5BBG0206 Molecular Biology Research Skills 2020-2021

Workshop 2.  How to use SnapGene software to design a new plasmid and create plasmid maps

The live on-line workshop 2 session consists of a live demonstration of how to use Snapgene
software to construct the plasmid sequence and map of pASKGH (from workshop 1).

Workshop 2 below is a step by step reproduction of the live demonstration with screen shots.

You have a choice

1. You can attempt to do Workshop 2 doing all the steps yourself in Snapgene following the
guidance and screen shots below before the workshop 2 session and then watch the live
demonstration to check you have understood the workshop content.
2. You can watch the live on-line workshop 2 demonstration and follow the steps using this
document then you can go through workshop 2 after this live session doing all the steps yourself in
Snapgene.

All three live on-line workshop sessions will be recorded and one of the recordings placed on the
5BBG0206 KEATS site.

Learning outcomes

After completing the workshop students should:

Be able to use SnapGene Viewer functions to design a new plasmid.
Be able to annotate key features on the DNA sequence and the plasmid map.

Introduction
There are a number of commercial software packages that allow you to manipulate DNA sequence
to design new DNA constructs e.g. plasmids. There is no clear market leader and different research
groups within the same institution often use different software. We have chosen to use SnapGene
Viewer in workshop 2. The full version of the SnapGene software is expensive ($295 for one
computer per year) but you can do a lot of work just using the free version called SnapGene Viewer.
Obviously, quite a few of the functions in the full package do not work in the free version (and if you
try to use them the free version will offer you a free trail of the full version), but there are still many
useful features you can use to help you design new DNA constructs and make useful plasmid maps.
This workshop takes you through some key SnapGene functions. As with any bioinformatics based
software the steps required to use it effectively takes practice, but eventually will become
instinctive.

Overview of SnapGene Viewer capability

1. create annotated plasmids maps and DNA sequence
2. identify ORFs
3. identify restriction enzyme sites
4. identify key plasmid features (promoters, selectable markers, common tags)
5. design basic primers for PCR and DNA sequencing

A few of disadvantages of using the SnapGene Viewer

1. The key thing you can’t do easily with the free version is join different bits of DNA together
or edit DNA, but this workshop shows you a way around this by editing DNA sequence in a
Word document.

1
 2. You need to save your SnapGene construct as a file on your own computer (otherwise you
will lose your work because you can’t save it on the Snapgene server using the few Sanpgene
Viewer). It might be a good idea to save different stages of your construct design as
differently named files i.e. xxxxx-1, xxxxx-2. Like all files it is essential you have saved copies
of all your work in two locations (own computer, OneDrive (cloud), memory stick, external
hard drive).

There are several ‘user friendly’ videos that help you use SnapGene. This is because most users will
not have been formally taught to use SnapGene in a class or by a person, they have taught
themselves how to use it.

https://www.SnapGene.com/
SnapGene home page (accessed January 2021).
You can download SnapGene Viewer onto your own computer (link top of page).

https://www.snapgene.com/support/tutorial-videos/
SnapGene Tutorial videos (accessed January 2021)

Other useful Bioinformatics programmes

http://nc2.neb.com/NEBcutter2/
NEBcutters is useful tool for finding out DNA fragment sizes if you cut a plasmid DNA sequence with
different combinations of restriction enzymes. You can’t do this as easily with free version of
SnapGene Viewer. Though you can work out DNA fragment sizes from the Snapegene Viewer
plasmid map and a bit of adding/subtracting.

https://blast.ncbi.nlm.nih.gov/Blast.cgi
You can use BLAST to quickly check if the DNA sequence codes for the right gene (blastn) or protein
(blastx).

http://bio.lundberg.gu.se/edu/translat.html
You can use this software to translate a DNA sequence into protein showing the three forward
frames. Ignore the top bit which is only showing you the top reading frame and look at the three
forward reading frames. Useful as a quick check you have picked the DNA sequence of an ORF
correctly.

https://www.bioinformatics.org/sms/rev_comp.html
This piece of software is copied on several different sites. I used it to get reverse primers I have
copied from a double stranded DNA sequence into the 5’ to 3’ direction as a single line of DNA
sequence (pick reverse option). Though you can do this in Snapgene as well.

https://www.ebi.ac.uk/Tools/sfc/emboss_seqret/
You can be used this software to change DNA or protein sequences to different bioinformatics
formats i.e. FASTA
Many ‘cut and paste’ boxes in bioinformatics software programmes will accept many formats, but if
the software is rejecting your input with a format error message you can use this tool to convert
your sequence to a format it recognises.

2
Contents page

Part 1. Accessing SnapGene Viewer................................................................................................4

A. Accessing SnapGene Viewer on a student college computer (for the few going to campus)...4
B. Accessing SnapGene Viewer on own computer........................................................................4
Part 2. Creating file with human growth hormone DNA sequence.................................................5
Part 3. Using human growth hormone DNA sequence file to design the gene specific part of PCR
primers............................................................................................................................................17
Part 4. Adding restriction enzyme sites to gene specific PCR primers to subclone human growth
hormone into of PCR primers pASK-IBA37plus..............................................................................20
Part 5. Creating file with DNA sequence pASK-IBA37plus.............................................................20
Part 6. Adding pASK-IBA37plus DNA sequences to Word document and creating DNA sequence
of pASKGH.......................................................................................................................................21
Part 7. Creating a new DNA file for pASKGH in SnapGene............................................................26
Part 8. Using SnapGene file of pASKGH to extract amino acid sequence of 6xHis tagged human
growth hormone to create a figure................................................................................................35
Part 9. Making sure your protein is in frame with the rest of the desired fusion protein...........36
Appendix.........................................................................................................................................37
Figure 1. DNA sequence of human growth hormone in FASTA format.......................................37
Figure 2. Amino acid sequence of human growth hormone in FASTA format............................37
Figure 3. The complete DNA sequence of pASK-IBA37plus.........................................................38

3
Part 1. Accessing SnapGene Viewer

A. Accessing SnapGene Viewer on a student college computer (for the few going to campus)
Log on to a student college computer.
Click on the Windows symbol in the bottom left hand corner of the screen.
Go to the Software centre
Search for SnapGene, click on SnapGene
Click install
Go to App menu (Window symbol bottom left) and click on SnapGene.

Note. You cannot download external software on a student college computer (which is why
SnapGene is available in the Software Centre).

B. Accessing SnapGene Viewer on own computer

Go to https://www.snapgene.com/snapgene-viewer/

Download and install the free software SnapGene Viewer (Windows or MacOS version)

4
Part 2. Creating file with human growth hormone DNA sequence

The aim of this demonstration is to create a new plasmid sequence for pASKGH (pASK-IBA37plus
with a human growth hormone insert that will express 6xHis tagged growth hormone in E. coli). You
already have some knowledge to help with the construction of this plasmid because this is the
plasmid designed in workshop 1.

pASK-IBA37plus information is also in the module handbook.

The DNA sequences we will be using are in the Appendix at the end of this worksheet. In a different
situation you would need find the relevant DNA sequences on a DNA database (like NCBI at
https://www.ncbi.nlm.nih.gov/ ) or manufactures web site (for commercially available plasmids).

a. Creating a new DNA file

Open SnapGene Viewer.
Click on New DNA file

You will see this.

Copy and paste the human growth hormone DNA sequence from the Workshop 2. Appendix Figure 1
into the box.
Select linear DNA (this is not the full plasmid sequence)
Name the file human growth hormone

5
Click OK

And also click add 1 feature. The SnapGene software automatically detects some features of the
DNA. At this stage keep these features, you can always edit out unwanted ones later.

You should see this.

This might be a good moment to save a version of this file on your own computer (etc.)
Save as human growth hormone 1

6
b. Finding the human growth hormone open reading frame

Now we are going to use an ORF tool to find open reading frames (we are aiming to identify only
DNA sequence that codes for growth hormone protein).
Click on the downward arrow (fourth tab from the top on the left hand side) click on ORFs only.

You should see this

Go to sequence (bottom left). You should see this.

Actually, this is not so useful because it is NOT picking out ORFs which start with Met and end with
stop codon for longest ORF.

7
We need to identify start of the ORF (met) that codes for human growth hormone.
Click on the downward arrow (fourth tab from the top on the left-hand side) click on translation
options.

Remove tick except DNA ends

Click OK

Better - now only identifying ORFs which start with Met and end with stop codons.

From NCBI web information about the DNA sequence we are using we know CDS 28..681 (see
Workshop 1, Figure 2). This ORF starts at nucleotide 28 and ends at nucleotide 681.

8
Does the longest ORF now correspond with the growth hormone amino acid sequence on NCBI site
(Appendix Figure 2)? Yes

Does the longest ORF corresponding with the CDS 28..681 on NCBI site (Workshop 1. Figure 2 or
Workshop 2. Appendix Figure 2)? Yes

c. Creating a feature of the human growth hormone open reading frame

On NCBI site (Appendix Figure 2) the CDS 28..681 Note. This includes the stop codon.
Move your mouse arrow along the bar until 28 comes up

Now holding down the left clicker on your mouse highlight sequence until you reach 681.

9
Now the DNA sequence (28 to 681) you want should be highlighted in blue. If it is not right click on
the blue highlight and start again.

Click on add feature

You will see this

10
Call the feature ‘human growth hormone’, select arrow and pick the colour of arrow and click OK.

You will see this. Snapgene has recognised the feature is a coding sequence (ORF).
Click translate that you agree the feature should include the amino acid sequence.

You will see this.

11
In map view it looks like this.

This might be a good moment to save a version of this file on your own computer.
Save as human growth hormone 2

d. Creating a map of human growth hormone with selected restriction enzymes

We can use Snapgene to check which restriction enzymes in the MCS of the target plasmid pASK-
IBA37plus cut within the human growth hormone ORF. You need to do this to identity which
restriction enzymes could be added to the PCR primers to allow direction subcloning of human
growth hormone into the MCS of pASK-IBA37plus. Suitable restriction enzymes must not cut within
the human growth hormone ORF. This has already been covered in workshop 1.

At the moment Snapgene is automatically adding all the restriction enzymes that cut 6 bp
recognition sequences to the human growth hormone map/sequence. From workshop 1 Figure 10
we already know

BamHI – does not cut

EcoRI – does not cut
EcoRV – does not cut
HindIII– does not cut
KpnI – does not cut
PstI – cuts once in human growth hormone ORF
SacII – does not cut
XhoI – does not cut

You can see only PstI cuts within the human growth hormone ORF in the screen shot above.

12
You can create a map of the human growth hormone with only the restriction enzymes in the MCS
of the target plasmid pASK-IBA37plus.

Select Enzymes and choose enzymes and click

You will see this. Click remove all.

Now you can add the restriction enzymes in the MCS of the target plasmid pASK-IBA37plus cut
within the human growth hormone ORF. As PstI is the only enzyme that cuts this DNA you can only
add PstI. You are not given option to add BamHI, EcoRI, EcoRV, HindIII, KpnI, SacII and XhoI because
these enzymes do not cut the DNA sequence.

13
You will see this.

You can also tidy this file up by removing the orange predicted ORF lines by clicking on ORF tool.

Then you see this.

This might be a good moment to save a version of this file on your own computer.
Save as human growth hormone 2

d. Transferring the nucleotide sequence for the human growth hormone to a Word file

Now open Microsoft Word and a new file.

Save this word document as pASKGH DNA sequence

14
Now we are going to copy and paste just the human growth hormone DNA sequence from
SnapGene into the Word document. We need this later to create the full DNA sequence of pASKGH
in the word document.

Create a heading in the Word document Human growth hormone sequence

Before we do this

Do you or don’t you want the human growth hormone stop codon?

The answer to this question you need to know which end of the protein you are fusing to the amino
acid tag (or reporter gene) to. You want the amino acid tag (or reporter gene) to form one
continuous open reading frame with the protein that contains no in-frame stop codons.

In this case the 6xHis tag in pASK-IBA37plus will be at the N terminal end of human growth hormone
so the answer is yes you want stop codon at the C terminal end of human growth hormone.

In the sequence view highlight the DNA sequence that is the is the human growth hormone ORF and
includes the stop codon. Move your mouse arrow along the bar until 28 comes up and hold down
the left clicker on your mouse highlight sequence until you reach 681.

Now click on edit (top right) and click on copy top strand bases.

15
Go to your Word document
And paste the copied DNA sequence under the heading Human growth hormone sequence
And highlight the sequence yellow (see below for example of how this should look in Word
document).

Human growth hormone sequence (from met to *)

atggctacaggctcccggacgtccctgctcctggcttttggcctgctctgcctgccctggcttcaaga
gggcagtgccttcccaaccattcccttatccaggctttttgacaacgctatgctccgcgcccatcgtc
tgcaccagctggcctttgacacctaccaggagtttgaagaagcctatatcccaaaggaacagaagtat
tcattcctgcagaacccccagacctccctctgtttctcagagtctattccgacaccctccaacaggga
ggaaacacaacagaaatccaacctagagctgctccgcatctccctgctgctcatccagtcgtggctgg
agcccgtgcagttcctcaggagtgtcttcgccaacagcctggtgtacggcgcctctgacagcaacgtc
tatgacctcctaaaggacctagaggaaggcatccaaacgctgatggggaggctggaagatggcagccc
ccggactgggcagatcttcaagcagacctacagcaagttcgacacaaactcacacaacgatgacgcac
tactcaagaactacgggctgctctactgcttcaggaaggacatggacaaggtcgagacattcctgcgc
atcgtgcagtgccgctctgtggagggcagctgtggcttctag

16
Part 3. Using human growth hormone DNA sequence file to design the gene specific part of PCR
primers

Now you need to know how you are going to subclone the human growth hormone into pASK-IBA37
before you can create the whole DNA sequence of pASKGH.

Lets start by using SnapGene to pick the gene specific parts of forward and reverse PCR primer
sequences using the human growth hormone SnapGene file.

a. Selecting gene specific sequence of forward primer

Holding down the left clicker on your mouse highlight sequence starting from the ATG (met) at the
beginning of the human growth hormone coding region.

I suggest you select between 25 -30 bases (you can always shorten it by removing bases from the 3’
end later). As you highlight it is giving you a Tm value. So 28 bases has a Tm of 71-72°C (though it is
not clear which Tm calculation formula they are using).

Now click on edit (top right) and click on copy top strand

17
Create title in your Word document and paste what you have copied underneath it

Gene specific part of the forward primer for hormone growth sequence

atggctacaggctcccggacgtccctgc

If you calculate the Tm using

Tm (°C) = 64.9°C + (41°C x ((number of G’s and C’s in the primer – 16.4)/N))

Or the basic Tm calculator at http://www.biophp.org/minitools/melting_temperature/demo.php

The Tm of the 28 base primer is 68.7 °C.

We designed forward and reverse primers to amplify human growth hormone in workshop 1 so the
above should be familiar.

b. Selecting gene specific sequence of reverse primer

Holding down the left clicker on your mouse highlight sequence starting from the TAG (stop) at the
end of the human growth hormone coding region.

I suggest you select between 25 -30 bases (you can always shorten it by removing bases from the 3’
end later). As you highlight it is giving you a Tm value. So, 29 bases has a Tm of 68°C.

18
Now click on edit (top right) and click on copy bottom strand and select 5’ to 3’ direction

Create title in your Word document and paste what you have copied underneath it

Gene specific part of the reverse primer for hormone growth sequence

ctagaagccacagctgccctccacagagc

If you calculate the Tm using

Tm (°C) = 64.9°C + (41°C x ((number of G’s and C’s in the primer – 16.4)/N))

Or the basic Tm calculator at http://www.biophp.org/minitools/melting_temperature/demo.php

The Tm of the 29 base primer is 67.2 °C

We designed forward and reverse primers to amplify human growth hormone in workshop 1 so the
above should be familiar.

19
Part 4. Adding restriction enzyme sites to gene specific PCR primers to subclone human growth
hormone into of PCR primers pASK-IBA37plus

a. Deciding which restriction enzyme sites to use to subclone human growth hormone into
pASK-IBA37plus.

From workshop 1 we have already decided possible restriction enzyme pairs are

BamHI and HindIII (as both 100% activity in buffer E)

EcoRI and SacII (as both 100% activity in buffer H)

EcoRI and XhoI (as both 100% activity in buffer H)

SacII and XhoI (as both 100% activity in buffer H)

From workshop 1 we have already decided for the BamHI and HindIII

BamHI needs to be added at the 5’ end of the forward primer (will be at N terminal end of human
growth hormone).

HindIII needs to be added to the 5’ end of the reverse primer (will be at C terminal end of human
growth hormone).

We are going to use BamHI and HindIII in the following demonstration

Part 5. Creating file with DNA sequence pASK-IBA37plus

a. Creating a new DNA file for pASK-IBA37plus in Snapgene

In SnapGene viewer
Click on New DNA file
Copy and paste the pASK-IBA37plus DNA sequence (Workshop 2 Appendix Figure 3) into the box
Select plasmid DNA (should be the default setting)
Name the file pASK-IBA37plus
Click OK
And click add all 11 features detected automatically

20
 This might be a good moment to save a version of this file on your own computer.
Save as pASK-IBA37plus-1
Part 6. Adding pASK-IBA37plus DNA sequences to Word document and creating DNA sequence of
pASKGH

Now we are going to transfer two different parts of the pASK-IBA37plus DNA into the Word
Document so we can use these to create the pASKGH DNA sequence.

a. Selecting the DNA sequence of pASK-IBA37plus that contains the sequence upstream of and
including the BamHI site (G↓GATCC).
Click on sequence (bottom left hand side)

Holding down the left clicker on your mouse highlight the sequence from 1 to the end of BamHI site
(at 249).

Now click on edit (top right) and click on copy top strand

21
Create suitable title in your Word document and paste what you have copied underneath it

Highlight the sequence green

DNA sequence 1- 249 pASKIBA37plus including BamHI

CCATCGAATGGCCAGATGATTAATTCCTAATTTTTGTTGACACTCTATCATTGATAGAGTTATTTTAC
CACTCCCTATCAGTGATAGAGAAAAGTGAAATGAATAGTTCGACAAAAATCTAGAAATAATTTTGTTT
AACTTTAAGAAGGAGATATACAAATGGCTAGCAGAGGATCGCATCACCATCACCATCACATCGAAGGG
CGCCGAGACCGCGGTCCCGAATTCGAGCTCGGTACCCGGGGATCC

b. Selecting the DNA sequence of pASK-IBA37plus that contians the sequence downstream of and
including the HindIII site (A↓AGCTT).

Holding down the left clicker on your mouse highlight the sequence from 295 to the end of the
plasmid sequence 3270 (includes HindIII site)

Now click on edit (top right) and click on copy top strand

22
Create suitable title in your Word document and paste what you have copied underneath it

Highlight the sequence blue

DNA sequence 295 to 3270 pASKIBA37plus including HindIII

AAGCTTGACCTGTGAAGTGAAAAATGGCGCACATTGTGCGACATTTTTTTTGTCTGCCGTTTACCGCT
ACTGCGTCACGGATCTCCACGCGCCCTGTAGCGGCGCATTAAGCGCGGCGGGTGTGGTGGTTACGCGC
AGCGTGACCGCTACACTTGCCAGCGCCCTAGCGCCCGCTCCTTTCGCTTTCTTCCCTTCCTTTCTCGC
CACGTTCGCCGGCTTTCCCCGTCAAGCTCTAAATCGGGGGCTCCCTTTAGGGTTCCGATTTAGTGCTT
TACGGCACCTCGACCCCAAAAAACTTGATTAGGGTGATGGTTCACGTAGTGGGCCATCGCCCTGATAG
ACGGTTTTTCGCCCTTTGACGTTGGAGTCCACGTTCTTTAATAGTGGACTCTTGTTCCAAACTGGAAC
AACACTCAACCCTATCTCGGTCTATTCTTTTGATTTATAAGGGATTTTGCCGATTTCGGCCTATTGGT
TAAAAAATGAGCTGATTTAACAAAAATTTAACGCGAATTTTAACAAAATATTAACGCTTACAATTTCA
GGTGGCACTTTTCGGGGAAATGTGCGCGGAACCCCTATTTGTTTATTTTTCTAAATACATTCAAATAT
GTATCCGCTCATGAGACAATAACCCTGATAAATGCTTCAATAATATTGAAAAAGGAAGAGTATGAGTA
TTCAACATTTCCGTGTCGCCCTTATTCCCTTTTTTGCGGCATTTTGCCTTCCTGTTTTTGCTCACCCA
GAAACGCTGGTGAAAGTAAAAGATGCTGAAGATCAGTTGGGTGCACGAGTGGGTTACATCGAACTGGA
TCTCAACAGCGGTAAGATCCTTGAGAGTTTTCGCCCCGAAGAACGTTTTCCAATGATGAGCACTTTTA
AAGTTCTGCTATGTGGCGCGGTATTATCCCGTATTGACGCCGGGCAAGAGCAACTCGGTCGCCGCATA
CACTATTCTCAGAATGACTTGGTTGAGTACTCACCAGTCACAGAAAAGCATCTTACGGATGGCATGAC
AGTAAGAGAATTATGCAGTGCTGCCATAACCATGAGTGATAACACTGCGGCCAACTTACTTCTGACAA
CGATCGGAGGACCGAAGGAGCTAACCGCTTTTTTGCACAACATGGGGGATCATGTAACTCGCCTTGAT
CGTTGGGAACCGGAGCTGAATGAAGCCATACCAAACGACGAGCGTGACACCACGATGCCTGTAGCAAT
GGCAACAACGTTGCGCAAACTATTAACTGGCGAACTACTTACTCTAGCTTCCCGGCAACAATTGATAG
ACTGGATGGAGGCGGATAAAGTTGCAGGACCACTTCTGCGCTCGGCCCTTCCGGCTGGCTGGTTTATT
GCTGATAAATCTGGAGCCGGTGAGCGTGGCTCTCGCGGTATCATTGCAGCACTGGGGCCAGATGGTAA
GCCCTCCCGTATCGTAGTTATCTACACGACGGGGAGTCAGGCAACTATGGATGAACGAAATAGACAGA
TCGCTGAGATAGGTGCCTCACTGATTAAGCATTGGTAGGAATTAATGATGTCTCGTTTAGATAAAAGT
AAAGTGATTAACAGCGCATTAGAGCTGCTTAATGAGGTCGGAATCGAAGGTTTAACAACCCGTAAACT
CGCCCAGAAGCTAGGTGTAGAGCAGCCTACATTGTATTGGCATGTAAAAAATAAGCGGGCTTTGCTCG
ACGCCTTAGCCATTGAGATGTTAGATAGGCACCATACTCACTTTTGCCCTTTAGAAGGGGAAAGCTGG
CAAGATTTTTTACGTAATAACGCTAAAAGTTTTAGATGTGCTTTACTAAGTCATCGCGATGGAGCAAA
AGTACATTTAGGTACACGGCCTACAGAAAAACAGTATGAAACTCTCGAAAATCAATTAGCCTTTTTAT
GCCAACAAGGTTTTTCACTAGAGAATGCATTATATGCACTCAGCGCAGTGGGGCATTTTACTTTAGGT
TGCGTATTGGAAGATCAAGAGCATCAAGTCGCTAAAGAAGAAAGGGAAACACCTACTACTGATAGTAT
GCCGCCATTATTACGACAAGCTATCGAATTATTTGATCACCAAGGTGCAGAGCCAGCCTTCTTATTCG
GCCTTGAATTGATCATATGCGGATTAGAAAAACAACTTAAATGTGAAAGTGGGTCTTAAAAGCAGCAT
AACCTTTTTCCGTGATGGTAACTTCACTAGTTTAAAAGGATCTAGGTGAAGATCCTTTTTGATAATCT
CATGACCAAAATCCCTTAACGTGAGTTTTCGTTCCACTGAGCGTCAGACCCCGTAGAAAAGATCAAAG

23
GATCTTCTTGAGATCCTTTTTTTCTGCGCGTAATCTGCTGCTTGCAAACAAAAAAACCACCGCTACCA
GCGGTGGTTTGTTTGCCGGATCAAGAGCTACCAACTCTTTTTCCGAAGGTAACTGGCTTCAGCAGAGC
GCAGATACCAAATACTGTCCTTCTAGTGTAGCCGTAGTTAGGCCACCACTTCAAGAACTCTGTAGCAC
CGCCTACATACCTCGCTCTGCTAATCCTGTTACCAGTGGCTGCTGCCAGTGGCGATAAGTCGTGTCTT
ACCGGGTTGGACTCAAGACGATAGTTACCGGATAAGGCGCAGCGGTCGGGCTGAACGGGGGGTTCGTG
CACACAGCCCAGCTTGGAGCGAACGACCTACACCGAACTGAGATACCTACAGCGTGAGCTATGAGAAA
GCGCCACGCTTCCCGAAGGGAGAAAGGCGGACAGGTATCCGGTAAGCGGCAGGGTCGGAACAGGAGAG
CGCACGAGGGAGCTTCCAGGGGGAAACGCCTGGTATCTTTATAGTCCTGTCGGGTTTCGCCACCTCTG
ACTTGAGCGTCGATTTTTGTGATGCTCGTCAGGGGGGCGGAGCCTATGGAAAAACGCCAGCAACGCGG
CCTTTTTACGGTTCCTGGCCTTTTGCTGGCCTTTTGCTCACATGACCCGACA

24
c. Now you need to join the three sequences you have pasted in your Word document together.
upstream pASK37plus then human growth hormone and downstream pASKIBA37plus to creat the
DNA sequence of pASKGH.

Be careful not to delete or add any nucleotides.

Create a new sensible title in your Word Dcocument.

Note. Highlighting the three sequences in different colours in Word is useful as you can now easily
identify where the parts of the DNA sequence of pASKGH come from.

DNA sequence of pASKGH

N.B. I am deliberately keeping plasmid in CAPITALS and growth hormone in lower case so I can see
which is which in Snapgene sequence viewer.

CCATCGAATGGCCAGATGATTAATTCCTAATTTTTGTTGACACTCTATCATTGATAGAGTTATTTTAC
CACTCCCTATCAGTGATAGAGAAAAGTGAAATGAATAGTTCGACAAAAATCTAGAAATAATTTTGTTT
AACTTTAAGAAGGAGATATACAAATGGCTAGCAGAGGATCGCATCACCATCACCATCACATCGAAGGG
CGCCGAGACCGCGGTCCCGAATTCGAGCTCGGTACCCGGGGATCCatggctacaggctcccggacgtc
cctgctcctggcttttggcctgctctgcctgccctggcttcaagagggcagtgccttcccaaccattc
ccttatccaggctttttgacaacgctatgctccgcgcccatcgtctgcaccagctggcctttgacacc
taccaggagtttgaagaagcctatatcccaaaggaacagaagtattcattcctgcagaacccccagac
ctccctctgtttctcagagtctattccgacaccctccaacagggaggaaacacaacagaaatccaacc
tagagctgctccgcatctccctgctgctcatccagtcgtggctggagcccgtgcagttcctcaggagt
gtcttcgccaacagcctggtgtacggcgcctctgacagcaacgtctatgacctcctaaaggacctaga
ggaaggcatccaaacgctgatggggaggctggaagatggcagcccccggactgggcagatcttcaagc
agacctacagcaagttcgacacaaactcacacaacgatgacgcactactcaagaactacgggctgctc
tactgcttcaggaaggacatggacaaggtcgagacattcctgcgcatcgtgcagtgccgctctgtgga
gggcagctgtggcttctagAAGCTTGACCTGTGAAGTGAAAAATGGCGCACATTGTGCGACATTTTTT
TTGTCTGCCGTTTACCGCTACTGCGTCACGGATCTCCACGCGCCCTGTAGCGGCGCATTAAGCGCGGC
GGGTGTGGTGGTTACGCGCAGCGTGACCGCTACACTTGCCAGCGCCCTAGCGCCCGCTCCTTTCGCTT
TCTTCCCTTCCTTTCTCGCCACGTTCGCCGGCTTTCCCCGTCAAGCTCTAAATCGGGGGCTCCCTTTA
GGGTTCCGATTTAGTGCTTTACGGCACCTCGACCCCAAAAAACTTGATTAGGGTGATGGTTCACGTAG
TGGGCCATCGCCCTGATAGACGGTTTTTCGCCCTTTGACGTTGGAGTCCACGTTCTTTAATAGTGGAC
TCTTGTTCCAAACTGGAACAACACTCAACCCTATCTCGGTCTATTCTTTTGATTTATAAGGGATTTTG
CCGATTTCGGCCTATTGGTTAAAAAATGAGCTGATTTAACAAAAATTTAACGCGAATTTTAACAAAAT
ATTAACGCTTACAATTTCAGGTGGCACTTTTCGGGGAAATGTGCGCGGAACCCCTATTTGTTTATTTT
TCTAAATACATTCAAATATGTATCCGCTCATGAGACAATAACCCTGATAAATGCTTCAATAATATTGA
AAAAGGAAGAGTATGAGTATTCAACATTTCCGTGTCGCCCTTATTCCCTTTTTTGCGGCATTTTGCCT
TCCTGTTTTTGCTCACCCAGAAACGCTGGTGAAAGTAAAAGATGCTGAAGATCAGTTGGGTGCACGAG
TGGGTTACATCGAACTGGATCTCAACAGCGGTAAGATCCTTGAGAGTTTTCGCCCCGAAGAACGTTTT
CCAATGATGAGCACTTTTAAAGTTCTGCTATGTGGCGCGGTATTATCCCGTATTGACGCCGGGCAAGA
GCAACTCGGTCGCCGCATACACTATTCTCAGAATGACTTGGTTGAGTACTCACCAGTCACAGAAAAGC
ATCTTACGGATGGCATGACAGTAAGAGAATTATGCAGTGCTGCCATAACCATGAGTGATAACACTGCG
GCCAACTTACTTCTGACAACGATCGGAGGACCGAAGGAGCTAACCGCTTTTTTGCACAACATGGGGGA
TCATGTAACTCGCCTTGATCGTTGGGAACCGGAGCTGAATGAAGCCATACCAAACGACGAGCGTGACA
CCACGATGCCTGTAGCAATGGCAACAACGTTGCGCAAACTATTAACTGGCGAACTACTTACTCTAGCT
TCCCGGCAACAATTGATAGACTGGATGGAGGCGGATAAAGTTGCAGGACCACTTCTGCGCTCGGCCCT
TCCGGCTGGCTGGTTTATTGCTGATAAATCTGGAGCCGGTGAGCGTGGCTCTCGCGGTATCATTGCAG
CACTGGGGCCAGATGGTAAGCCCTCCCGTATCGTAGTTATCTACACGACGGGGAGTCAGGCAACTATG
GATGAACGAAATAGACAGATCGCTGAGATAGGTGCCTCACTGATTAAGCATTGGTAGGAATTAATGAT
GTCTCGTTTAGATAAAAGTAAAGTGATTAACAGCGCATTAGAGCTGCTTAATGAGGTCGGAATCGAAG
GTTTAACAACCCGTAAACTCGCCCAGAAGCTAGGTGTAGAGCAGCCTACATTGTATTGGCATGTAAAA

25
AATAAGCGGGCTTTGCTCGACGCCTTAGCCATTGAGATGTTAGATAGGCACCATACTCACTTTTGCCC
TTTAGAAGGGGAAAGCTGGCAAGATTTTTTACGTAATAACGCTAAAAGTTTTAGATGTGCTTTACTAA
GTCATCGCGATGGAGCAAAAGTACATTTAGGTACACGGCCTACAGAAAAACAGTATGAAACTCTCGAA
AATCAATTAGCCTTTTTATGCCAACAAGGTTTTTCACTAGAGAATGCATTATATGCACTCAGCGCAGT
GGGGCATTTTACTTTAGGTTGCGTATTGGAAGATCAAGAGCATCAAGTCGCTAAAGAAGAAAGGGAAA
CACCTACTACTGATAGTATGCCGCCATTATTACGACAAGCTATCGAATTATTTGATCACCAAGGTGCA
GAGCCAGCCTTCTTATTCGGCCTTGAATTGATCATATGCGGATTAGAAAAACAACTTAAATGTGAAAG
TGGGTCTTAAAAGCAGCATAACCTTTTTCCGTGATGGTAACTTCACTAGTTTAAAAGGATCTAGGTGA
AGATCCTTTTTGATAATCTCATGACCAAAATCCCTTAACGTGAGTTTTCGTTCCACTGAGCGTCAGAC
CCCGTAGAAAAGATCAAAGGATCTTCTTGAGATCCTTTTTTTCTGCGCGTAATCTGCTGCTTGCAAAC
AAAAAAACCACCGCTACCAGCGGTGGTTTGTTTGCCGGATCAAGAGCTACCAACTCTTTTTCCGAAGG
TAACTGGCTTCAGCAGAGCGCAGATACCAAATACTGTCCTTCTAGTGTAGCCGTAGTTAGGCCACCAC
TTCAAGAACTCTGTAGCACCGCCTACATACCTCGCTCTGCTAATCCTGTTACCAGTGGCTGCTGCCAG
TGGCGATAAGTCGTGTCTTACCGGGTTGGACTCAAGACGATAGTTACCGGATAAGGCGCAGCGGTCGG
GCTGAACGGGGGGTTCGTGCACACAGCCCAGCTTGGAGCGAACGACCTACACCGAACTGAGATACCTA
CAGCGTGAGCTATGAGAAAGCGCCACGCTTCCCGAAGGGAGAAAGGCGGACAGGTATCCGGTAAGCGG
CAGGGTCGGAACAGGAGAGCGCACGAGGGAGCTTCCAGGGGGAAACGCCTGGTATCTTTATAGTCCTG
TCGGGTTTCGCCACCTCTGACTTGAGCGTCGATTTTTGTGATGCTCGTCAGGGGGGCGGAGCCTATGG
AAAAACGCCAGCAACGCGGCCTTTTTACGGTTCCTGGCCTTTTGCTGGCCTTTTGCTCACATGACCCG
ACA

26
Part 7. Creating a new DNA file for pASKGH in SnapGene

a. Creating a new DNA file for pASKGH in SnapGene.

In SnapGene viewer
Click on New DNA file
Copy and paste the pASKGH DNA sequence from the Word document into the box
Select plasmid DNA (should be the default setting)
Name the file pASKGH
Click OK
And click add all 12 features detected automatically. Note. the features automatically detected do
not include the full growth hormone ORF.

This might be a good moment to save a version of this file on your own computer.
Save as pASKGH-1

The default plasmid map/sequence needs a bit of editing to select key restriction enzyme sites and
to add the ORF that codes for the full growth hormone.

b. Lets start with adding the human growth hormone ORF as a feature.

You could use ‘word count’ to count characters in the pASKGH DNA sequence in the Word
document. From this I worked out the human growth hormone ORF is between 249 (ATG….) and
903 in the pASKGH DNA sequence.
Or
You could use the BamHI and HindIII sites that have been automatically added to the map when we
created the file.
Or
In this case Snapgene is pointing out the amino acids/DNA sequence in-frame with the hGH signal
sequence (ORF) to you could use this as a guide.
Or
In this case the DNA sequence we copy pasted into Snapgene has the human growth hormone in
lower case and the plasmid backbone in capitals (handy).

There is more than one way to do this - do what you are comfortable with once you are familiar
with the software.

27
Whatever method you use to identify the ORF highlight to human growth hormone sequence in
blue.

Now we have added the ‘blue highlighted range’ we can edit it to be a feature of the plasmid.
Click on features and then on add feature.

Name the feature Human growth hormone, pick arrow (right direction), select CDS and tick translate
this feature in sequence view, pick colour. Click OK to add feature to plasmid.

28
You should see the following.

Go to map (bottom left hand side) The map of pASKGH now looks like this.

This might be a good moment to save a version of this file on your own computer (etc.)
Save as pASKGH-2

At the moment the map looks a bit odd because the 6xHis and Xa cleavage site and growth hormone
are not joined together but the sequence view shows they are all in the same reading frame.

c. Now I want to join up 6xHis, Xa and the growth hormone ORF in one block/ORF and identify the
first Met (start codon) upstream of the 6xHis tag (and downstream of the RBS). I have decided to do
this by creating a single feature which I will call 6xHis tagged human growth hormone.

N.B. you might need to add 1 or two nucleotides to the PCR primer get the 6xHis tag in-frame with
the growth hormone, but in this case it is already in frame and forms one CDS.

29
Go to sequence view.
Holding down the left clicker on your mouse highlight sequence starting from the ATG (met) which is
just after the RBS upstream of the 6xHis tag and ending the stop codon of human growth hormone
coding region. N.B. highlighting 160 to 903 on sequence.

Click on features and then on add feature. Name 6xHis tagged human growth hormone, pick arrow,
pick CDS, tick translate and pick colour. Click OK.

The map view looks like this.

30
This might be a good moment to save a version of this file on your own computer.
Save as pASKGH-3

d. Now let’s have sensible number of useful restriction enzymes sites on the map.
Click on Enzymes and choose enzymes.
I chose commonly use restriction enzymes sites in the MCS and PstI because it cuts within the
human growth hormone DNA sequence.

BamHI, EcoRI, HindIII, KpnI, PstI, and SacI

NB. BamHI and HindIII are the sites used to sub-clone the growth hormone into pASK-IBA37plus.

Remove all enzymes. The add desired restriction enzyme sites – one by one - click ok.

31
Now the map looks like this.

This might be a good moment to save a version of this file on your own computer.
Save as pASKGH-4

e. Now add the PCR primers as features. Scientists do not normally annotate their plasmid maps
with primer sequences, but this is helpful practice for figures you need to produce for the
Experimental Design coursework.

From workshop 1

Forward primer aaaggatccatggctacaggctcccggacgtccctgc with BamHI

Reverse primer aaaaagcttctagaagccacagctgccctccacagagc with HindIII

A. Forward primer.
Go to the sequence view. Highlight the forward primer (RE site and gene specific parts).

32
Click primer and then add primer.

Select top strand (as this is sequence of forward primer)

You will see this. Change name to forward primer, add the extra three 5’ aaa, and click add primer
to template.

33
Then sequence view will look like this.

B. Reverse primer.
Reverse primer aaaaagcttctagaagccacagctgccctccacagagc with HindIII

Go to the sequence view. Highlight the reverse primer (RE site and gene specific parts).

Click primer and then add primer.

Select bottom strand (as this is sequence of reverse primer)
Change primer name to reverse primer, add the extra three 5’ aaa, and click add primer to template

34
Then sequence view will look like this.

The map view looks like this

Why is the primer 5’ aaa not in the pASKGH plasmid sequence?

1. When you use PCR to amplify the human growth hormone from pCR4-TOPO-GH the double
stranded PCR product looks like this (forward and reverse primer sequences highlighted)
5’aaaGGATCCatggctacaggctcccggacgtccctgc..cgctctgtggagggcagctgtggcttctagAAGCTTttt3’
3’tttCCTAGGtaccgatgtccgagggcctgcagggacg..gcgagacacctcccgtcgacaccgaagatcTTCGAAaaa5’

2. Prior to the ligation reaction you cut the growth hormone PCR product with BamHI and HindIII so
the DNA looks like this (5’aaa ends removed).

3. In the ligation reaction you join the BamHI HindIII cut growth hormone DNA to the BamHI HindIII
cut pASK-IBA37plus plasmid.

35
Part 8. Using SnapGene file of pASKGH to extract amino acid sequence of 6xHis tagged human
growth hormone to create a figure

Now I want to use SnapGene file pASKGH copy the amino acid sequence of the 6xHis tagged human
growth hormone protein open reading frame and use this to create a figure of the amino acid
sequence of the in a Word document 6xHis tagged human growth hormone protein.

In SnapGene pASKGH file right click on the 6xHis tagged growth hormone feature, select copy
feature translation and copy 1-letter amino acids and then paste the amino acid sequence in your
Word document.

Paste the copies amino acid sequence into Word document should look like this

MASRGSHHHHHHIEGRRDRGPEFELGTRGSMATGSRTSLLLAFGLLCLPWLQEGSAFPTIPLSRLFDNAMLRAHRL
HQLAFDTYQEFEEAYIPKEQKYSFLQNPQTSLCFSESIPTPSNREETQQKSNLELLRISLLLIQSWLEPVQFLRSVFANS
LVYGASDSNVYDLLKDLEEGIQTLMGRLEDGSPRTGQIFKQTYSKFDTNSHNDDALLKNYGLLYCFRKDMDKVETFL
RIVQCRSVEGSCGF*

Use three different colours to highlight

1. The amino acid sequence that corresponds to human growth hormone.

2. The amino acid sequence that corresponds to the 6xHis tag.
3. Any additional amino acids that are neither human growth hormone or the 6xHis tag.

MASRGSHHHHHHIEGRRDRGPEFELGTRGSMATGSRTSLLLAFGLLCLPWLQEGSAFPTIPLSRLFDNAMLRAHRL
HQLAFDTYQEFEEAYIPKEQKYSFLQNPQTSLCFSESIPTPSNREETQQKSNLELLRISLLLIQSWLEPVQFLRSVFANS
LVYGASDSNVYDLLKDLEEGIQTLMGRLEDGSPRTGQIFKQTYSKFDTNSHNDDALLKNYGLLYCFRKDMDKVETFL
RIVQCRSVEGSCGF*

NOTE. There are no stop codons in the middle of the above amino acid sequence, it is one open
reading frame (ORF) from the first Methionine to the stop codon.

36
Part 9. Making sure your protein is in frame with the tag or reporter gene to create a fusion
protein

From workshop 1 and 2

By ‘chance’, as we have used BamHI, if you place the first ATG (first methionine) of the growth
hormone next to the BamHI site you do create an in-frame fusion between the N terminal 6xHis tag
and the human growth hormone. But this is not always the case.

Forward primer aaaggatccatggctacaggctcccggacgtccctgc with BamHI

In this theoretical example you would need to add an extra nucleotide between the ATG and the
BamHI site in the forward primer to get the growth hormone in frame with the 6xHis tag.

Theoretical forward primer aaaggatccaatggctacaggctcccggacgtccctgc with BamHI

Please NOTE (for experimental design coursework)

37
If the DNA sequence you are adding to a plasmid was not in-frame with the N-terminal tag or
reporter gene you would need to add one or two nucleotides between the restriction enzyme site
and the ATG (first methionine) on the forward primer to ensure your protein and the amino acid
sequence of the tag or reporter protein are the same open reading frame otherwise your fusion
protein will not be translated correctly.

If the DNA sequence you are adding to a plasmid was not in-frame with the C-terminal tag or
reporter gene you would need to add one or two nucleotides between the restriction enzyme site
and the XXX (codon for end amino acid) on the reverse primer to ensure your protein and the amino
acid sequence of the tag or reporter protein are the same open reading frame otherwise your fusion
protein will not be translated correctly.

There will be an additional presentation at the end of workshop 2 session called

Making sure you plasmid will create a in-frame fusion protein

38
Appendix
Figure 1. DNA sequence of human growth hormone in FASTA format

Taken from https://www.ncbi.nlm.nih.gov/nuccore/50960726

gtcctgtggacagctcacctagctgcaatggctacaggctcccggacgtccctgctcctg
gcttttggcctgctctgcctgccctggcttcaagagggcagtgccttcccaaccattccc
ttatccaggctttttgacaacgctatgctccgcgcccatcgtctgcaccagctggccttt
gacacctaccaggagtttgaagaagcctatatcccaaaggaacagaagtattcattcctg
cagaacccccagacctccctctgtttctcagagtctattccgacaccctccaacagggag
gaaacacaacagaaatccaacctagagctgctccgcatctccctgctgctcatccagtcg
tggctggagcccgtgcagttcctcaggagtgtcttcgccaacagcctggtgtacggcgcc
tctgacagcaacgtctatgacctcctaaaggacctagaggaaggcatccaaacgctgatg
gggaggctggaagatggcagcccccggactgggcagatcttcaagcagacctacagcaag
ttcgacacaaactcacacaacgatgacgcactactcaagaactacgggctgctctactgc
ttcaggaaggacatggacaaggtcgagacattcctgcgcatcgtgcagtgccgctctgtg
gagggcagctgtggcttctagctgcccgggtggcatccctgtgacccctccccagtgcct
ctcct

Figure 2. Amino acid sequence of human growth hormone in FASTA format

Taken from https://www.ncbi.nlm.nih.gov/nuccore/50960726

MATGSRTSLLLAFGLLCLPWLQEGSAFPTIPLSRLFDNAMLRAHRLHQLAFDTYQEFEEA
YIPKEQKYSFLQNPQTSLCFSESIPTPSNREETQQKSNLELLRISLLLIQSWLEPVQFLR
SVFANSLVYGASDSNVYDLLKDLEEGIQTLMGRLEDGSPRTGQIFKQTYSKFDTNSHNDD
ALLKNYGLLYCFRKDMDKVETFLRIVQCRSVEGSCGF

39
Figure 3. The complete DNA sequence of pASK-IBA37plus
The highlighted DNA sequence was used to create figure 4.
(taken from https://www.iba-lifesciences.com/isotope/2/2-1437-000-Sequence-pASK-IBA37plus.txt)

CCATCGAATGGCCAGATGATTAATTCCTAATTTTTGTTGACACTCTATCATTGATAGAGTTATTTTACCACTCCC
TATCAGTGATAGAGAAAAGTGAAATGAATAGTTCGACAAAAATCTAGAAATAATTTTGTTTAACTTTAAGAAGGA
GATATACAAATGGCTAGCAGAGGATCGCATCACCATCACCATCACATCGAAGGGCGCCGAGACCGCGGTCCCGAA
TTCGAGCTCGGTACCCGGGGATCCCTCGAGGTCGACCTGCAGGGGGACCATGGTCTCTGATATCTAACTAAGCTT
GACCTGTGAAGTGAAAAATGGCGCACATTGTGCGACATTTTTTTTGTCTGCCGTTTACCGCTACTGCGTCACGGA
TCTCCACGCGCCCTGTAGCGGCGCATTAAGCGCGGCGGGTGTGGTGGTTACGCGCAGCGTGACCGCTACACTTGC
CAGCGCCCTAGCGCCCGCTCCTTTCGCTTTCTTCCCTTCCTTTCTCGCCACGTTCGCCGGCTTTCCCCGTCAAGC
TCTAAATCGGGGGCTCCCTTTAGGGTTCCGATTTAGTGCTTTACGGCACCTCGACCCCAAAAAACTTGATTAGGG
TGATGGTTCACGTAGTGGGCCATCGCCCTGATAGACGGTTTTTCGCCCTTTGACGTTGGAGTCCACGTTCTTTAA
TAGTGGACTCTTGTTCCAAACTGGAACAACACTCAACCCTATCTCGGTCTATTCTTTTGATTTATAAGGGATTTT
GCCGATTTCGGCCTATTGGTTAAAAAATGAGCTGATTTAACAAAAATTTAACGCGAATTTTAACAAAATATTAAC
GCTTACAATTTCAGGTGGCACTTTTCGGGGAAATGTGCGCGGAACCCCTATTTGTTTATTTTTCTAAATACATTC
AAATATGTATCCGCTCATGAGACAATAACCCTGATAAATGCTTCAATAATATTGAAAAAGGAAGAGTATGAGTAT
TCAACATTTCCGTGTCGCCCTTATTCCCTTTTTTGCGGCATTTTGCCTTCCTGTTTTTGCTCACCCAGAAACGCT
GGTGAAAGTAAAAGATGCTGAAGATCAGTTGGGTGCACGAGTGGGTTACATCGAACTGGATCTCAACAGCGGTAA
GATCCTTGAGAGTTTTCGCCCCGAAGAACGTTTTCCAATGATGAGCACTTTTAAAGTTCTGCTATGTGGCGCGGT
ATTATCCCGTATTGACGCCGGGCAAGAGCAACTCGGTCGCCGCATACACTATTCTCAGAATGACTTGGTTGAGTA
CTCACCAGTCACAGAAAAGCATCTTACGGATGGCATGACAGTAAGAGAATTATGCAGTGCTGCCATAACCATGAG
TGATAACACTGCGGCCAACTTACTTCTGACAACGATCGGAGGACCGAAGGAGCTAACCGCTTTTTTGCACAACAT
GGGGGATCATGTAACTCGCCTTGATCGTTGGGAACCGGAGCTGAATGAAGCCATACCAAACGACGAGCGTGACAC
CACGATGCCTGTAGCAATGGCAACAACGTTGCGCAAACTATTAACTGGCGAACTACTTACTCTAGCTTCCCGGCA
ACAATTGATAGACTGGATGGAGGCGGATAAAGTTGCAGGACCACTTCTGCGCTCGGCCCTTCCGGCTGGCTGGTT
TATTGCTGATAAATCTGGAGCCGGTGAGCGTGGCTCTCGCGGTATCATTGCAGCACTGGGGCCAGATGGTAAGCC
CTCCCGTATCGTAGTTATCTACACGACGGGGAGTCAGGCAACTATGGATGAACGAAATAGACAGATCGCTGAGAT
AGGTGCCTCACTGATTAAGCATTGGTAGGAATTAATGATGTCTCGTTTAGATAAAAGTAAAGTGATTAACAGCGC
ATTAGAGCTGCTTAATGAGGTCGGAATCGAAGGTTTAACAACCCGTAAACTCGCCCAGAAGCTAGGTGTAGAGCA
GCCTACATTGTATTGGCATGTAAAAAATAAGCGGGCTTTGCTCGACGCCTTAGCCATTGAGATGTTAGATAGGCA
CCATACTCACTTTTGCCCTTTAGAAGGGGAAAGCTGGCAAGATTTTTTACGTAATAACGCTAAAAGTTTTAGATG
TGCTTTACTAAGTCATCGCGATGGAGCAAAAGTACATTTAGGTACACGGCCTACAGAAAAACAGTATGAAACTCT
CGAAAATCAATTAGCCTTTTTATGCCAACAAGGTTTTTCACTAGAGAATGCATTATATGCACTCAGCGCAGTGGG
GCATTTTACTTTAGGTTGCGTATTGGAAGATCAAGAGCATCAAGTCGCTAAAGAAGAAAGGGAAACACCTACTAC
TGATAGTATGCCGCCATTATTACGACAAGCTATCGAATTATTTGATCACCAAGGTGCAGAGCCAGCCTTCTTATT
CGGCCTTGAATTGATCATATGCGGATTAGAAAAACAACTTAAATGTGAAAGTGGGTCTTAAAAGCAGCATAACCT
TTTTCCGTGATGGTAACTTCACTAGTTTAAAAGGATCTAGGTGAAGATCCTTTTTGATAATCTCATGACCAAAAT
CCCTTAACGTGAGTTTTCGTTCCACTGAGCGTCAGACCCCGTAGAAAAGATCAAAGGATCTTCTTGAGATCCTTT
TTTTCTGCGCGTAATCTGCTGCTTGCAAACAAAAAAACCACCGCTACCAGCGGTGGTTTGTTTGCCGGATCAAGA
GCTACCAACTCTTTTTCCGAAGGTAACTGGCTTCAGCAGAGCGCAGATACCAAATACTGTCCTTCTAGTGTAGCC
GTAGTTAGGCCACCACTTCAAGAACTCTGTAGCACCGCCTACATACCTCGCTCTGCTAATCCTGTTACCAGTGGC
TGCTGCCAGTGGCGATAAGTCGTGTCTTACCGGGTTGGACTCAAGACGATAGTTACCGGATAAGGCGCAGCGGTC
GGGCTGAACGGGGGGTTCGTGCACACAGCCCAGCTTGGAGCGAACGACCTACACCGAACTGAGATACCTACAGCG
TGAGCTATGAGAAAGCGCCACGCTTCCCGAAGGGAGAAAGGCGGACAGGTATCCGGTAAGCGGCAGGGTCGGAAC
AGGAGAGCGCACGAGGGAGCTTCCAGGGGGAAACGCCTGGTATCTTTATAGTCCTGTCGGGTTTCGCCACCTCTG
ACTTGAGCGTCGATTTTTGTGATGCTCGTCAGGGGGGCGGAGCCTATGGAAAAACGCCAGCAACGCGGCCTTTTT
ACGGTTCCTGGCCTTTTGCTGGCCTTTTGCTCACATGACCCGACA

40