sequence TogeneratetheSimilaritygraph(uppergraph),specificvalues(ina01range)are
similarity graph assignedtoeachresidueatagivenalignmentpositionineachalignedsequence,
dependingonwhethertheresidueisidentical,similar,orweaklysimilartothe
correspondingresidueoftheconsensussequence.Thevalues(1(identical),0.5
(similar),and0.2(weaklysimilar)foreachresidueatagivenpositionaretotaled;the
sumisdividedbythenumberofthesequencesinthealignment,normalizingthe
resultingvalue.
Sequence TheComplexitygraph(lowergraph)iscalculatedasasumofallpairwiseresidue
complexity graph substitutionscoresatagivenalignmentpositiondividedbythenumberofpairsinthe
alignment.Thescoresaretakenfromtheresiduesubstitutionmatrixusedfor
alignmentcalculation.

8 Alignment pane
Magnify the graphs InWindows,clickintheGraphspaneandusethemousewheeltochangethe

horizontalscaleofthegraphtozoominonparticularfeatures.Ascrollbarwillappear
belowthegraphasyoumagnify.
Onthehorizontalscale,eithernumericalpositionsinthesequenceorresiduescanbe
shown,dependingonthedegreeofzoominginthegraphicspane.
Select a region Dragyourcursoroveraregionofagraphtohighlightthatsameregioninthe

Alignmentpane.
Alignment pane
TheAlignmentpanedisplaysalignedsequencesandtheresultingconsensussequence.
Thetoprowinthepaneconsistsofthealignmentconsensus.Consensusresiduesare
thosethatappearmostcommonlyataparticularsite.
Note: Youcansavetheconsensussequenceasaseparatesequencemolecule;clickon
SaveConsensusintheProjectPropertiespane.
Select a region DragyourcursoroverasequenceregionintheAlignmentpanetohighlightthatsame

regionintheGraphspane.
Dot Plot
Note: TheViewDotPlotbuttonisonlyavailablewhentwofragmentsareselectedin
theFragmentsList.
TheDotPlotisamethodforcomparingtwosequencestofindallpossiblematchesof
residues.Thismethodcanalsobeusedtofinddirectorinvertedrepeatsinproteinand
DNAsequences.ItcanpredictregionsinRNAthatareselfcomplementaryand
thereforemightformadoublestrandedregionorsecondarystructure.
IntheDotPlotmethodofsequencecomparison,onesequence(A)islistedontheX
axisofthegraphandtheothersequence(B)islistedontheYaxis.Startingwiththe
firstpositionsinAandB,theprogramslidesthewindowofncharactersalongthe
sequences,performingacomparisonofadjacentpositionsinthewindows.Ifthe
similarityofresiduesineachpositionisaboveacertaincutoff,adotisplacedinthe
matrixinthepositiondefinedbythestartingpositionsofthewindowforboth
sequences.Adiagonallinesegmentindicatesthatthetwosequencesmatch
consistentlyoveranextendedregion.
AlargerwindowsizeisgenerallyusedforDNAsequencesthanproteinsequences
sincethenumberofrandommatchesismuchgreaterforDNA.

Dot Plot 8
Display the Dot Plot WithtwofragmentsselectedintheFragmentslist,clickonViewDotPlottoopenthe

DotPlotViewerandviewasimilarityplotoftheselectedmolecules.

8 Dot Plot

9 3D Molecule Viewer
The3DMoleculeViewerisatoolforvisualizingproteinstructuresinthree
dimensions.ItcanopenProteinDataBank(PDB)fileswiththefileextension*.pdb.
The3DMoleculeViewerusesJMol,anopensourceJavaviewerforchemical
structuresin3DthathasbeenintegratedintoVectorNTIExpress.Formore
informationaboutJMol,visitwww.jmol.org.
Download 3D Structure Files

YoucansearchforPDBfiles(withthe*.pdbfileextension)inpublicdatabasesusing
theEntrezquerytoolinVectorNTIExpress.ClickonthePublicDatabaseSearch
buttononthemaintoolbar,andforthedatabasetype,enterstructure.
Savedownloaded*.pdbfilesinanappropriatefilelocationandaccessthemusingthe
toolasdescribedbelow.
Open a molecule in 3D Molecule Viewer

1. ToopenaPDBmoleculeinthe3DMoleculeViewer,clickonthe3DMolecule
Viewertoolonthemaintoolbar,orselectitfromtheDiscovermenu.
2. IntheOpendialog,selecta*.pdbfiletoopenit.
Toopenadifferentmolecule,selectFile>Open>3DMolecule.
Note: ThePDBfileyouopenwillremainopeninVectorNTIExpressuntilyouclose
thewindow,andyoucannavigatebacktoitbyclickingthe3DMoleculeViewer
button.

3D Molecule Viewer
9 Elements of the 3D Molecule Viewer window
Elements of the 3D Molecule Viewer window

The3DMoleculeViewerwindowconsistsofthreepanes:
Molecule Display
pane
Outline pane
Properties pane
Graphicspanedisplaysthethreedimensionalstructureofthemolecule.
Propertiespanelistsanypropertiesassociatedwiththemolecule
Outlinepaneliststheaminoacidsinthesequence
TherightclickmenuintheMoleculeDisplaypanecontainsinformationaboutthe
moleculeandtoolsformanipulatingthegraphicrepresentationofthestructure,
exportingmoleculedatainvariousformats,andoptimizingthedisplay.
Magnify and rotate the molecule

IntheMoleculeDisplaypane:
Tozoominorout,rotatethewheelonyourmouseorselectZoomfromtheright
clickmenu.
Torotatethemoleculeinthreedimensionalspace,dragyourcursorinthepane.
Torepositionthemoleculewithinthepanewithoutrotation,holdtheCtrl+Alt
keysanddragyourcursorinthepane.
Tobeginspinningthemoleculeinthreedimensionalspace,selectSpin>Onfrom
therightclickmenu,andadjustthesettingsontheSpinmenu.SelectSpin>Off
tostopspinning.

Highlight an amino acid or a chain 9
Highlight an amino acid or a chain

ClickwithinthestructureintheMoleculeDisplaywindowtohighlighta
particularaminoacid.Clickonitagaintounhighlightit.
ClickonanaminoacidintheOutlinepanetohighlightitintheMoleculeDisplay
pane.
ClickonachainintheOutlinepanetohighlightthatentireaminoacidchainin
thestructure.
Additional menu operations

TherightclickmenuintheMoleculeDisplaywindowcontainsalltheJMolcommands
forviewingandmanipulatingthestructure.Foradditionalinformationaboutthese
commands,visitwww.jmol.org.

3D Molecule Viewer
9 Additional menu operations

10 Sim4 and Spidey Analysis
Sim4andSpideyaretoolsforaligningexpressednucleicacidsequences(e.g.,mRNA)
withgenomicsequencesinonlinedatabases.VectorNTIExpressincludesthesetools
foranalyzingmoleculesstoredinthedatabase.
Sim4
Sim4isasimilaritybasedtoolforaligninganexpressednucleicacidsequences(EST,
cDNA,mRNA)withagenomicsequence.FormoreinformationaboutSim4,seehttp:/
/www.hgmp.mrc.ac.uk/.
Sim4employsthefollowingmultistageBLASTbasedtechnique:
Sim4detectsallpossibleexactmatchesofWmersbetweenthetwosequencesand
extendsthemtomaximalscoringgapfreesegments(exoncores).
Exoncoresareextendedintotheadjacent,asyetunmatchedfragmentsusing
greedyalignmentalgorithms.Heuristicsareusedtofavorconfigurationsthat
conformtothesplicesiterecognitionsignals.Ifnecessary,theprocessisrepeated
withlessstringentparametersontheunmatchedfragments.
Sim4functionssimilarlytoBLAST,butperformsamorethoroughmRNAalignment
search.
Launch Sim4 ToopentheSim4Analysistool:

analysis tool InDatabaseExplorer,rightclickonamoleculeoruseCtrl+clickorShift+clickto
selectmultiplemoleculesandrightclick.Intherightclickmenu,selectAnalysis
Monitor>Sim4Analysis.
WithamoleculeopeninMoleculeEditor,clickontheSim4Analysisbuttonin
theMoleculeEditortoolbar.
TheAnalysisMonitorwillopen,withtheSim4Analysissettingsselectedandthe
selectedmolecule(s)listed.

Sim4 and Spidey Analysis
10 Sim4
TheanalysiswindowhasanAnalysisJobspanecontainingtheanalysissettings,and
anAnalysisListpanelistingthemoleculesloadedinthetool.
Analysis Jobs Molecule and strand to analyze

settings IntheAnalysisJobspane:
SelectthemoleculefromtheNamedropdownlist.
SpecifytheStrandtoanalyze.TheBothoptionsearchesbothstrandsandreports
thebestresult.
Sim4 parameters
WordSizeThenumberofDNAbasesinasizeunit.Thelargerthewordsize,
thefasterandlesssensitiveSim4becomes.
LimitofScoreDropoffThetriggerforstoppingungappedextension.
SearchforSmallExonsIfYesisselected,anadditionalsearchforsmallexonsis
performed.
DiagonalDistanceTheupperboundaryofdiagonaldistancewithin
consecutiveHSPsinanexon.
AllowAmbiguityCharactersIfchecked,allowsthefollowingambiguity
characters:ABCDGHKMNRSTVWXY.Ifunchecked,onlythefollowing
charactersareallowed:ACGTNX.
WeightFactorforLinkingHSPsThemultiplicationfactorusedwhen
calculatingthescoreofachainofHSPs.
DefaultsResetsSim4parameterstotheoriginaldefaultvalues.
HSP Score Threshold

FirstStageThethresholdforHSPsforthefirststageofcomparison.Ifnovalue
isspecified,thedefaultvalueisused.
SecondStageThethresholdforHSPswhenaligningtheextendedand
unmatchedportions.Ifnovalueisspecified,thedefaultvalueisused.
Submit the job Whenyouhavemadeyourselections,clickonSubmit.

Spidey 10
Whenanalysisiscomplete,aCompletedcheckboxwillappearnexttothejobnamein
theAnalysisMonitor.
View analysis TheAnalysisMonitorcontainsalistofalltheanalysesperformedbyVectorNTI

results Express,includingSim4analyses.ClickonAnalysisMonitoronthemaintoolbarto
openit.
Toviewtheanalysisforaparticularmolecule,youcan:
OpenthemoleculeintheMoleculeEditor,andclickonAnalysisResults.
OpentheAnalysisMonitoranddoubleclickontheparticularanalysis.
Spidey
SpideyisanmRNAtogenomicalignmentprogram.Ittakesasinputasinglegenomic
sequenceandasetofmRNAaccessionsorFASTAsequences.Allprocessingisdone
onemRNAsequenceatatime.ThefirststepforeachmRNAsequenceisahigh
stringencyBLASTagainstthegenomicsequence.TheBLASTalignmentsaresortedby
scoreandthenassignedintowindowsbyarecursivefunction.Formoreinformation
aboutSpidey,seewww.ncbi.nlm.nih.gov/spidey/.
Afterthegenomicwindowsareconstructed,theinitialBLASTalignmentsarefreed
andanotherBLASTsearchisperformed,thistimewiththeentiremRNAagainstthe
genomicregiondefinedbythewindow,andatalowerstringencythantheinitial
search.Spideythenusesagreedyalgorithmtogenerateahighscoring,non
overlappingsubsetofthealignmentsfromthesecondBLASTsearch.Thisconsistent
setisanalyzedcarefullytomakesurethattheentiremRNAsequenceiscoveredbythe
alignments.
OncethemRNAiscompletelycoveredbythesetofalignments,theboundariesofthe
alignmentsareadjustedsothatthealignmentsabuteachotherpreciselyandsothat
theyareadjacenttogoodsplicedonorandacceptorsites.Topositiontheexon
boundaries,theadjacentexonalignmentoverlapregionplusafewbasepairsoneach
sideisexaminedforsplicedonorsites,usingfunctionsthathavedifferentsplice
matricesdependingontheorganismchosen.Thetopfewsplicedonorsites(byscore)
arethenevaluatedastohowmuchtheyaffecttheoriginalalignmentboundaries.The
sitethataffectstheboundariestheleastischosen,andisevaluatedastothepresenceof
anacceptorsite.Thealignmentsaretruncatedorextendedasnecessarysothatthey
terminateatthesplicedonorsiteandsothattheydonotoverlap.
FordetailsonSpideyanalysis,seehttp://www.ncbi.nlm.nih.gov/IEB/Research/Ostell/
Spidey/index.html.
Launch Spidey ToopentheSpideyAnalysistool:

analysis tool InDatabaseExplorer,rightclickonamoleculeoruseCtrl+clickorShift+clickto
selectmultiplemoleculesandrightclick.Intherightclickmenu,selectAnalysis
Monitor>SpideyAnalysis.
WithamoleculeopeninMoleculeEditor,clickontheSpideyAnalysisbuttonin
theMoleculeEditortoolbar.

10 Spidey
TheSpideyAnalysistoolwillopenwiththeselectedmolecule(s)listed.
TheanalysiswindowhasanAnalysisJobspanecontainingtheanalysissettings,and
anAnalysisListpanelistingthemoleculesloadedinthetool.
Analysis Jobs Molecule and organism to analyze

settings IntheAnalysisJobspane:
SelectthemoleculefromtheNamedropdownlist.
SpecifytheOrganismtoanalyzeforyourgenomicsequence.
Spidey Parameters MinimummRNAGenomicIdentityPercentidentitycutoffforgenemodels.

MinimumLengthofmRNACoveredmRNAlengthcoveragecutoffinpercent
forgenemodels.
LimitNumberofGeneModelstoSetsthemaximumnumberofgenemodels
Spideyanalysisreturns.
Evalue
1stPassTheEvaluecutofffortheinitialhighstringencyalignment.The
higherthevalue,thelessstringentandfastertherun.
2ndPassTheEvaluecutoffforthelowstringencyBLASTsearchwithina
genomicwindow,basedonthehighstringencyresult.
3rdPassTheEvaluecutoffforaverylowstringencyBLASTsearchtofind
hitsformRNAgaps.
DivergentSequencesIfchecked,searchparametersareadjustedtotolerate
mismatchesandgapsforinterspeciesalignment.
UseLargeIntronSizesIfchecked,muchlargermaximalintronsizesare
allowed.Checkingthisoptionincreasescomputationtimesignificantly.
DefaultsSetsSpideyparameterstooriginaldefaultvalues.
Submit the job Whenyouhavemadeyourselections,clickonSubmit.

Whenanalysisiscomplete,aCompletedcheckboxwillappearnexttothejobnamein
theAnalysisList.

Spidey 10
View analysis TheAnalysisMonitorcontainsalistofalltheanalysesperformedbyVectorNTI

results Express,includingSpideyanalyses.ClickonAnalysisMonitoronthemaintoolbarto
openit.
Toviewtheanalysisforaparticularmolecule,youcan:
OpenthemoleculeintheMoleculeEditor,andclickonAnalysisResults.
OpentheAnalysisMonitoranddoubleclickontheparticularanalysis.

10 Spidey

11 Clone2Seq
Clone2Seqsimplifiesthetwofragmentrestrictionandligationcloningprocess.Using
Clone2Seq,youwillbeabletocloneasingleDNAorRNAinsertintoavectorwithout
havingtogothroughmultiplestepsofthemoleculeconstructionwizard.
Launch Clone2Seq TolaunchClone2Seq:

ClickontheClone2Seqbutton onthemaintoolbar.
SelectFile>New>Clone2SeqProjectfromthemainmenu.
Clone2Seq window TheClone2Seqwindowconsistsoftwomoleculepanes,andaleftpanecontainingthe

FragmentListandMoleculePropertieslists.
TheleftmostmoleculepanecontainstheDNA/RNAmoleculethatisfirstfragmentin
theclone,andtherighthandpanecontainsthesecondfragment.
Foreachmolecule,therestrictionsites(selectedbyRestrictionAnalysisinthe
MoleculeEditor)willbedisplayedinthemoleculepane,andsequenceendsoflinear
moleculeswillbeshownintheViewSequencefieldbelowthepane.
Figure1 Clone2Seq Window
Select molecules 1. ClickonOpenMoleculebelowthelefthandpanetoselectthefirstmolecule.

2. Repeatthisoperationintherighthandpanetoselectthesecondmolecule.
Note: ClickonClearFragmenttoremoveamoleculefromthepane.

Clone2Seq
11
Note: Themoleculeinthelefthandpanewillalwaysbethefirstfragmentoftheclone
andthemoleculeintherighthandpanewillalwaysbethesecondfragmentofthe
clone.Thisisdonetomaintainthedirectionalityoftheresultingconstruct.
Molecule NotethefollowingmoleculerequirementsforClone2Seqassembly:
requirements Twolinearmoleculeswithbluntendsmaybeclonedasis.
Twolinearmoleculeswithoverhangsmusthavematchingoverhangs.Thiscanbe
accomplishedviarestrictiondigestion,asdescribedinGeneratingmolecule
fragmentsbelow,orbymodifyingthefragmentends,asdescribedinModifying
fragmentends.
Circularmoleculesmustbelinearizedonacutsite,asdescribedinGenerating
MoleculeFragmentsbelow.
Display restriction ClickonLeftMoleculePropertiesorRightMoleculeProperties(belowtheFragment

sites in molecules List)todisplayorhideselectedrestrictionsitesinthemolecules.Theselected
restrictionsiteswillbedisplayedinthemoleculepanes.
Note: TheonlyrestrictionsiteslistedwillbethoseidentifiedbyRestrictionAnalysisin
theMoleculeEditor.Ifthedesiredrestrictionsitesarenotlisted,openthemoleculesin
theMoleculeEditor,performRestrictionAnalysiswiththedesiredrestriction
enzymes,andthenreloadthemoleculesinClone2Seq.SeeChapter2,Molecule
Editoronpage39formoreinformationonRestrictionAnalysis.
Generate molecule TogenerateamoleculefragmentfromalinearorcircularmoleculeinClone2Seq:

fragments 1. Clickonarestrictionsiteinthedesiredmoleculetogenerateasinglecutsite,or
shiftclickontworestrictionsitestoselecttheregionbetweenthem(theregion
willappearselectedinthewindow,asshownbelow).

11
Note: Whenselectingrestrictionenzymesthatgenerateoverhangs,besuretoselect
enzymesinbothmoleculesthatwillgeneratecomplementaryoverhangs.
Alternatively,youcanmodifythefragmentendsasdescribedbelow.
2. ClickonCut&AddtoFragmentbelowthemoleculepane.
3. ThefragmentwillbeaddedtotheFragmentList.
Note: Fragmentsinthelistarenamedforthemolecule,aswellastheregionthatwas
cut.Forexample,ADRA1A_1_608isafragmentoftheADRA1Amoleculetakenfrom
the1608bpregion.
4. Toselectthefragmentforcloning,rightclickonthefragmentinthelistandselect
SelectasleftmoleculeorSelectasrightmolecule.
5. Thefragmentwillbeaddedtotheappropriatepane,andthecutendswillbe
displayedbelowthatpane.
6. Repeatthisprocedureforthesecondmolecule.
Edit the Fragment RightclickonafragmentintheFragmentListandselectDeleteFragmenttodeleteit,

List orSaveFragmenttoDatabasetosaveitasamoleculeinthedatabase.

Clone2Seq
11
Modify fragment 1. Tomodifytheendsofamoleculeorfragment,clickonModifyEnds,thenselect

ends thedesiredmodificationusingthedropdownlist.
2. Themodificationwillbepreviewedinthedialog.ClickonOKtomakethe
modification.
Assemble the 1. Whenyouhavemadeyourselections,clickontheAssemblebutton.Notethat

molecule thisbuttonwillnotbeavailableunlessbothmoleculesarelinear.
2. IntheConstructdialog,enteranamefortheassemblyandselectthedesired
moleculetypeandstartpositionandwhethertheresultingassemblyiscircularor
linear.

11
3. ClickonConstruct.Theassembledmoleculewillbedisplayedinapreview
window.
Note: Youcanusethetoolsinthewindowtomagnifythemoleculeordisplayitas
linearorcircular.
4. ClickonSavetosavethemoleculetothedatabase.
Multiple fragment ToclonemultiplefragmentsusingClone2Seq,firstcreateaclonefromtwofragments

cloning asdescribedabove,thengenerateaclonefromtheclonedmoleculeandthenext
fragment.Repeatasnecessary.

Clone2Seq
11

12 Gateway Cloning
GatewayCloningTechnologyisarapidandhighlyefficientmethodforthecloning
andsubcloningofDNAsegments.Thissystemisbasedonthewellcharacterized
bacteriophagelambdabasedsitespecificrecombinationsystem(attLxattRattBx
attP).
GatewayCloningisa2stepprocess.Inthefirststepasequenceofinterestcontaining
attBsitesisrecombinedwithadonorvectorcontainingattPsitesintoanentryclone,
creatingattLsitesintheprocess.ThesecondsteprecombinestheattLcontainingentry
clonewithadestinationvectorcontainingattRsites,generatinganexpressionclone
thatcanbepropagatedandexpressedinarangeofhostcellsforagivenexperiment.
For more FordetailedinformationonGatewayCloning,seetheGatewayTechnologyUserGuide

information ortheGatewayTechnologywithClonaseIIUserGuide,availablefordownloadfrom
www.lifetechnologies.com/manuals.
Gateway Cloning Workflow diagram

Workflow BP Reaction
att B att B att P att P att L att L att R att R

gene ccdB gene ccdB
BP Clonase
attB-flanked donor entry By-product

PCR product or vector clone
expression clone
LR Reaction
att L att L att R att R att B att B att P att P
gene ccdB gene ccdB
LR Clonase
Entry destination expression By-product

clone vector clone
Step 1. Create an entry clone

ThestandardmethodforcreatinganentrycloneintheVectorNTIExpressGateway
CloningToolinvolvesamplifyingasequenceormoleculeofinterestwithattB
containingprimersdesignedbythesoftware,thenperformingBPrecombinationwith
adonor(pDONR)vectortogenerateanentryclone.
PCR product (flanked by attB sites) + pDONR vector (with attP) Entry Clone
(with attL)
Youcanalsocreateanentryclonebythefollowingmethods:
SelectanexistingattBcontainingDNAmoleculeFromwithintheGateway
CloningTool,youcanselectanalreadyexistingattBcontainingDNAmoleculein
thedatabase,suchasaGatewayExpressionCloneorapCMVSPORT6library,
forrecombinationwithadonorvector,tocreatetheentryclone

Gateway Cloning
12 Gateway Cloning Tool
ConstructanentryclonebyalternativemoleculeconstructionmethodsYou
canconstructyourownentrycloneusingotherVectorNTIExpressmolecule
constructionmethods,ensuringthattheclonecontainstherequiredattL1and
attL2sites(labeledasfeaturesasdescribedinAddEntryClonestoUseinLR
Reaction),thensavethemoleculeinthedatabaseandselectitdirectlyinthe
GatewayCloningtool.
Step 2. Create an expression clone

UsingtheGatewayCloningTool,entryclonescreatedfromanyoftheabovemethods
arerecombinedwithdestination(pDEST)vectorsinanLRrecombinationreactionto
generateexpressionclones,whichcandriveexpressionofthesequenceofinterest
whentransformedintohostcells.
Gateway Cloning Tool

Open the Gateway TheGatewayCloningToolcontainssettingsandfunctionsforassemblinga
Cloning Tool Gatewayconstructusingtheworkflowdescribedabove,andforcreatingand
managingGatewayCloningprojects.
Thereareseveralwaystoopenthetool:
ClickontheGatewayCloningbuttononthemaintoolbar.
SelectFile>New>GatewayCloningProjectfromthemainmenu.
WithamoleculeopeninMoleculeEditor,rightclickintheGraphicsor
SequencepaneandselectLaunchGateway.
LoadanexistingGatewayCloningprojectasdescribedinCreate,save,and
loadprojectsonpage124.
Thetoolwindowiscomposedofthefollowingpanes:
Gateway Project Thispanedisplaysthenameofthecurrentproject,andincludescontrolsforediting,

pane saving,andclosingprojects.Italsolistsanygeneratedmoleculesforthecurrent
project.

Gateway Cloning Tool 12
ClickontheGatewayProjectbuttontodisplaythispane.
Task List pane Thispanedisplaysthelistoftasksintheselectedproject.ClickontheTaskbuttonto

displaythislist.
AsyounavigatethroughtheGatewayCloningworkflow,theTaskListdisplaysthe
currenttask,andallowsyoutomovebetweentasksbyclickingonadifferenttask.

Gateway Cloning
12 Create, save, and load projects
Current Task pane Thispanedisplaysthecommandsandsettingsforthecurrentlyselectedtaskinthe

project.AsyounavigatethroughtheTaskListworkflow,thefunctionsinthispane
willchange.
Create, save, and load projects

Thetoolsforsaving,editing,andclosingprojectsarelocatedintheGatewayProject
pane:
TosaveanewGatewayCloningproject,clickonEditProjectintheGateway
Projectpane.Inthedialog,enteranameandanydescriptionfortheproject.
Tosavechangestoaproject,clickontheSaveProjectbutton.
Tocloseaproject,clickontheCloseProjectbutton.Ifthereareunsavedchanges,
youwillbepromptedtosavetheprojectbeforeclosing.

Gateway Cloning workflow 12
Toloadanexistingproject,inDatabaseExplorer,gototheProjectslist,double
clickontheProjectsfolder,selecttheCloningProjectssubfolderfromtheLocal
Database,anddoubleclickontheGatewayCloningprojectinthelisttoopenit.
Gateway Cloning workflow

TheTaskListdisplaysthedefaultGatewayCloningworkflow:
AmplifyFragmentstoUseinBPReaction
RecombineEntryClonesbyBP
PreviewEntryClones
AddEntryClonestoUseinLRReaction
CreateExpressionClonesbyLR
PreviewExpressionClones
Thissectiondescribeseachtaskintheworkflow.
Amplify fragments TheAmplifyFragmentstoUseinaBPReactiontaskisthedefaulttaskdisplayed

to Use in a BP whenyoufirstopentheGatewayCloningTool.
reaction Thefirststepinthistasktoselectthefragment(s)youwanttoamplifybyPCRforuse
inaBPcloningreaction.ThesefragmentswillbelistedintheFragmentstoAmplify
list.
Load molecules or fragments in the Fragments to Amplify list

WithamoleculeopeninMoleculeEditor,rightclickintheGraphicsorSequence
paneandselectLaunchGatewaytoloadtheentiresequenceinthelist.
WithamoleculeopeninMoleculeEditor,selectaportionofthesequenceinthe
GraphicsorSequencepane,rightclick,andselectLaunchGatewaytoloadonly
thatpartofthesequenceinthelist.

Gateway Cloning
12 Gateway Cloning workflow
WiththeGatewayCloningwindowopen,makesuretheAmplifyFragmentsto
UseinBPReactiontaskisselectedandclickontheAddbuttonunderFragments
toAmplifytoselectacompletemoleculefromthedatabase.
Tochangetheregionstoamplifyintheselectedmolecules,typeanewrangeinthe
FromandTofieldsintheFragmentstoAmplifysubpane.
Amplification settings
Withthefragment(s)loaded,selectthedesiredamplificationsettingsunderPCR
AmplificationSettings.Thestandardoptionsaredescribedbelow.
Standard Settings
Tm(C) Enter limits in degrees Celsius for primer
melting temperature (Tm) (temperature at
which 50% of primer is a duplex) and the
difference between Tm for sense and
antisense primers.
%GC Enter limits in degrees Celsius for primer
melting temperature (Tm) (temperature at
which 50% of primer is a duplex) and the
difference between Tm for sense and
antisense primers.
Primer Length Defaults to 20-25, recommended for
Gateway Primers
DNA/RNA button Select the type of nucleotide sequence.
Add GGGG-attBx 5 Extensions The default attB extensions are for single
fragment cloning: attB1 for the sense primer
and attB2 for the antisense primer. Select
from the dropdown list to replace the
defaults with other attB sequences for
creating Entry Clones for MultiSite Gateway
Cloning projects.
Add generated primers to oligo list Select this checkbox to add the primers you
generate to the oligo list
Note: Foradditionalamplificationsettings,clickonAdvanced.Theadvanced
amplificationsettingsareidenticalforallPCRprimers,andaredescribedinChapter3,
PrimerDesign.

Amplify
Whenyouhavemadeyourselections,clickontheAmplifybutton.Thenexttaskpane
willbedisplayed,andthegeneratedPCRproduct(s)willappearlistedintheBP
Insertssubpane,andalsolistedintheGatewayProjectpane.
Recombine Entry IntheRecombineEntryClonesbyBPtaskpane,youcanmodifythelistoffragments

Clones by BP withattBsitesandselectadonor(pDONR)vectororvectorswithwhichtocreate
entryclones.
BP Inserts
ThefragmentsyouamplifiedwithattBsitesarelistedintheBPInsertslistatthetopof
thepane.
ToaddapreviouslyamplifiedfragmentwithattBsites,orafragmentdesigned
withattBsitesbyanothermeans(e.g.,restrictionligation),clickontheAdd
buttonandselectthemoleculefromthedatabase.
Toremoveamoleculefromlist,selectitandclickonRemove.
Tocleartheentirelist,clickonClearAll.

Gateway Cloning
pDONR Vector
ThepDONRvectorisatypeofGatewayCloningvectorthatcontainsattPsites,
whicharerecombinedwiththefragmentscontainingattBsitestocreateentryclones.
att B att B att P att P att L att L att R att R
gene ccdB gene ccdB
BP Clonase II
attB-flanked PCR donor entry by-product
product or attB vector clone
expression clone
AvarietyofpDONRvectorsaresoldbyLifeTechnologies,andinsilicosequencesfor
theseareinstalledaspartofthedefaultVectorNTIExpressinstallation.
IMPORTANT! ToberecognizedasapDONRvectorinVectorNTIExpress,amolecule
mustcontainthecorrectattP1andattP2sequences,andthesesitesmustbelabeledas
featuresinthemoleculewiththefeaturenamesattP1andattP2.SeeChapter2,
MoleculeEditoronpage39forinformationonidentifyingandnamingfeatures.
ToselectapDONRvector:
ClickontheAddbuttonandselectthepDONRvectorfromthedatabase.
Toremoveavectorfromlist,selectitandclickonRemove.
Create the Entry Clone

Whenyouhavemadeyourselections,clickonCreateEntryClone.ThePreviewEntry
Clonestaskpanewillopen.

Preview Entry ThePreviewEntryClonestaskpanelistsalltheentryclonescreatedfromtheattB

Clones containingfragment(s)andthedonorvector(s)youselected,andincludesapreview
windowforviewinganentryclone.
EntryclonescontainattL1andattL2sites,andareusedtogenerateexpressionclones
viaanLRreaction.
IntheEntryCloneslist:
SelectacloneandclickonSavetoDatabasetosaveitasaDNAmoleculeinthe
database.
DoubleclickonaclonetodisplayitinthePreviewwindow.
InthePreviewwindow:
Usingthemagnifyingtoolstozoominandoutofthemolecule.
Displaythemoleculeaslinearorcircularbyclickingontheappropriatebutton.
Add Entry Clones Aftercreatingentryclones,clickonAddEntryClonestoUseinLRReactioninthe

to Use in LR TaskListtoproceedtothenexttaskintheworkflow.
Reaction Anyentryclonesthatyougeneratedfromtheprevioustasksintheworkflowwillbe
listedinthiswindow.
Toselectneworadditionalentryclonesinthedatabase,clickontheAddbutton
andselectfromthedialogbox.

Gateway Cloning
IMPORTANT! ToberecognizedasanentrycloneinVectorNTIExpress,amolecule
mustcontainthecorrectattL1andattL2sequences,andthesesitesmustbelabeledas
featuresinthemoleculewiththefeaturenamesattL1andattL2.SeeChapter2,
Toremoveanentryclonefromthelist,selectitandclickontheRemovebutton.
Create Expression IntheCreateExpressionClonesbyLRtaskpane,youcanmodifythelistofentry

Clones by LR clonesandselectadestination(pDEST)vectororvectorswithwhichtocreate
expressionclones.
Entry Clones
Anyentryclonesthatyougeneratedfromtheprevioustasksintheworkflowwillbe
listedintheEntryCloneslist.
Toselectneworadditionalentryclonesinthedatabase,clickontheAddbutton
andselectfromthedialogbox.
Toremoveanentryclonefromthelist,selectitandclickontheRemovebutton.
Toclearthelist,clickonClearAll.
pDEST Vector
ThepDESTvectorisatypeofGatewayCloningvectorthatcontainsattRsites,which
arerecombinedwiththefragmentscontainingattLsitestocreateexpressionclones.
att L att L att R att R att B att B att P att P
gene ccdB gene ccdB
LR Clonase II
entry destination expression by-product
clone vector clone
AvarietyofpDESTvectorsaresoldbyLifeTechnologies,andinsilicosequencesfor
theseareinstalledaspartofthedefaultVectorNTIExpressinstallation.
IMPORTANT! ToberecognizedasapDESTvectorinVectorNTIExpress,amolecule
mustcontainthecorrectattR1andattR2sequences,andthesesitesmustbelabeledas
featuresinthemoleculewiththefeaturenamesattR1andattR2.SeeChapter2,

ToselectapDESTvector:
ClickontheAddbuttonandselectthepDONRvectorfromthedatabase.
Create the Expression Clone

Whenyouhavemadeyourselections,clickonCreateExpressionClone.ThePreview
ExpressionClonestaskpanewillopen.
Preview ThePreviewExpressionClonestaskpanelistsalltheexpressionclonescreatedfrom
Expression Clones theentryclonesandthedestinationvector(s)youselected,andincludesapreview
windowforviewinganexpressionclone.
IntheExpressionCloneslist:
SelectacloneandclickonSavetoDatabasetosaveitasaDNAmoleculeinthe
database.
DoubleclickonaclonetodisplayitinthePreviewwindow.
InthePreviewwindow:
Usingthemagnifyingtoolstozoominandoutofthemolecule.
Displaythemoleculeaslinearorcircularbyclickingontheappropriatebutton.

Gateway Cloning

13 TOPO Cloning
TOPOTechnologyisafast,efficientwaytoclone.ThekeytoTOPOCloningisthe
enzymeDNAtopoisomeraseI,whosebiologicalroleistocleaveandrejoinDNA
duringreplication.Toharnessthisactivity,vectorsarelinearizedandeachendis
conjugatedwithtopoisomeraseonthe3phosphate.ThisenablesfastligationofDNA
sequenceswithcompatibleends.After5minutesatroomtemperature,theenzymeis
released,theligationiscompleteandtherecombinantmoleculeisreadyfor
transformationintoE.coli.
ManyLifeTechnologiesexpressionvectorsareadaptedforonestepTOPOCloning
ofPCRproductsinbothdirectionalandnondirectionalformats.Othervectorscontain
attrecombinationsequencesexteriortotheTOPOcloningsitessothatclonedinserts
arereadyforentryintotheTOPOsystem.
TOPOvectorscanbegroupedintothreecategories,basedonthenatureoftheirends:
ZeroBluntvectorshavetwobluntendsandcanacceptbluntendedDNA
fragments,includingampliconsproducedbyaproofreadingpolymerase.Inserts
areclonedinbothorientations.
TAvectorshavetwoendswith3Toverhangs.TheycanacceptproductsofPCR
amplificationwithaTaqpolymerase,whoseterminaltransferaseactivityadds3
Aoverhangstotheamplicon.Insertsareclonedinbothorientations.
Indirectionalvectorsoneterminalisbluntendedandtheotherhasa5GGTG
overhangonthebottomstrand.PCRproductsaregeneratedwitha5CACC
extensionononeendandthisstrandwhenunwoundispreferentiallyannealedto
thevectoroverhang.Morethan90%oftheclonesareinthecorrectorientation
andthetimespentinscreeningcoloniesistherebyreduced.
Thepresenceoftopoisomeraseenzymealsohelpsprotectvectorendsfrom
degradation,particularlyfromcontaminatingnucleasesthatmaybepresentinligase
preparations.Moreover,theavoidanceofrestrictionsitecutbackforcloningPCR
productsmeansthatinternalcleavagesitesarenotaproblem.
TOPO Cloning in Anylinear,doublestrandedDNAsequenceofinterestmaybeclonedintoaTOPO

Vector NTI vectorusingVectorNTIExpress.Inaddition,linearsequenceswith3Aoverhangs,
theproductsofPCRamplificationwithaTaqDNApolymerase,maybeclonedinto
Express
TOPOTAvectors.SuchTaqgeneratedmoleculescanbegeneratedinsilicousingthe
TOPOCloningtool.
TOPO Cloning Tool

Open the TOPO TheTOPOCloningToolcontainssettingsandfunctionsforassemblingaTOPO
Cloning Tool constructusingtheworkflowdescribedabove,andforcreatingandmanagingTOPO
Cloningprojects.
Thereareseveralwaystoopenthetool:

TOPO Cloning
13 TOPO Cloning Tool
ClickontheTOPOCloningbuttononthemaintoolbar.
SelectFile>New>TOPOCloningProjectfromthemainmenu.
paneandselectLaunchTOPOCloning.
LoadanexistingTOPOCloningprojectasdescribedinCreate,save,andload
projectsonpage136.
Thetoolwindowiscomposedofthefollowingpanes:
TOPO Project TheTOPOProjectpanedisplaysthenameofthecurrentproject,andincludes

pane controlsforediting,saving,andclosingprojects.Italsolistsanygeneratedmolecules
forthecurrentproject.
ClickontheTOPOProjectbuttontodisplaythispane.

TOPO Cloning Tool 13
Task List pane TheTaskListpanedisplaysthelistoftasksintheselectedproject.ClickontheTask

buttontodisplaythislist.
AsyounavigatethroughtheTOPOCloningworkflow,theTaskListdisplaysthe
currenttask,andallowsyoutomovebetweentasksbyclickingonadifferenttask.
Current Task pane Thispanedisplaysthecommandsandsettingsforthecurrentlyselectedtaskinthe

project.AsyounavigatethroughtheTaskListworkflow,thefunctionsinthispane
willchange.

TOPO Cloning
13 Create, save, and load projects
Create, save, and load projects

Thetoolsforsaving,editing,andclosingprojectsarelocatedintheTOPOProject
pane:
TosaveanewTOPOCloningproject,clickonEditProjectintheTOPOProject
pane.Inthedialog,enteranameandanydescriptionfortheproject.
Tosavechangestoaproject,clickontheSaveProjectbutton.
Tocloseaproject,clickontheCloseProjectbutton.Ifthereareunsavedchanges,
youwillbepromptedtosavetheprojectbeforeclosing.
Toloadanexistingproject,inDatabaseExplorer,gototheProjectslist,openthe
CloningProjectsfolderintheLocalDatabase,anddoubleclickontheTOPO
Cloningprojectinthelisttoopenit.
TOPO Cloning workflow

TheTaskListdisplaysthedefaultTOPOCloningworkflow:
AmplifyFragmentstoUseinTOPOReaction
CreateTOPOClones
PreviewClones
Thissectiondescribeseachtaskintheworkflow.
Amplify fragments TheAmplifyFragmentstoUseinTOPOReactiontaskisthedefaulttaskdisplayed

to Use in TOPO whenyoufirstopentheTOPOCloningTool.
reaction

TOPO Cloning workflow 13
Thefirststepinthistasktoselectthefragment(s)youwanttoamplifybyPCRforuse
inaTOPOcloningreaction.ThesefragmentswillbelistedintheFragmentslist.
Load molecules or fragments in the Fragments list

paneandselectLaunchTOPOCloningtoloadtheentiresequenceinthelist.
WithamoleculeopeninMoleculeEditor,selectaportionofthesequenceinthe
GraphicsorSequencepane,rightclick,andselectLaunchTOPOCloningtoload
onlythatpartofthesequenceinthelist.
WiththeTOPOCloningwindowopen,makesuretheAmplifyFragmentsto
UseinTOPOReactiontaskisselectedandclickontheAddbuttonunder
Fragmentstoselectacompletemoleculefromthedatabase.
Tochangetheregionstoamplifyintheselectedmolecules,typeanewrangeinthe
FromandTofieldsintheFragmentstoAmplifysubpane.
PCR Amplification Settings

Withthefragment(s)loaded,selectthedesiredamplificationsettingsunderPCR
AmplificationSettings.Thestandardoptionsaredescribedbelow.
Standard settings
Tm(C) Enter limits in degrees Celsius for primer melting
temperature (Tm) (temperature at which 50% of primer is
a duplex) and the difference between Tm for sense and
antisense primers.
%GC Enter limits in degrees Celsius for primer melting
temperature (Tm) (temperature at which 50% of primer is
a duplex) and the difference between Tm for sense and
antisense primers.
Primer Length Defaults to 20-25, recommended for TOPO Primers
DNA/RNA button Select the type of nucleotide sequence.
Add generated primers to oligo Select this checkbox to add the primers you generate to
list the oligo list
Cloning termini
IntheoptionsunderCloningtermini:
ChooseBlunttogenerateanampliconwith2bluntends.Thesewillbetheexact
boundariesoftheselection.TheseampliconsarebestusedwithZeroBlunt
TOPOvectors.

TOPO Cloning
13 TOPO Cloning workflow
ChooseTAtogenerateanampliconaswouldbeproducedbyamplificationwith
aTaqpolymerase.Theprimersinsuchacasewillannealtotheexactboundaries
oftheselection.However,terminaltransferaseactivityintheenzymewilladd3
Aoverhangstoeachendoftheamplicon.
ChoosedirectionalforcloninginTOPOdirectionalvectors,e.g.pENTRD/
TOPO.Theampliconwillbegeneratedusingprimers,oneofwhichincludesa
5CACCextension.
Advanced
Foradditionalamplificationsettings,clickonAdvanced.Theadvancedamplification
settingsareidenticalforallPCRprimers,andaredescribedinChapter3,Primer
Design.
Amplify
Whenyouhavemadeyourselections,clickontheAmplifybutton.Thenexttaskpane
willbedisplayed,andthegeneratedPCRproduct(s)willappearlistedintheInserts
subpane,andalsolistedintheTOPOProjectpane.

TOPO Cloning workflow 13
Create TOPO IntheCreateTOPOClonestaskpane,youcanmodifythelistoffragmentsand

Clones selectavectororvectorswithwhichtocreateTOPOclones.
Inserts
ThefragmentsyouamplifiedarelistedintheInsertslistatthetopofthepane.
Toaddapreviouslyamplifiedfragmentorafragmentdesignedwiththe
necessaryoverhangsbyanothermeans,clickontheAddbuttonandselectthe
moleculefromthedatabase.
Toremoveamoleculefromlist,selectitandclickonRemove.
Vectors
Toselectavector:
ClickontheAddbuttonandselectthevectorfromthedatabase.
Create the TOPO Clone

Whenyouhavemadeyourselections,clickonCreateTOPOClone.ThePreview
Clonestaskpanewillopen.

TOPO Cloning
13 TOPO Cloning workflow

14 GeneArt Cloning
Introduction
UsingVectorNTIExpress,youcancreateGeneArtassembliesfromDNAmolecules
inthedatabase.Simplyselectthefragmentstobeassembledandthesoftwarewill:
Analyzethesequencesforhomologiesbetweenthefragments
DesignPCRprimerstocreatethenecessaryendhomologies
DesignstitchingoligosforuseinGeneArtHighOrderassemblies
Displaytheassembledmoleculewiththespecifiedprimersand/orstitchingoligos
inasinglemoleculefile
FormoreinformationaboutGeneArttechnology,visitourwebsiteat
www.lifetechnologies.comandsearchforGeneArt.Detailedtechnicalinformation
foreachtypeofGeneArtassemblymethodisavailableinthefollowinguserguides:
GeneArtSeamlessCloningandAssemblyKitUserGuideandGeneArtHighOrderGenetic
AssemblySystemUserGuide.Theseareavailablefordownloadfrom
www.lifetechnologies.com/manualsandaresuppliedwitheachkit.
GeneArt GeneArtSeamlessCloningTechnologyisahighlyefficient,vectorindependent
Seamless Cloning systemforthesimultaneousandseamlessassemblyofuptofourDNAfragmentsplus
avectortotalingupto13kbinlength(includingthevector).Thesystemallowsthe
Overview
cloningoftheDNAfragmentsintovirtuallyanylinearizedE.colivector,doesnot
requirepreexistingrecombinationsitesoranyextraDNAsequences,andeliminates
theneedforextensiveenzymatictreatmentsoftheDNAsuchasrestrictionand
ligation.Asingleproprietaryenzymemixturerecognizesandpreciselyassemblesthe
DNAfragmentssharinga15basepair(bp)endhomologythatyoucancreatebyPCR
amplification.
GeneArt High TheGeneArtHighOrderGeneticAssemblySystemisahighlyefficient,vector

Order Assembly independentsystemforthesimultaneousassemblyofupto10DNAfragmentsplusa
vectortotalingupto110kbinlength(includingthevector).Thesystemrelieson
Overview
yeastsabilitytotakeupandrecombineDNAfragmentswithhighefficiency.This
process,termedtransformationassociatedrecombination,greatlyreducesinvitro
handlingofDNAandeliminatestheneedforenzymatictreatmentsofDNAsuchas
restrictionandligationwhileallowingprecisefusionsofDNAsequences.

GeneArt Cloning
14 How GeneArt Assembly Works
How GeneArt Assembly Works

InGeneArtAssembly,multipleDNAfragmentsplusavectorcanbejoinedusing
overlappingsequencehomologybetweenfragmentendstosplicethefragments
together.Ifhomologydoesnotalreadyexistbetweenfragmentends,VectorNTI
ExpresswillautomaticallydesignPCRprimersthatyoucanusetoaddhomologyto
thefragmentsviaPCRamplification.Alternatively,forHighOrderAssembly,upto
threesetsofstitchingoligosmaybedesignedtocreatesplicesacrossfragments
withouthomology.
WhenthefragmentsandvectorareassembledinVectorNTIExpress,thesoftware
performsahomologycheckbasedontherulesfortheGeneArtAssemblymethod
youareusing.Thehomologyofthesequenceendswillbeanalyzed,andinHigh
OrderAssemblytheinternalsequencesofthefragmentswillalsobeanalyzedto
ensurethatanyinternalhomologieswillnotinterferewithfragmentsplicing.Then
anyrequiredPCRprimersand/or,inthecaseofHighOrderAssembly,stitching
oligoswillbedesigned.
TherulesforhomologyaredescribedindetailintheGeneArtSeamlessCloningand
AssemblyKitUserGuideandGeneArtHighOrderGeneticAssemblySystemUserGuide.
Seamless Cloning: Workflow diagram and an example of PCR primers designed to generate a
15-bp fragment end homology:
Assembled
Linear vector Recombine DNA fragments
circular construct
DNA fragments and linear vector
15-bp homology
5'-GTG AAT TCG GGC TCG TTA GGA CTA...-3' Forward Primer
for Fragment 2
9-nt 6-nt
Reverse Primer
for Fragment 1 3'-...ATA CCC CAC TTA AGC CCG AGC-5'
Sequences specific Sequences specific

to Fragment 1 to Fragment 2
High Order Assembly: Workflow diagram and an example of PCR primers designed to generate a
30-bp fragment end homology:
Stitching
DNA fragments
oligos
Assembled construct
Linear vector
Transformation-associated recombination
30-nt homology
Forward Primer
5'-TCG AGC GCA TTT CGC CGT AAA GCG AGT TTA TTA GGA CTA...-3' for Fragment 2
15-nt 15-nt
Reverse Primer
for Fragment 1 3'-...GTG AAT TCG AGC TCG CGT AAA GCG GCA TTT CGC TCA AAT-5'
Sequences specific Sequences specific

to Fragment 1 to Fragment 2

Open the GeneArt Assembly Tool 14
Open the GeneArt Assembly Tool

YoucanopentheGeneArtAssemblyWizardfromtheMoleculeEditorwitha
moleculeloadedorfromthemaintoolbarwithnomoleculeselected.
Note: AllmoleculesusedinGeneArtassemblymustbelinear.
From the molecule 1. WithalinearDNAmoleculeloadedintheMoleculeEditor,selectthepartofthe

editor sequenceyouwanttocloneormakenoselectiontoclonethewholemolecule.
Note: Theselectedmoleculeorsequencemustbe>100bp.
RightclickandselectLoadinGeneArtorclickontheGeneArtCloningbutton
onthemaintoolbar.
2. Selectthestrandyouwanttoanalyze(DirectorComplementary).
3. TheGeneArtAssemblyWizardwillopenwiththemoleculeorsequenceloaded.
From the main 1. Withnomoleculeselected,clickontheGeneArtCloningbuttononthemain

toolbar with no toolbar,orselectDesign>GeneArtCloning.
molecule selected 2. TheGeneArtAssemblyWizardwillopenwithnosequenceloaded.
GeneArt Assembly Wizard with molecule selected

GeneArt Cloning
14 GeneArt Assembly Wizard
GeneArt Assembly Wizard

TheGeneArtAssemblyWizardwindowliststhemoleculesandfragmentstobe
assembledandincludessettingsforassembly.
Assembly settings ThetoolincludesdifferentoptionsforSeamlessCloningorHighOrderAssembly.
1. SelecttheappropriateAssemblyoptionfromthedropdownlist.
2. SelecttheMoleculeFormyouwantforthefinalassembly:CircularorLinear.
Add and organize TheAssemblyWizardhastoolsforaddingandremovingfragmentsanddesignating

fragments theirorderofassembly.
Fragment requirements
AfragmentforGeneArtAssemblycanbeanymoleculeinthedatabasethatis100
basepairsinlength.
Add fragments
Toaddafragment,clickontheAddFragmentbuttonandselectamoleculefromthe
database.
Themoleculewillappearaddedtothelistinthetool.
Select the vector

OnefragmentineachGeneArtassemblymustdesignatedasthevector.Thevector
formsthebasefragmentontowhichotherfragmentsareadded.Thevectorisalways
thefirstfragmentlistedintheWizard,andisflaggedwithaVintheOrdercolumn.

GeneArt Assembly Wizard 14
Tochangethefragmentdesignatedasthevector,clickinsidetheVectorcolumnand
thatfragmentwillbemovedtothetopofthelist.
Vector icon
Re-order fragments
ThefragmentswillbeassembledintheorderinwhichtheyrelistedintheGeneArt
tool,startingwiththevector.TheorderofthefragmentsisreflectedintheOrder
column,whichdesignatesthevectorwithaVandnumberstherestofthefragments
inorder.
Toreorderthenumberedfragments,selectafragmentinthelistandthenclickonthe
UporDownbuttontochangeitsposition.Notethatyoucannotmoveafragmentinto
theVectorpositionusingthebuttonsselectthevectorasdescribedabove.
Figure2 Clicking on the Up button to move a selected fragment up in the list.
Fragment orientation
TheorientationofeachfragmentinthefinalassemblyisindicatedintheOrientation
column(ForwardorReverse).Youcanchangetheorientationbyclickinginthis
column.
Remove a fragment
Toremoveafragment,clickonitinthelisttoselectitandthenclickonRemove
Fragment.
Design PCR VectorNTIExpressSoftwarecanautomaticallydesignPCRprimersforadding

primers to create homologytotheendsofafragmentforGeneArtassembly.InthePCRreaction,each
primerwilladdbasestotheamplifiedfragmenttocreatethenecessaryhomologywith
end homology
theadjacentfragment.
Note: Ifthefragmentsalreadyhavetherequiredendhomology,primerdesignis
unnecessary.

GeneArt Cloning
ThisprimerdesignoptionisavailableforbothSeamlessCloningandHighOrder
Assembly,thoughthelengthofthePCRprimersvariesbetweenassemblymethods,
basedontheirdifferentendhomologyrequirements.Theprimerdesignrulesare
describedindetailintheGeneArtuserguides.ThePCRprimerdesignswillbesaved
withthefinalassembledmolecule.
TodesignPCRprimerstocreatethenecessaryhomologyforafragment:
1. ClickintheAmplifycolumnforthatfragment.
2. TheNwillchangetoaYforthatfragment.
Design stitching ForHighOrderAssembly,inadditiontoprimerdesigntocreateendhomology,upto

oligos (High Order threesetsofstitchingoligosmaybedesignedtocreatesplicesacrossfragments
withouthomology.Theseoligolinkerscreateabridgeacrossadjacentfragmentsto
Assembly only)
promoterecombinationaljoininginyeast.
Note: AlimitofthreesetsofstitchingoligosmaybeusedinasingleHighOrderAssembly,
andifstitchingoligosareused,atotaloffivefragmentsplusvectormaybeincludedin
theassembly.(ThisdiffersfromaHighOrderAssemblywithoutstitchingoligos,
whichcanincludeupto10fragmentsplusvector.)
ThestitchingoligorulesaredescribedindetailintheGeneArtHighOrderGenetic
AssemblySystemUserGuide.Theoligodesignswillbesavedwiththefinalassembled
molecule.
Toselectstitchingoligosforaparticularfragment:
1. WithHighOrderAssemblyselected,clickintheStitchcolumnforthatfragment.
2. TheNwillchangetoaYforthatfragment.
Create the 1. WhenyouhaveselectedyourdesiredsettingsintheGeneArtAssemblyWizard,

assembled clickontheNextbutton.
molecule 2. Specifyanameforthemoleculeinthedatabase,aswellasnamesforanyPCR
primersand/orstitchingoligos.

GeneArt Assembly Wizard 14
3. ThefinalassemblywillbedisplayedintheMoleculeEditor.
ThesourcefragmentswillbelistedintheFeatureMapunderMiscFeature.
ClickonaPCRPrimerorStitchOligointheGraphicspanetohighlightthat
featureinthesequence.
ThePCRprimersandstitchingoligosarelistedintheAnalysisResultspane,and
willbeaddedtotheOrderinglist.

GeneArt Cloning

15 Parts Assembler
UsingVectorNTIExpressSoftware,youcanassemblestandardDNApartsthat
encodebasicbiologicalfunctionsfrommoleculesusingdefinedassemblystandards.
ThestandardDNAsequencesdefinedinVectorNTIExpresshavebeendevelopedvia
anopentechnicalstandardssettingprocessledbytheBioBricksFoundation.
Atitsmostbasiclevel,apartisanyDNAsequencewithadefinedbiologicalfunction
(e.g.,apromoterregion,asequenceencodingaprotein,etc.).Tojointwoparts
together,upstreamanddownstreamsequencescontainingsitesforspecificrestriction
enzymesareaddedtoeachpart.Thisallowsforthecreationoflargerpartsbychaining
togethersmalleronesinanydesiredorder.Intheprocessofchainingpartstogether,
therestrictionsitesareremoved,allowingthefurtheruseofthoserestrictionenzymes
withoutbreakingthenew,largerassemblyapart.Tofacilitatethisassemblyprocess,
eachpartitselfmaynotcontainanyoftheserestrictionsites.
Additional VectorNTIExpressPartsAssembleriscompatiblewithBioBrickpartstandards.For
Information about generalinformationabouttheBioBricksFoundation,visitwww.biobricks.org.For
informationaboutpartsandassemblystandards,includinginstructionsandtutorials,
Parts and
visithttp://openwetware.org/wiki/The_BioBricks_Foundation:Standards/Technical/
Standards Resources.
TheDNAsequencesofthousandsofpublicdomainstandardbiologicalpartsare
availablethroughtheRegistryofStandardBiologicalPartsathttp://partsregistry.org.
Foradetaileddescriptionoftheassemblystandards,visithttp://openwetware.org/
wiki/The_BioBricks_Foundation:Standards/Technical/Formats.
Note: BioBrickisatrademarkoftheBioBricksFoundation,Inc.TheBioBrick
trademarkisusedhereinmerelyforthepurposeoffairuse.TheBioBricksFoundation,
Inc.isnotaffiliatedwithLifeTechnologiesCorporation.TheBioBricksFoundation,Inc
hasnotauthorized,sponsoredorotherwiseendorsedthisproductorouruseofthe
BioBricktrademarkherein.InformationaboutBioBrickandtheBioBricks
Foundation,Inc.canbefoundatwww.biobricks.org.
Using the Parts Assembler

ToopenthePartsAssembler:
ClickonthePartsAssemblerbuttononthemaintoolbar.
SelectFile>New>PartsAssemblerProjectfromthemainmenu.
Selecting Parts ThePartsAssemblerwindowisdividedintotwopanesoneforeachpartyouwantto

combine.

Parts Assembler
15 Using the Parts Assembler
1. Toselectthefirstpartthatyouwanttoassemble,clickontheOpenMolecule
buttonbeneaththeleftpane,andselectthemoleculefromthedatabase.The
moleculemustbecompatiblewithadefinedassemblystandard,asdescribedin
thefollowingsection.
2. Selectthesecondmoleculeintherightpane.
Whenyouselectamolecule,itssequencewillbedisplayedinthepaneandthe
assemblystandardsthatarecompatiblewiththatsequencewillbelistedatthebottom
ofthepane.Ifthemoleculedoesnotconformtoanyofthestandards,youwillreceive
awarningmessage.
Restrictions on Anymoleculeinthedatabasecanbeselectedasapart,aslongitconformsoneofthe
Parts assemblystandardsshowninthefollowingtable.Toconformtoanassemblystandard,
themoleculemustnotcontaintherestrictionenzymedigestionsiteslistedinthetable
forthatstandard.Somestandardsalsoincludeadditionalrules.Formoreinformation
aboutassemblystandards,visithttp://openwetware.org/wiki/
The_BioBricks_Foundation:Standards/Technical/Formats.
Assembly Standard Parts must not contain the following restriction sites
10 EcoR I, Not I, Xba I, Spe I, Pst I, Nhe I, Pvu II, Xho I, Avr II, Sap I
12 EcoR I, Spe I, Nhe I, Not I, Pst I
20 EcoR I, Xba I, Spe I, Sbf I
21 EcoR I, Bgl II, BamH I, Xho I
23 EcoR I, Not I, Xba I, Spe I, Pst I (in addition, sequences must be
in frame without start or stop codons, and may not begin with
TC)
25 EcoR I, Not I, Xba I, NgoMI (aka NgoMIV), AgeI, Spe I, Pst I
Note: Ifyouareplanningtocreateandsharepartswithotherindividualsandgroups
usingthesestandards,werecommenddesigningthemsothattheycontainnoneofthe
restrictionsiteslistedforanyoftheassemblystandards,toensuremaximum
portability.
Assembly Settings Whenyouhaveselectedtwomolecules,clickontheAssemblebuttonatthebottomof

thewindow.

Using the Parts Assembler 15
TheConstructandAssembledialogwillopenwithoptionsforassembly.
EnteranameforyournewconstructintheNamefield.
Device Type
Adeviceinisanyconstructionthatperformsaparticularbiologicalfunction.The
DeviceTypesectionofthedialogallowsyoutocharacterizethetypeofdeviceyouare
constructing.ThisinformationwillbeincludedintheGeneralDescriptioninformation
oftheassembledmolecule.
Thedropdownlistincludesstandardtypesofdevices.Alternatively,youcanselect
Unassignedfromthelist,orenteryourowndevicedescriptionbyselectingtheOther
checkboxandtypinginthefield.
Chassis
Partsaretypicallyintegratedintothegenomeofaparticularorganisma.k.a.a
chassisforpropagationandfunctionality.Youcanspecifythegenomeofthe
organismthatyouwillbeusingbyselectingtheappropriatecheckboxorenteringtext
intheOtherfield.ThisselectedchassiswillbelistedintheGeneralDescription
informationoftheassembledmolecule.
Assembly Direction and Standard

Selectthedirectionofeachmoleculetouseforthefinalassembly,andselectthe
assemblystandardtousefromthedropdownlist.
Note: Ifthereisnosharedassemblystandardthatwillworkwithbothmolecules,this
dropdownlistwillbeblankandnoassemblywillbepossible.
Completing and WhenyouclickonOKintheConstructandAssembledialog,theassembledmolecule

previewing the willopeninaPreviewwindow.
assembly Youcanusethetoolsinthewindowtomagnifytheassembly,orviewitasalinearor
circularmolecule.

Parts Assembler
15 Viewing the assembly in Molecule Editor
ClickonSaveinthePreviewwindowtosavetheassemblyasamoleculeinthe
database.
Viewing the assembly in Molecule Editor

ToviewtheassembledmoleculeintheMoleculeEditor,opentheDatabaseExplorer
andopenthemolecule.
Theassembledmoleculeconsistsofthetwooriginalsequencesjoinedtogetherwitha
standardbridgingsequenceorscarcreatedbytheoverlapoftherestrictiondigested
ends.Thescarsequenceisdeterminedbytherestrictionenzymesusedintheassembly
standard,asdescribedonthewebpagesmentionedearlier.
TheoriginalpartsarelistedintheComponentslistinthePropertiespane.
ThescarislistedintheFeatureMap,anddisplayedasfeatureintheGraphics
pane.
TheGeneralDescriptionincludestheinformationyouenteredaboutthe
assembledpart,includingdevicetypeandchassis.

16 Contig Assembly using
ContigExpress
ContigExpressisaprogramforassemblingandeditingsequencingfragments,either
intheformoftextsequencesorchromatogramsfromautomatedsequencers,into
longercontiguoussequencesorcontigs.
ContigExpressusesCAP3todrivetheassemblyprocess.Thiswidelyusedsequence
assemblyprogramcanusequalityvaluescores(QVs)inendstrimming,contig
constructionandconsensuscalculation.ItalsoproducesexcellentresultswhenQVs
areunavailable.Itiscapableofusingforwardreverseconstraintstoevaluatecontigs,a
featurethathelpsinaccurateplacementofrepetitivesequencefragments.Itcanalso
identifyanddiscardchimericsequencingreadsthatfrequentlyresultfromlane
trackingerrors.OneofthemajorstrengthsofCAP3isitsconsensusgeneration
algorithmbasedonweightedsumofQVs.
ContigExpressanalysiscanbesavedasaContigExpressProject,whichcontainsthe
fragments,theirassemblies,andassemblyoptions.InContigExpress,fragmentscanbe
editeddirectly,withthechromatogramsinfullview.Changesaretrackedanda
historyismaintained.ThecontigsgeneratedcanbesavedtotheVectorNTIExpress
database.
About this chapter

Thischapterhasthefollowingsections:
OpenContigExpress ................................................. 153
ManageContigExpressprojects....................................... 155
Addfragmentstoassemble ........................................... 156
Fragmenttrimming .................................................. 156
Assemblysettings................................................... 160
Performtheassembly ................................................ 164
Save,delete,andrenamecontigs...................................... 165
Viewtheassembly................................................... 166
Translatetheconsensussequence ..................................... 168
Editthecontigsequence.............................................. 169
Open ContigExpress
ToopentheContigExpresstool:
ClickontheContigExpressbuttononthemaintoolbar.

Contig Assembly using ContigExpress
16 Open ContigExpress
LoadanexistingContigExpressprojectasdescribedinManageContigExpress
projectsonpage155.
ContigExpress TheContigExpresswindowconsistsofthefollowingpanes:
window ContigProjectpane:Inthispane,youopen,save,andcloseprojects;navigate
tasks;andadd,organize,andassembleyoursequencingfragments.Thispane
includesthefollowingfeatures:
Projectmanagementbuttons
TaskList
Contig/FragmentTree
Contigandfragmentmanagementbuttons
FragmentSequenceViewer:Displaysthesequenceoftheselectedfragmentand
anytrimmedbases,aswellasachromatogramofthefragment
Trimming/assemblysettings(seepages156164):Settingsfortrimmingand
assemblingfragments
ContigViewer(shownonpage166):Displaysthecontigassemblyingraphical
form,withtheoverlappingassembledfragmentsequencesandchromatograms
TheContigViewerincludesthefollowingsubpanes:
GraphPane
AlignmentPane
Fragment Sequence Viewer
Contig Project pane
Manage project buttons
Task List
Contig/Fragment Tree
Manage fragments/
contigs buttons

Manage ContigExpress projects 16
Manage ContigExpress projects

Thetoolsforsaving,editing,andloadingprojectsarelocatedintheContigProject
pane:
Save and rename a Tosaveanewproject,clickonNewProjectinthepane.Inthedialog,entera

project nameandanydescriptionfortheproject.
Tosavechangestoanexistingproject,clickontheSaveProjectbutton.
Tochangeaprojectnameordescription,clickonEditProject.
Open a project Toloadanexistingprojectinthedatabase,inDatabaseExplorer,gotothe

Projectslist,doubleclickontheProjectsfolder,selecttheContigAssembly
ProjectsfolderfromtheLocalDatabase,anddoubleclickontheContigExpress
projectinthelisttoopenit.
Toloadanexistingprojectthathasbeensavedasa.cepxor.cepfile,clickonthe
LoadProjectsbuttonintheProjectPropertiespaneandselecttheprojectfromthe
Opendialog.
Close a project Tocloseaproject,clickontheCloseProjectbutton.Ifthereareunsavedchanges,you

willbepromptedtosavetheprojectbeforeclosing.
Export a project Toexportaprojectasa.cepxfile,clickontheExportProjectbuttonandenteraproject

nameintheExportContigExpressProjectdialog.
Navigate the Task ClickonastepintheTaskListtodisplaythesettingsforthatstep.

List

16 Add fragments to assemble
Add fragments to assemble

ThetoolsforaddingthesequencefragmentstoassemblearelocatedbelowtheContig/
FragmentTree.
Asyouaddfragments,theyaredisplayedintreeformatwithselectionboxesthat
allowtoselectindividualfragmentsforsubsequenttrimmingandassembly.
Add fragments Toaddindividualfragments:

ClickAddfromFiletoselectasupportedfiletype:
GenBank(*.gb)
FASTA(*.txt)
ABI(*.abi)
AB1(*.ab1)
StadenSCF(*.scf)
LegacyContigExpressprojectfile,containingmultiplefragments(*.cep)
ClickAddfromDatabasetoselectDNAmoleculesfromthelocaldatabase.
Note: PresstheCtrlorShiftkeystoselectmultiplefiles/molecules.
Remove fragments Toremovefragmentsfromtheassemblyproject,selectthecheckboxesnexttothe

fragmentnamesinthetreeandclickontheDeletebutton.Thisremovesfragments
fromtheprojectonly,anddoesnotdeletethemfromthedatabase.
Rename fragments Torenameafragment,rightclickonthefragmentnameinthelistandselectEdit.

Enteranewnameforthefragmentinthedialogbox.
Fragment trimming
Fragmenttrimmingisanoperationperformedonfragmentstooptimizesequencing
resultsandcontigassembly.Trimmingisperformedonfragmentendstoremove
unresolvedorpoorqualitynucleotidesbasedonchromatogramresults.Itisalsoused
toremoveresiduevectorsequencesfromthefragments.

Fragment trimming 16
Select fragments Toselectordeselectfragmentsfortrimming,clickthecheckboxnexttoeach

to trim fragmentnameintheContig/FragmentTree.
Toselectordeselectallthefragmentsinthelist,clickthetoplevelFragments
checkbox.
Trim ends ClickonTrimendsintheTasklistoftheContigProjectpanetoselectthesettingsthat

determinehowfragmentendsaretrimmed.
5 end settings
Removeblocksofoffscalepeaks>=than___consecutivebasesremovesthe
definednumberofconsecutivebasesthatarebelowacceptablecriteria.
Scanningentirefragment,trimuntil5end(____bases)contains<___
ambiguitiestrimspoorlyresolvedbases.
Scanningentirefragment,trimuntil5end(____bases)contains<___bases
withQV<____trimsbasesbasedonthedefinedchromatogramquality.
Trimatleast__basesisanarbitrarysettingthatmaybebaseduponthefactthat
yourprimershavetails.
3 end settings
Frommaxpeak,trimfromfirstblockof___consecutivebases<___%ofmax
valuetotheendremovesbasesinthedefinedregionofthesequencewhose
peaksdonotmeetthe%valueyoudefine.
Scanentirefragment,trimuntilremaining3end(____bases)contains<___
ambiguitiestrimspoorlyresolvedbases.
Scanentirefragment,trimuntil3end(____bases)contains<___baseswith
QV<____trimsbasesbasedonthedefinedchromatogramquality.
Trimatleast__basesisanarbitrarysettingthatmaybebaseduponthefactthat
yourprimershavetails.
Post trim settings

Maximumremaininglength____basessetsthemaximumlengthofthe
fragmentthatmustbeleftaftertrimming
Removeleadingandtrailingambiguitiesremovespoorlyresolvednucleotides
thatmaybeleftaftertrimming
RemovepolyA/T(morethan____consecutive)removesthesenucleotidesthat
maybepresentifthesequencewasflipped,producingapolyT5end.

16 Fragment trimming
Preview the trimmed ends

1. WiththefragmentstobetrimmedselectedintheContig/FragmentTree,clickon
Calculatetopreviewthetrimmingresults.
2. ClickonafragmentnameintheContig/FragmentTreetodisplaythefragmentin
theFragmentpanewiththetrimmedregionshighlighted.
TrimlocationsaredisplayedasrednucleotidesintheFragmentpane.
Trim vector Ifyouaresequencinginsertsincloningvectors,vectorcontaminationtrimmingallows

contaminations youtotrimthevectorresiduesthathavebeenamplifiedaspartthesequencing
process,leavingonlythesequenceofinterestintheresultingcontigassembly.
ClickonTrimvectorcontaminationsintheTasklistoftheContigProjectpaneto
selectthesettingsthatdeterminehowvectorresiduesaretrimmed,andthevectorfile
containingthesequencetobetrimmed.
Note: Whenvectortrimmingisperformed,thevectorsequenceanditsreverse
complementisautomaticallyused,sothatforwardandreversesequencingreadscanbe
trimmedusingonesetup.
Vector trimming settings

TheCommonSettingsinthispaneareasfollows:
Minimumvectoroverlapistheminimumnumberofbasesinthefragmentthat
overlapwiththoseontheclone.Thissettingmustbe5orgreater.
Minimumvectoroverlapwithambiguitiesincludespoorlyresolvedresidues
Vectormatchthresholdisthe%requiredtomatch
Removeadditionalfrom5/3endreferstotheadditionalbasestobe
removed

Fragment trimming 16
Select the vector sequence

ToaddavectorsequencetothelistofsequencestotheVectorList:
ClickonImporttoimportavectorsequencefroma.fastaor.gbfile,or
ClickonDatabasetoselectafileinthedatabase.
ClickonRemovetoremoveafilefromthelist.
Note: Thevectorsequencemaybeanexistingsequencefile,oryoumaywanttocreate
asequencefilecontainingthespecificresiduestobetrimmed.
Preview the trimmed vector contaminations

1. SelectthefragmentstobetrimmedintheContig/FragmentTree.Makesurethe
vector(s)youselectedwereusedtoamplifytheselectedfragments.
2. ClickonTrimtopreviewthetrimmingresultsfortheselectedfragments.
3. ClickonafragmentnameintheContig/FragmentTreetodisplaythefragmentin
theFragmentpanewiththetrimmedregionshighlighted.
TrimlocationsaredisplayedasrednucleotidesintheFragmentpane.
Additional notes on vector trimming
Trimming contamination from TOPO cloning projects

TOPOvectorsarerepresentedintheVectordatabaseastheyaresuppliedand
used,i.e.,aslinearmolecules.Inthisform,itisimpossibletodefineapolylinker
crossingbothcloningtermini.Werecommendtemporarilyconvertingthevectorto
circular(DNA/RNAmenu>Edit>DNA/RNAMoleculetab)inordertodefinea
suitablepolylinker.
ManyTOPOvectorsarehighlysymmetricaboutthe2cloningtermini.Useof
thedefaulttrimmingparametersmaytrimtheentireread.Werecommendvalues
of>=15fortheoverlapsizeand>=85forthematchthreshold.
Trimming an intronic sequence from sequencing reads of amplified exons

Youcanusevectortrimmingoperationstoremoveintronicsequencesfromamplified
exons.Intheseworkflows,thesequencingreadsareoftenprimedfromsitesinthe
intronregions.Todothis,itisnecessaryistocreateapolylinkerthatjoinstogether
directstrandsequencefrombeforeandaftertheexonunderinvestigation.
1. UsingVectorNTIExpressSoftware,openafilecontainingtheannotatedexon
boundedbyintronsequence.

16 Assembly settings
2. Makeacontinuousselectionfrom50basesbeforeto50basesaftertheexon,i.e.
theexonaswellas5and3intronsequence.
3. Usetheselectiontocreateanewlinearmolecule.Nameittoindicatethatitisan
exonpolylinker.
4. Opentheexonpolylinkerfile,selecttheexonanddeleteit.Thensavethefile
containingthetwoconcatenatedintronsequences.
5. SelectthefileintheTrimvectorcontaminationstaskandproceedwithtrimming.
Assembly settings
ClickontheAssemblecontigintheworkflowtheviewtheassemblysettings.
Assembly tab
Therearethreemaincheckboxestocontrolsomeimportantaspectsofthesequence
assemblyprocess,asshowninthetablebelow:
Assembly Options
Use Quality Values When available, base quality values (QVs) are used to trim
poor-quality ends, compute overlaps between reads, construct
multiple sequence alignments, and generate a consensus
sequence. Use of such scores is optional; when they are
unavailable, assembly will still proceed. Default ON.
Detect Chimeric Reads Chimeric reads consist of pieces from different parts of the
sequence region, usually generated as artifacts. They are
identified based on overlap conflicts and are excluded from the
construction of contigs. The mechanism of detecting chimeric
reads can be enabled or disabled with Detect Chimeric
Reads check box. Default: ON

Assembly settings 16
Assembly Options
Use Forward-Reverse When reads from both ends of subclones are available,
Constraints constraints are satisfied if they lie on opposite strands of a
double-stranded DNA molecule and within a specified
minimum and maximum range. This corrects assembly errors
due to misplacement of reads containing repeat sequences
and minimizes occurrence of singletons. A few unmet
constraints are allowed. However, if a sufficient number of
constraints are not satisfied by a join AND they all suggest an
alternative one supported by the overlap information, the
alternative join will be made. For most small- and moderate-
sized projects, it is not necessary to use this feature, unless in
a situation involving large region of repeats. Default: OFF.
TheAssemblyTabcontainssomedetailedsettingsrelatedtotheuseofforward
reverseconstraints.AllthesesettingsaredisabledwhenUseForwardReverse
Constraintsisunchecked,andareenabledandeditablewhenchecked.
Forwardandreversereadsmustbenamedidenticallyuptothedelimiter(default=
dot),andmustcontainpairedsuffixesthereafter(e.g.,.s&.ror.x&.y).Suffixesmay
beaddedanddeleted.Onlytheletterimmediatelyfollowingthedelimiteris
recognizedasthesuffix.Otherlettersfollowingit,ifany,donotcontributetothe
identityasforwardversusreverseendsequence;however,theydodistinguishone
forward/reversepairfromanother,thusmakingtwosequencepairswiththematching
reverse/forwardendsequence.Minimumandmaximumdistancesmayalsobeedited.
Note: Takecarewhendefiningfilenamesatthesequencer,especiallywhenaprojectis
loadedinbatchesoverseveraldays.Entrieswithmisplacedsuffixes(e.g.
<filename>F.<projectName>,insteadof<filename>.F<projectName>)willbe
overlookedbytheconstraintsfeature,althoughtheymaybeincludedinanassembly.
Min.Dist.MinimumDistancebetweentheforwardandreversereads.Default:
0.
Max.Dist.MaximumDistancebetweentheforwardandreversereads.Default:
6000.
Delimitertheletterseparatingmainpartandsuffixpartinreadnames.Incases
thattherearemultipleoccurrencesofthedesignateddelimiter,therightmost
delimiterisselected.
Thevaluesofminimumandmaximumdistanceareuniformlyappliedtoallsequence
pairs.
Clipping Tab
Sequenceclippingbasedonqualityandsimilarityisanessentialstepintheoverlap
computationandassemblyprocessandensuresvalidityandcorrectness.Youcan
controlandfinetunetheclippingprocessthroughadjustingtheparametersonthe
Clippingtab.

16 Assembly settings
Note: Theclippingisirreversibleanditerativewithineachproject.Whenmultiple
assembliesaremadewithinaproject,eachroundofclippingappliestothesequences
thathadbeenclippedinthelastroundofassembly.Thisimpliesthatalaterattemptto
assemblemayaffecttheexistingassemblies.Insuchcases,ContigExpressgeneratesa
warningmessage.Ifyouchoosetoproceed,clickontheYesbutton.Theaffected
contigswillbedissolved.Ifyouchoosenottoproceed,clickontheNobuttonandthe
currentassemblywillbestopped.
UseAutomaticClippingEndsofsequencingreadsareusuallyunreliableand
lowinQVvalues.CAP3comeswithamechanismtodetectandclipthesepoor
endregionsbasedonsequencesimilaritieswithorwithoutQVs.Clippingisdone
beforethecomputationofoverlaps.Note:trimmingisavailableinContigExpress,
inadditiontoCAP3clipping,andcanbecarriedoutbeforeloadinginput
sequencesintoassemblyprojects.DefaultON.
MatchScoreFactorAlsousedincalculatingSimilarityScoreforcomputing
overlaps,butonlyeditableintheClippingTab.MatchScoreFactorisapositive
integertoawardeachmatchbetweentwobasesfromthepairofsequencesbeing
comparedduringthebandedSmithWatermanalignment.Default:2.
MismatchScoreFactorAlsousedincalculatingSimilarityScoreforcomputing
overlaps,butonlyeditableintheClippingTab.MismatchScoreFactorisa
negativeintegertopenalizeeachmismatchbetweentwobasesfromthepairof
sequencesbeingcomparedduringthebandedSmithWatermanalignment.
Default:5.
GapPenaltyFactorAlsousedinthecomputationofoverlaps,butonlyeditable
intheClippingTab.GapPenaltyFactorisapositiveintegertopenalizeeachgap
extendedduringthebandedSmithWatermanalignment.Default:6.
QualityCutoffforClippingQualityCutoffforClippingappliestotheclipping
ofapoorendregionforeachreadwhenQVsareprovided.Itisnotusedwhen
QVsarenotavailable.Thespecifiedvalueisusedtofindthelowqualityendsof
reads,wherethequalityvalueofabaseisconsideredlowifitislessthanthis
value.Default:12.
ClippingRangeClippingRangeappliestotheclippingofapoorendregionfor
eachreadregardlessofwhetherQVsareavailable.Thevalueisusedtoextendthe
rangesforclippingfurtherawayfromtheendsbasedonthelowqualitypositions
ateachendasdeterminedwiththeQualityCutoffforClippingvalue.Thelarger
thevalueofClippingRange,themoreextensivetheclippingforpoorendregions.
Default:250.
MinNumGoodReadsatClipPositionThisisthedepthofgoodcoverageat
theclipposition.Itappliestotheclippingofapoorendregionforeachread
regardlessofwhetherQVsareavailable.Dependingontheactualdepthof
coverage,theMinimumNumberofGoodReadsatClipPositionparameter
determinestheexactclippingpositionwithintheclippingregion.Thelargerthis
valueis,themoreextensivetheclippingbecomes.Default:3.
Overlap Tab
Overlapsbetweenreadsarecomputedimmediatelyfollowingsequenceclipping.This
stepservesasthebasisforcontigconstruction.Inordertoensurethequality,each
overlapisevaluatedwithafewmeasures.Youcanchangetherigorofoverlap
computation.Theparametersareexplainedbelow:
OverlapLengthCutoffMinimumlengthrequiredforthelengthinbasepairof
alltheoverlaps.Default:40.

Assembly settings 16
OverlapPercentIdentityCutoffMinimumpercentidentityoftheoverlap.
Default:0.80.
OverlapSimilarityScoreCutoffMinimumsimilarityscoreforanoverlap.The
scoreiscomputedasthesumofmatch,mismatchorgapscoresforeachpairof
basesweightedbyQVs.Default:900.
BaseQualityCutoffforDifferenceDeterminestheminimumoverlapquality
byexaminingthedifferencesoftheoverlapatbasesofhighqualityvalues.Thisis
usefulonlywhenQVsareavailable.TheBaseQualityCutoffforDifferencevalue
defineshighqualitybases.Default:20.
MaxQVSumatDifferenceAppliesonlywhenQVsareavailable.Eachoverlap
isgivenadifferencescorebasedontheQVsandtheBaseQualityCutofffor
Difference.AnoverlapwithadifferencescoreovertheMaxQVSumatDifference
valueisexcludedfromcontigconstruction.Default:200.
MaxClearanceforErrorRateAppliesevenandespeciallywhenQVsarenot
available.Iftheerrorrateoftheoverlapisgreaterthanthesumofthoseofthe
overlappingfragmentsplusthisvalue,theoverlapisnotusedforassembly.The
smallerthevalue,thebetterthequalitycontroloftheoverlaps.Default:30.
BandExpansionSizeThisparameterspecifiesbandexpansionsize.The
programautomaticallydeterminesaminimumbandofdiagonalsforan
overlappingalignmentbetweentwosequencereads.Thebandisthenexpanded,
ineachdirection,byanumberofbasesspecifiedhere.Thisaffectsthe
computationofbothpotentialoverlapsandtrueoverlaps.Default:20.
MaxGapLengthThisisthemaximumgaplengthallowedinanoverlap.
Default:20.
MaxNumWordMatchesThisparametercontrolsthefastmethodforfinding
potentialoverlapsbetweenapairofsequences.Foreachwordinoneread,at
mostMaxNumWordMatchesoccurrencesofthewordintheotherreadare
consideredfornongappedextension.Alargervalueforcestheprogramto
considermorewordmatchesattheexpenseoftimeandcomputermemory.
Default:300.
MaxOverhangPercentLengthThisparametercontrolsthedifferentoverhang
regionsbeforeorafterthealignedregion.Itisdefinedas100timesthetotallength
ofthedifferentoverhangregionsdividedbythelengthoftheoverlap.Overlaps
withavaluegreaterthanthemaximumcutoffarenotusedforassembly.Default:
20.
Contig Tab
ContigTabincludesparametersaffectingconstructionofcontigandmultiplesequence
alignments,thustheconsensussequences.QVsareusedextensivelyduringthis
process,iftheyareavailable.Ifunavailable,theprogramassignsaQVof10foreach
base.Threeoftheparameters,MatchScoreFactor,MismatchScoreFactorandGap
PenaltyFactor,areeditableonlyonContigTab.
MatchScoreFactorMatchScoreFactorisapositiveintegertoawardeachmatch
betweentheexistingalignmentandthesequencebeingaddedwhencalculating
thescoreofglobalalignments.Default:2.
MismatchScoreFactorMismatchScoreFactorisanegativeintegertopenalize
eachmismatchbetweentheexistingalignmentandthesequencebeingadded
whencalculatingthescoreofglobalalignments.Default:5.

16 Perform the assembly
GapPenaltyFactorAlsoGapPenaltyFactorisapositiveintegertopenalizeeach
gapextendedwhencalculatingthescoreofglobalalignmentsbetweenthe
existingalignmentandthesequencebeingadded.Default:6.
MinNumConstraintsCorrectionThisistheminimumdifferencebetweenthe
numbersofconstraintssatisfiedinthecurrentassemblyandinthealternative
assembly.Adifferencegreaterthanthisvalue,ifthecontigisalsosupportedbyan
alternativesetofoverlaps,resultsinthealternativejoin.
Min.NumConstraintsLinkingThisistheminimumnumberofconstraintsfor
reportingalinkbetweentwocontigs.
Lite Settings Tab

InContigExpress,youcancreatetwodifferenttypesofcontigs,FullContigsorLite
contigs.OntheLiteSettingstab,youcanspecifythetypeofcontigsyouwantto
assembleintheproject.
InFullcontigsmode,chromatogramdataareretrievableandsequenceediting
performedintheContigViewerarereflectedintheindividualfragmentfiles.
Litecontigsdisregardfragmentchromatogramdataandthereisnodynamic
associationbetweenaLiteContigsanditscomponentfragments.Editingdoneon
LiteContigsisNOTreflectedintheoriginalfragmentsequences(original
sequencesremainunedited).AssemblyinLiteContigmodereducesmemory
consumptionandis,therefore,thepreferredcontigtypeforassemblinglarge
projects.

Lite Settings Parameters
Discard chromatogram Check this box to discard sequence file chromatogram data
data on import when the sequences are imported. Check this box before you
import your sequence reads. Note: you can also discard
chromatograms for all fragments after import using Project >
Discard All Chromatograms command.
Full Contigs only Check this box to perform assembly in Full Contig mode. You
can retrieve chromatograms and maintain dynamic links
between the contigs and their component reads.
Lite Contigs only Check this box to perform assembly in Lite Contig mode. All
linkage between the contigs and sequence reads will be lost
and no chromatogram is retrievable in the Contig Viewer.
Lite contigs if selected Creates Lite Contigs during the assembly process only if the
more than <#> fragments selected number of fragments is greater than the number
specified; otherwise creates full/ regular contigs.
Perform the assembly

Afteryouhaveselectedthetrimmingandassemblysettings,selectthefragmentsyou
wanttoassembleintheContig/FragmentTreeandclickonAssemble.
Assemblymaytakesometime,dependingonhowmanyfragmentsyouselected.

Save, delete, and rename contigs 16
Whenassemblyiscomplete,theassembledcontig(s)andanyunassembledfragments
willbelistedintheContig/FragmentTree(shownbelow)anddisplayedintheContig
Viewer(seenextpage).
Note: Ifacontigcannotbecreatedfromthefragments,analertboxwillbedisplayed.
Note: AlthoughVectorNTIExpressdoesnothaveanexactuppersizelimitfora
ContigExpressproject,yourprojectsizemaybelimitedbyavailablecomputer
resources.Ifyoudoencounteranoutofmemorysituation,youshouldconsider
assemblyinLiteContigMode(seeLiteSettingsTabonpage164).Manyatimesthe
outofmemoryproblemoccursduetothepresenceoftoomanyassembliesinthe
ContigExpressProject.Inthiscase,youareadvisedtodeletesomeoftheseassemblies
asdescribedbelow,savetheproject,andrestartContigExpress.Limitingthenumber
ofassembliesinaprojectisalwaysagoodideawithlargeprojects.Ifyoudecidetouse
LiteContigModebutdontwanttolosethelinkagebetweenyourcontigsand
fragments,youcandiscardchromatogramdata(seeLiteSettingsTabonpage164)
andtradetheabilitytoinvokechromatogramsforalargercapacity.
Save, delete, and rename contigs

MostofthesecommandsarelocatedbelowtheContig/FragmentTree.
Save contig TosaveallthecontigassembliesasseparateDNA/RNAmoleculesinthe

assemblies database,clickonSaveContigbelowtheContig/FragmentsList
Tosavethecontigassembliesaspartoftheproject,clickonSaveProjectatthetop
oftheContigProjectpane.
Delete a contig Todeleteaparticularcontigassembly,selectthecheckboxforthecontigintheContig/

assembly FragmentListandclickonDelete.
Re-assemble Youcanselectfragmentsincontigsandreassemblethem.Selectthecheckboxesnext
contigs tothedesiredfragmentsintheContig/FragmentListandclickonAssemble.
Theexistingassemblieswillbedeletedandreplacedwiththenewones.

16 View the assembly
Rename a contig Torenameacontig,rightclickonthecontignameinthelistandselectEdit.Entera

newnameinthedialogbox.
View the assembly

ClickoncontignameintheContig/FragmentListtodisplaytheassemblyinthe
ContigViewer.
Contig Viewer Graph Pane
Click on contig name in tree
Contig Viewer Alignment pane
TheContigViewercontainstwopanes:
TheGraphPanedisplaysagraphicalrepresentationofthecontigassembly
TheAlignmentPaneshowstheassembledfragmentsequences,chromatogram
data,andtranslatedsequence.
Contig Viewer TheContigViewerGraphPanecontainshorizontalarrowsrepresentingtherelative

Graph Pane positionsofthefragmentsformingthecontig.Thearrowheadsindicatewhetherthe
respectivefragmentisinthedirectorcomplementarystrand,withthenamesofthe
fragmentsdisplayedabovethefragmentlines.

View the assembly 16
Contig coverage bar

TheContigCoveragebarspansthelengthofthecontigandcontainssegmentsof
varyingpatterns/colorsthatrepresenttheamountandtypeoffragmentcoveragein
thatsegment.
Contig Coverage Bar
Thepatternsandcolorsare:
Singlefragmentgraybarwithslants
Twofragmentsinthesamedirectionredcrosshatching
Twofragmentsindifferentdirectionsbluecheckerboard
Multiplefragmentsinbothdirectionssolidbluebar
Contig Viewer TheContigAlignmentPanedisplaysthenucleotidesequencesofthefragmentsthat

Alignment pane formthecontig,withoverlappingregionsalignedappropriatelyanddisplayed
relativetotheirpositionsinthecontig.
Theconsensussequenceisdisplayedbelowthefragments,andchromatogramsforthe
fragmentsaredisplayed.Translationscanbeidentifiedanddisplayed.Youcaneditthe
sequenceshereandseehowyouractionsarereflectedinthecontigalignmentand
consensus.
UsethehorizontalandverticalscrollbarsintheAlignmentPanetoviewthefragment
sequences,overlappingregions,andconsensussequence.
Scale
Fragment
sequence
Overlapping region
Chromatogram
data
Consensus
sequence
Positions with Ns Ambiguities or gaps

are flagged with * are flagged with +

16 Translate the consensus sequence
Symbols in the sequence

Intheconsensussequence,asterisks(*)indicatepositionscontainingNsandplus
symbols(+)indicateambiguityand/orgaps.
Inthefragmentsequence,gapsareindicatedbyahyphen()symbol.
Highlight a region
DragyourcursoroveraregionintheGraphPanetohighlightthatregioninthe
AlignmentPane,andviceversa.
Find Next/Previous Ambiguous Symbol

Tosearchtheconsensussequenceforambiguouspositionsorsymbols(NorR),
clickontheFindNextAmbiguousorFindPreviousAmbiguousbuttonstotheright
oftheGraphPane,orrightclickontheconsensussequenceandselectthecommands
fromtherightclickmenu.
Ifanambiguouspositionorsymbolisfound,itspositionisselectedanddisplayed.If
therearenomoreambiguoussymbolsinthespecifieddirection,analertpopsup.
Hide or show chromatograms

Chromatogramsaredisplayedforeachfragmentinthecontig.Thecoloredpeaksin
thechromatogramcorrespondthecoloredbasesbelowtheeditablesequenceinthe
AlignmentPane.
TohidethechromatogramforafragmentintheAlignmentPane,rightclickon
thefragmentsequenceandselectHide<fragmentname>.
Toredisplaythechromatogram,rightclickandselectShow<fragmentname>.
Tohideorshowallchromatograms,rightclickintheAlignmentPaneandselect
HideallchromatogramsorShowallchromatograms.
Translate the consensus sequence

Totranslatetheconsensussequence,rightclickonthesequenceandselect
Translation,orclickontheTranslationbuttononthetoolbartotherightoftheGraph
Pane.
ThetranslatedsequenceappearsbelowtheconsensussequenceintheAlignment
Pane.
Note: Ambiguouspositionsintheconsensusaretranslatedasasingle,definedamino
acidifcodondegeneracypermits(i.e.GGN=Gly).Gapsintheconsensusareignored
inthetranslations.

Edit the contig sequence 16
Edit the contig sequence

YoucaneditthecontigsequenceintheAlignmentPanebyclickingwithintheblack
textsequenceofaparticularfragment(abovethecoloredtext)orclickinginthe
consensussequenceandtypingIUBcharactersorpressingtheDeleteorBackspace
keys.
Alleditingchangesinthefragmentscauseanimmediaterecalculationandredisplay
oftheconsensussequence.Ifnecessary,editingchangesinthecontigconsensusare
reflectedimmediatelyinthefragmentsequencesthatcomprisethecontig.
Note: Sincefragmentsinacontigcanalsobepresentincontigsfromotherassemblies
intheproject,sucheditsareallowedonlyafteryouconfirmthatyouareawarethat
anyotherassembliescontainingthefragmentmaybedismissed.
Insertions YoucanenterA,T,G,C,Nandotheracceptableambiguousnucleotidedesignations
specifiedbyIUBcodes(refertoAppendixA,SymbolsandFormats:IUB(IUPAC)
AmbiguityCodesandASCIIFormat).
Insertionsmadeintheconsensuswillbeupdatedtoallfragmentsthatmaptothat
positionintheconsensus.
Note: Gapswillappearinthefragmentchromatogramsasyouaddsymbols.
Deletions IntheAlignmentPane,positionthecursorimmediatelyaheadofthedesiredposition
inafragmentortheconsensusandpresstheDeletebuttononthekeyboardoneor
moretimes.Youcanalsoselectacontinuousstretchofbasesandusethekeyboard
Deletebuttontoremovethem.Deletionsinafragmentwillupdatetheconsensus.
Deletionsmadeintheconsensuswillbeupdatedtoallfragmentsthatmaptothat
positionintheconsensus.
Sincefragmentsinacontigcanalsobepresentincontigsfromotherassembliesinthe
project,sucheditsareallowedonlyafteryouconfirmthatyouareawarethatanyother
assembliescontainingthefragmentmaybedismissed.
Saving sequence Sequencechangesaresavedwiththeproject,orifyousavethecontigasaseparate

changes molecule.Theydonotchangethefragmentmoleculeitselfinthedatabase.

16 Edit the contig sequence

A Symbols and Formats: IUB (IUPAC)
Ambiguity Codes and ASCII Format
Format for ASCII Sequence Files

AnASCIIsequencefilemustobeythefollowingrules:
Itmustbeaplain(ASCII)textfile.
Thefilemustcontainthenucleotide(aminoacid)sequencearrangedinlines.
Eachlinemaycontainthefollowing:
Nucleotide(aminoacid)symbolsandwhitespace,or
Anumberfollowedbywhitespaceandnucleotide(aminoacid)symbols
(therefore,similartoGenBankformat),inwhichcasethenumberwillbeignored,
or
Anumberonly,inwhichcasethenumberwillbeinterpretedasablockof
unknownnucleotides(aminoacids)ofthecorrespondinglength.
IUB Formats recognized by Vector NTI Express

Thefollowingcharacters,definedbytheInternationalUnionofBiochemistry(IUB),
areusedtorepresentnucleotidesthroughoutVectorNTIExpress:
Symbol Meaning
A adenine
T thymine
C cytosine
G guanine
R purine(A or G)
Y pyrimidine(C or T)
W A or T
S C or G
M A or C
K T or G
B C, G, or T
D T, G, or A
H C, A, or T
V C, G, or A
N unknown nucleotide

Symbols and Formats: IUB (IUPAC) Ambiguity Codes and ASCII Format
A IUB Formats recognized by Vector NTI Express
Aminoacidsarerepresentedbystandard1(or3)lettercodes:
1-Letter Symbol 3-Letter Symbol Meaning

E Glu Glutamic acid
G Gly Glycine
H His Histidine
I Ile Isoleucine
L Leu Leucine
K Lys Lysine
M Met Methionine
F Phe Phenylalanine
P Pro Proline
S Ser Serine
T Thr Threonine
W Trp Tryptophan
Y Tyr Tyrosine
V Val Valine
B Asx Asparagine or Aspartic acid

B Primer Tm Calculations
General Information
VectorNTIExpresscalculatesandreportstwodifferentmeltingtemperaturesfor
DNA/RNAoligonucleotides,ThermodynamicTm(Therm.Tm)and%GCTm.
Usefulness of Thermodynamic Tm Versus %GC Tm

VectorNTIExpressreportsboththeThermodynamicand%GCTmvalues,regardless
ofthelengthoftheoligo.However,generallyonlyoneofthereportedvalues
shouldbeconsidereduseful,dependingonthelengthoftheoligoasfollows:
Therm.Tmusefulforoligosthataregreaterthanabout710residuesandless
thanabout35residueslong
%GCTmusefulforoligosgreaterthanabout35residueslong
Note: Foroligosthatare710residuesorshorter(cutofflengthdependsonthebase
contentoftheparticularoligobeinganalyzed),VectorNTIExpressreportsaTherm.
Tmvalueofzero.
Effects of Primer (Probe) and Salt Concentration on Tm

Calculations
Tmcalculationsarehighlydependentonprimerandsaltconcentrations;varyingthese
concentrationscangreatlyaffecttheTmforanygivenprimer.Therefore,itis
importantthatyouadjusttheprimerandsaltconcentrationsappropriatelysothat
accurateTmvaluesaregenerated.
Note: InVectorNTIExpress,thedefaultparametersforprimerandsaltconcentration
are250pMand50mM,respectively,forcalculatingTmvalues.OtherTmcalculators
commonlyuseadefaultprobeconcentrationof50nM.Becauseofthis,VectorNTI
ExpressdefaultparameterTmvaluesmaynotcorrespondtothedefaultTmvalues
calculatedusingotherprograms.BeforecomparingVectorNTIExpressTmvalues
withthosegeneratedbyotherTmcalculators,makesurethattheparametersare
adjustedappropriately.
%GC Tm Calculation
The%GCTmcalculation1doesnotrelyonthethermodynamicpropertiesoftheoligo
(i.e.dHo,dSoanddGvalues).Theformulafor%GCTmisasfollows:
Tm=81.5+16.6(log[Na+])+0.41(%GC)675/probelength

Primer Tm Calculations
B Thermodynamic Tm Calculation
Note: [Na+]isinmolarunits.
Example: ForoligoGTGCGAGGCAGCTGCGGTAAat50mMsalt:
Tm=81.5+16.6(log(0.05))+0.41(65)675/20
=81.5+16.6(1.30)+26.6533.75
=81.521.58+26.6533.75
=52.82C
Thermodynamic Tm Calculation
TheThermodynamicTmcalculationisbasedontheNearestNeighbortheoryofDNA/
RNAduplexstability.Briefly,thistheorystatesthattheoverallduplexstability(and,
hence,themeltingtemperature)ofanoligonucleotidecanbepredictedfromthe
primarysequencebasedontherelativestabilityandtemperaturedependentbehavior
ofeverydinucleotidepairintheoligo2.Inpractice,enthalpy(dH)andfreeenergy
(dG)valuesforeachofthe10possibleWatsonCrickDNApairwiseinteractionsare
usedtocalculatepairwiseentropy(dS)valuesviathefollowingstandardequation:
dG=dHTdS
Note: TistemperatureinK.
ThepairwisedHanddSvaluesarethensummedtocalculateoverallvaluesforthe
oligounderconsideration.Theoverallvaluesareusedinthefollowingformula3to
calculatetheThermodynamicTm:
Therm.Tm=dH273.15+16.6(log[Na+])dS+dSo+R(ln(c/4))
Notes: dSoistheentropyassociatedwithhelixinitiation(10.8cal/molperK).
RistheUniversalGasConstant(1.987cal/molperK).
cistheconcentrationoftheprobe,inmolarunits.
Thefactor273.15correctsforabsolutetemperaturesothatthefinalTmisinC.
ThepairwisedHanddSvaluesforDNAusedinVectorNTIExpressaretakenfrom
reference2.Thosevalues,alongwiththecorrespondingdGvaluesat25C,appearin
thefollowingtable:
Interaction dH kcal/mol dS cal/mol per K dG kcal/mol
AA/TT -9.1 -24.0 -1.9
AT/TA -8.6 -23.9 -1.5
TA/AT -6.0 -16.9 -1.0
CA/GT -5.8 -12.9 -2.0
GT/CA -6.5 -17.3 -1.3
CT/GA -7.8 -20.8 -1.6
GA/CT -5.6 -13.5 -1.6
Table1 DNA Nearest Neighbor thermodynamics

Thermodynamic Tm Calculation B
CG/GC -11.9 -27.8 -3.6
GC/CG -11.1 -26.7 -3.1
GG/CC -11.0 -26.6 -3.1
XX/XX -6.0 -16.9 -1.0
Table1 DNA Nearest Neighbor thermodynamics (continued)
Notes: Allvaluesrefertothedisruptionofaduplexat1MNaCl,25CandpH7.
TheunitsfordHanddGarekcal/molofinteraction,whereasthosefordSare
cal/Kpermolofinteraction.
Example Theoligo5GTGCGAGGCAGCTGCGGTAA3isparsedasfollows:
TotaldH=164.7kcal/mol
ThetotaldSreportedbyVectorNTIExpressisthesumofthepairwisevalues
aboveandtheentropyassociatedwithhelixinitiation(dSo).Thus,forthe
exampleoligoabove:
TotaldS=405.7+(10.8)=416.5cal/molperK
The total dG is the sum of the pairwise dG values for the oligo plus a helix initiation
free energy term (dGo) that is added to better reflect experimentally determined free
energy values for tested oligos. The value of the helix initiation free energy term (dGo)
depends on the base composition of the oligo2 as follows:
+5.0kcal/molforoligoscontaininganyGCbasepairs
+6.0kcal/molforoligoscomposedexclusivelyofATbasepairs
Therefore,fortheexampleoligo:
TotaldGo=43.7kcal/mol(sumofthepairwisedGovalues)+5kcal/mol(freeenergy
term)=38.7kcal/mol
The 3 End dG is calculated using the number of 3 pairwise dG values specified in
the 3 End Length (bp) box, and is not further adjusted.
Using Vector NTI Expresss default probe and salt concentrations (250 pM and 50 mM,
respectively) and the values for dH and dS calculated above, Therm. Tm can be
calculated as follows:
dH - 273.15 + 16.6 Log Na
ThermTm = ---------------------------------------------------------
dS + dS o + R ln c---
4
164.7
= --------------------------------------------------------------------------------------------------------- 273.15 + 16.6 Log 0.05
12
250 10
0.4165 + 0.001987 ln ----------------------------
4

B Oligos Containing IUB Ambiguity Characters
164.7
= ---------------------------------------------------------------------------
- 273.15 + 16.6 1.301
0.4165 + 0.001987 23.49
164.7
= -------------------------------------------------
- + 273.15 21.60
0.4165 0.04667
= ---------------
164.7- 294.75
4632
= 355.57 294.75
= 60.82C
VectorNTI Express adjusts the %GC and Therm. Tm values accordingly, based on the
input formamide concentration.
Oligos Containing IUB Ambiguity Characters

VectorNTIExpresscananalyzeoligosthatcontainIUBnucleotideambiguity
characters(i.e.R,Y,W,S,M,K,B,D,H,VandN).Inthecaseofambiguitycharacters,
VectorNTIExpressusesaveragepairwisedHanddSvaluesforcalculatingtheTm.
Forexample,forthedinucleotidepairCB,VectorNTIExpressaveragestheCC,CG
andCTthermodynamicparameters(Table<Blue><Emphasis> 1)toobtainaverage
pairwisedHanddSvaluesforCB.Itthensumstheaveragepairwise
thermodynamicparametersandcalculatestheTherm.Tmvaluesaccordingtothe
equationdescribedabove(referThermodynamicTmCalculationonpage174).
Inthecaseof%GCTm,VectorNTIExpressappliestheappropriate%GCcontribution
representedbyeachambiguitysymboltothestandard%GCTmformula(see%GC
TmCalculationonpage173).Forexample,aBambiguitysymbolcontributesonly
twothirdstheamountofaGorCresiduetooverallGCcontent.
RNA Oligos
RNAoligosuseadifferentsetofpairwisethermodynamicvaluesthanDNAoligos.
PairwisethermodynamicvaluesforRNAaresummarizedinthefollowingtable:
Interaction dH kcal/mol dS cal/mol/K dG kcal/mol
AA/UU -6.6 -18.4 -1.1
Table2 RNA Nearest Neighbor thermodynamics

Primer/Probe Tm Values B
AU/UA -5.7 -15.5 -1.1
UA/AU -8.1 -22.6 -1.4
CA/GU -10.5 -27.8 -2.2
GU/CA -10.2 -26.2 -2.4
CU/GA -7.6 -19.2 -1.9
GA/CU -13.3 -35.5 -2.7
CG/GC -8.0 -19.4 -2.2
GC/CG -14.2 -34.9 -3.8
GG/CC -12.2 -29.7 -3.3
XX/XX -6.0 -16.9 -1.0
Table2 RNA Nearest Neighbor thermodynamics (continued)
Notes: Allvaluesrefertothedisruptionofaduplexat1MNaCl,25C,andpH7.
TheunitsfordHanddGarekcal/molofinteraction,whereasthosefordSare
cal/Kpermolofinteraction.
ThedSvalueforRNAoligosisadjustedby10.8cal/Kpermoltoreflectthe
entropyassociatedwithhelixinitiation,asitisforDNAoligos.
ThedGvalueisadjustedby+3.4kcal/moltoaccountforhelixinitiation.Note
thatthisadjustmentisNOTdependentonthebasecompositionoftheRNAoligo
asitisforDNAoligos(referThermodynamicTmCalculationonpage174).
Primer/Probe Tm, ForoligosdesignedusingVectorNTIExpresssPCRPrimers,SequencingPrimersand

TaOpt and Similarity HybridizationProbesfeatures,theoligoTm,productTaOptandoligopercentbinding
similarityarereportedintheTextPaneoftheMoleculeViewingwindow.
Calculations
Primer/Probe Tm Values
Tmsfordesignedprimers/probesarereportedisasfollowsinVectorNTIExpress:
Therm.Tmisreportediftheoligoislessthanorequalto35residues
%GCTmisreportediftheoligois36residuesorgreater
ForPCRproducts,VectorNTIExpressreportsthe%GC;theassumptionbeingthat
themajorityofPCRproductsarelargerthan35residues.
TaOpt Values
ThePCRproductTaOpt(optimalannealingtemperatureforamplificationofthe
fragment)inCiscalculatedusingthefollowingformula3:
TaOpt=((0.3)Tmprimer+(0.7)Tmproduct14.9)C
Notes: TmprimeristheTmofthelessstableprimerofthepair

B Primer/Probe Similarity Values
TmproductistheTmofthePCRproduct
Primer/Probe Similarity Values

WhendesigningPCR,sequencingorhybridizationprimers,VectorNTIExpress
reportstheoverallsimilarityinpercentofanoligotoitsbindingsitebasedonthe
oligosnucleotidecomposition.ForoligoscontainingIUBambiguitysymbols,three
similarityvaluesarereported:
MinimumSimilarity
MaximumSimilarity
AverageSimilarity
ForMinimumSimilarity,allambiguitiesareclassedascompletemismatches(i.e.,they
areassignedvaluesof0ateachposition).Forexample,a20mercontaining2Rsand2
NshasaMinimumSimilarityof80%.
ForMaximumsimilarity,allambiguitiesareconsideredidenticaltotheircognate
nucleotides(i.e.theyareassignedvaluesof1ateachposition),sotheMaximum
Similarityisalways100%.
ForAveragesimilarity,VectorNTIExpressweightseachambiguousnucleotide
dependingonwhetheritrepresents2,3,or4possiblenucleotides.Forexample,Ns
haveascoreof0.25,Rsof0.5,Bsof0.33,etc.Therefore,forthe20merdescribedabove,
theAverageSimilarityis85%.
References
BaldinoJr.,F.,Chesselet,M.F.,andLewisM.E.(1989)Highresolutioninsitu
hybridizationhistochemistry,MethodsEnzymol.168:761777.
Breslauer,K.,J.,Frank,R.,Blocker,H.,andMarky,L.A.(1986)PredictingDNAduplex
stabilityfromthebasesequence,Proc.Natl.Acad.Sci.USA83:37463750.
Rychlik,W.,Spencer,W.J.,andRhoads,R.E.(1990)Optimizationoftheannealing
temperatureforDNAamplificationinvitro,NucleicAcidsRes.18:64096412.
Sugimoto,N.,Nakano,S.,Yoneyama,M.,andHonda,K.(1996)Improved
thermodynamicparametersandhelixinitiationfactortopredictstabilityofDNA
duplexes,NucleicAcidsRes.24:45014505.
Freier,S.M.,Kierzek,R.,Jaeger,J.A.,Sugimoto,N.,Caruthers,M.H.,Nielson,T.,and
Turner,D.H.(1986)ImprovedfreeenergyparametersforpredictionsofRNAduplex
stability,Proc.Natl.Acad.Sci.USA83:93739377.

Index
Numerics expectationvalue 83
filtermasks 84
3DMoleculeViewer 105
programdescriptions 81
websitereference 81
A wordsize 84
Alignmultiplesequences.SeeAlignX BLASTsearch 81
AlignX Algorithms 81
Algorithms 99 Databases 82
Alignmentpane 102 Parametersforscoringandfiltering 83
Alignmentsettings 99 Results 85
ClustalWoptions 99
Complexitygraph 101 C
DotPlot 102
Cameratool 31
Graphspane 101
Managingprojects 96 Cloning
Multiplesequencealignmentoptions 100 GatewayCloning 121
GENEART 141
Opening 95
Pairwisealignmentoptions 99 TOPOCloning 133
Selectingfragmentstoalign 97 collaborativeresearch 35
Similaritygraph 101 connecttoaworkgroupshareddatabase 35
Analyses Contigassembly
DNA/RNA,BioAnnotator 73 SeealsoContigExpress 153
AnalysisMonitor ContigAssemblyprojects
Sim4analysis 109 export 27
Spideyanalysis 111 ContigExpress 153
analysisresults Ambiguousbases,finding 168
manage 34 Assemblysettings 160
ASCIIFormat 171 Assembly,viewing 166
Chromatograms,hiding/showing 168
Clippingsettings 161
B Contigconsensussequence 167
BackTranslation 51 ContigViewerAlignmentPane 167
Backtranslationofproteins 75 ContigViewerGraphPane 166
BioAnnotator Coveragebar 167
Analysistypes 73 Editingasequence 169
Bioannotator 71 Fragments,adding 156
BioniformaticWebresources 30 Litesettings 164
Opening 153
BLASTresults
Overlapsettings 162
export 27
Projects,exporting 155
BLASTSearch
Projects,managing 155
description 81
Savingcontigsasmolecules 165
<Title of Publication in footer> 179

Index
TaskList 155 CharactersUsed 171

Translatetheconsensussequence 168 Fileformatforimporting 171
Trimmingends 157 DNA/RNAandproteinmolecules
Trimmingvectorsequences 158 export 27
Windowfeatures 154 DNA/RNAmoleculedata 13
Workflow 155 DNA/RNAphysiochemicalproperties 71
copymolecules 30 duplicatedatabaseobjects
create databaseobjects
projects 33 duplicate 27
Createfeaturefromselection 42
Creating
features,GenomBench 93
E
editenzymes,oligos,gelmarkers 28
Entrezsearch 86
D Databases 87
data Results 87
group 26 enzymes
database 82 export 27
Databasearchives 14 properties,edit 28
DatabaseExplorer 13 Expectationvalue 83
Datatypes 13 export
Enzymes 19 data 27
Fileformats 21
Gelmarkers 19
Importingdata 21
F
Molecules,creating 17 FeatureMap 42
Oligos 19 Fileformats 21
Opening 14 fileformats 28
Openingfiles 16 Filetypesandextensions 16
Operations 17 Formats
Searchdatabase 23 ASCII 171
Subsets 16 IUBcodes 171
Subsets,creating 23
databaseobjectproperties 27
G
databaseobjects
addtosubsets 26 GatewayCloning 121
delete 27 gelmarkers
rename 26 properties,edit 29
sort 26 Gelmarkers,creating 19
Databasesearch 23 Genesynthesis 75
Database,local 13 GENEART 141
delete Assemblytool 143
projectsubsets 34 Fragments,addingandediting 144
deletedatabaseobjects 27 Highorderassembly 141
Deleting PCRprimers 145
features,GenomBench 94 Seamlesscloning 141
Stitchingoligos 146
disconnectfromashareddatabase 37
GeneArtsynthesis 79
DNAmolecules,partsassembly 149
GenomBenchFeatureMapPane
DNA/RNA
180 <Title of Publication in footer>

Index
creatingfeatures 93 Molecules,importing 21
deletingfeatures 94 MotifFinder 54
GenomBenchOverviewPane Mutations.SeeRegenerator
description 92
groupdata 26
N
Groupdatainsubsets 25
NationalCenterforBiotechnologyInformation(NC
BI),registerwith 38
H
H3Heading3 O
ClearaLocalDatabasesubset 24
OligoDuplexAnalysis 48
DeletethecontentsofaLocalDatabasesubset 25
DismissLocalDatabasesubsets 25 oligos
ViewasummaryofaLocalDatabasesubset 25 export 27
properties,edit 29
Oligos,creating 19
I Open
imagecapture 30, 31 applicationsinDatabaseExplorer 30
import open
fileformats 28 projects 33
Importing ORFFinder 45
ASCII 171
Importingdata 21
P
InterProdatabaseanalysis 54
PartsAssembler 149
IUBCodes 171
Parts,standardbiological 149
PCRprimers
J userdefined 61
JMol.See3DMoleculeViewer preferences,display 37
Primerdesign 57
L Analysisconditions 60
PCRprimers 60
Launching
ResultsinMoleculeEditor 58
BLASTSearch 81
Results,saving 59
Lowcomplexitysegments 84
Settings,savingandloading 58
Primers
M similarityvalues 178
manage TaOpt 177
BLASTresultsandanalysisresults Tmvalues 177
BLAST results printmoleculedata 33
manage 34 PRINTSdatabaseanalysis 52
Projectsdatabase 33 projects
Moleculefeature,creating 42 create,open,edit 33
molecules Projectsdatabase 33
copy 30 properties,databaseobjects 27
printdata 33 PROSITEdatabaseanalysis 52
save 32 ProteinDataBank(PDB)files 105
Molecules,creating 17 ProteinDomainAnalysis 52
Molecules,creatingoropening 39
<Title of Publication in footer> 181

Index
R TOPOCloning 133
TOPOcloning
Regenerator 75
generatingaclone 133
RemoteDatabase 35
Translationtool,MoleculeEditor 47
RenameaLocalDatabasesubset 24
RestrictionAnalysis 43
Restrictionenzymes,creating 19 U
ReverseSelectiontoComplementary 42 useNCBIWebservices 38
S V
savemolecules 32 viewers,DatabaseExplorer 15
screenshots,take 30, 31
Searches,BLASTandEntrez 81 W
Searchingthedatabase 23
WebanalysesofDNAorproteinsequences 50
Sequencealignment.SeeAlignX
Webresources 38
Sequenceanalysis 71
Sequencemodificationsforgenesynthesis 75
Sequence,enteringorediting 41
sequences,copy 31
setdisplaypreferences 37
shareddatabase 35
SilentMutationAnalysis 49
Sim4analysis 109
Similarity
betweenambiguousnucleotides 178
snapshots,take 30, 31
sortdata
data
sort 26
Spideyanalysis 111
subsets
addobjectsto 26
Subsets,creating 23
T
TaOpt 177
ThreeDimensionalMoleculeViewer.See3DMole
culeViewer
Tm
%GCcalculation 173
ambiguousoligos 176
generalinformation 173
primers/probes 177
references 178
RNAoligos 176
TaOpt 177
Therm.Tmcalculation 174
182 <Title of Publication in footer>

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USER GUIDE

Vector NTI Express Software

For Research Use Only. Not for use in diagnostic procedures.

Limited Use Label License: For Research Use Only

About This Guide . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 11

CHAPTER 1 Database Explorer . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 13

Vector NTI ExpressSoftware User Guide 3

Print molecule data . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 33

CHAPTER 2 Molecule Editor . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 39

4 Vector NTI ExpressSoftware User Guide

Selecting enzymes for analysis . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 44

CHAPTER 3 Primer Design . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 57

Vector NTI ExpressSoftware User Guide 5

Analyses descriptions and parameters . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 73

CHAPTER 6 BLAST and Entrez Searches . . . . . . . . . . . . . . . . . . . . . . . . . 81

CHAPTER 8 Align Multiple Sequences . . . . . . . . . . . . . . . . . . . . . . . . . . . 95

6 Vector NTI ExpressSoftware User Guide

CHAPTER 9 3D Molecule Viewer . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 105

CHAPTER 10 Sim4 and Spidey Analysis . . . . . . . . . . . . . . . . . . . . . . . . . 109

Vector NTI ExpressSoftware User Guide 7

Spidey Parameters . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 112

CHAPTER 11 Clone2Seq . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 115

CHAPTER 12 Gateway Cloning . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 121

CHAPTER 13 TOPO Cloning . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 133

8 Vector NTI ExpressSoftware User Guide

CHAPTER 14 GeneArt Cloning . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 141

CHAPTER 15 Parts Assembler . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 149

CHAPTER 16 Contig Assembly using ContigExpress . . . . . . . . . . . . . . 153

Vector NTI ExpressSoftware User Guide 9

Select fragments to trim . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 157

APPENDIX A Symbols and Formats: IUB (IUPAC) Ambiguity Codes and

APPENDIX B Primer Tm Calculations . . . . . . . . . . . . . . . . . . . . . . . . . . . 173

10 Vector NTI ExpressSoftware User Guide

Note on screen ThisguideincludesscreencapturesfortheMicrosoftWindowsversionofthe

Vector NTI ExpressSoftware User Guide 11

12 Vector NTI ExpressSoftware User Guide

About this chapter

Vector NTI ExpressSoftware User Guide 13

Open the Database Explorer

14 Vector NTI ExpressSoftware User Guide

Components of the TheDatabaseExplorerscreenconsistsofthefollowingcomponents:

Drag frames to resize viewers

Types of databases TherearethreetypesofdatabasesintheDatabaseExplorer:

Vector NTI ExpressSoftware User Guide 15

16 Vector NTI ExpressSoftware User Guide

Database Explorer operations

Create DNA/RNA and protein molecules

Vector NTI ExpressSoftware User Guide 17

Create a DNA/RNA TocreateaDNA/RNAmolecule:

18 Vector NTI ExpressSoftware User Guide

Create a Protein Tocreateaproteinmolecule:

Create gel markers, oligos, and enzymes

Create a gel Tocreateagelmarker:

Create an oligo Tocreateanoligo:

Vector NTI ExpressSoftware User Guide 19

Create an enzyme Tocreateanenzyme:

Cleavage Point on Complementary Strand = 10

20 Vector NTI ExpressSoftware User Guide

Import molecules Youcanimportmolecules(includingtheirfeaturetables)fromDDBJ,EMBL,FASTA,

File formats ThefollowingfileformatscanbeimportedintoVectorNTIExpress:

Vector NTI ExpressSoftware User Guide 21

Import files Therearetwowaystoimportfiles:bydragginganddroppingfilesinWindows

Drag and drop files into a subset

Import files via the main menu