pET-30a-c(+) Vectors

TB095 12/98
The pET-30a-c(+) vectors carry an N-terminal HisTag/thrombin/STag/enterokinase configuration plus an optional C-terminal HisTag sequence. Unique sites are shown on the circle map.
Note that the sequence is numbered by the pBR322 convention, so the T7 expression region is reversed on the circular map. The cloning/expression region of the coding strand transcribed by T7
RNA polymerase is shown below. The f1 origin is oriented so that infection with helper phage will
produce virions containing single-stranded DNA that corresponds to the coding strand. Therefore,
single-stranded sequencing should be performed using the T7 terminator primer (Cat. No. 69337-3).

Cat. No.
69909-3
69910-3
69911-3

pET-30a DNA
pET-30b DNA
pET-30c DNA

Xho I(158)
Not I(166)
Eag I(166)
Hind III(173)
Sal I(179)
Sac I(190)
EcoR I(192)
BamH I(198)
EcoR V(206)
Nco I(212)
Kpn I(238)
Bgl II(241)
Bpu1102 I(80)
Nsp V(268)
Nde I(346)
Xba I(384)

pET-30a(+) sequence landmarks

419-435
418
327-344
249-293
158-217
140-157
26-72
826-1905
3339
4048-4860
4956-5411

Dra III(5180)

6-5411)
(495
gin
i
r
o
f1
Pvu I(4479)
Sgf I(4479)
Sma I(4353)

Sph I(651)

Mlu I(1176)
Bcl I(1190)

lacI (826-1905)

The maps for pET-30b(+) and pET-30c(+)

are the same as pET-30a(+) (shown) with
the following exceptions: pET-30b(+) is a
5421bp plasmid; subtract 1bp from each site
beyond BamH I at 198. pET-30c(+) is a
5423bp plasmid; add 1bp to each site
beyond BamH I at 198.

SgrA I(495)

kan (
404
8-4
86
0)

T7 promoter
T7 transcription start
HisTag coding sequence
STag coding sequence
Multiple cloning sites
(Nco I - Xho I)
HisTag coding sequence
T7 terminator
lacI coding sequence
pBR322 origin
Kan coding sequence
f1 origin

Nru I(4136)

pET-30a(+)
(5422bp)

BstE II(1357)
Apa I(1387)

BssH II(1587)

Eco57 I(3825)

Hpa I(1682)

AlwN I(3693)

or

(3

BssS I(3450)

33

9)

PshA I(2021)

BspLU11 I(3277)
Sap I(3161)
Bst1107 I(3048)
Tth111 I(3022)

Bgl I(2240)
Fsp I(2258)
Psp5 II(2283)

T7 promoter primer #69348-3

pET upstream primer #69214-3

T7 promoter

lac operator

rbs

Xba I

AGATCGATCTCGATCCCGCGAAATTAATACGACTCACTATAGGGGAATTGTGAGCGGATAACAATTCCCCTCTAGAAATAATTTTGTTTAACTTTAAGAAGGAGA
Nde I

STag Nsp V

HisTag

Bgl II

TATACATATGCACCATCATCATCATCATTCTTCTGGTCTGGTGCCACGCGGTTCTGGTATGAAAGAAACCGCTGCTGCTAAATTCGAACGCCAGCACATGGACAGCCCAGATCTG
MetHisHisHisHisHisHisSerSerGlyLeuValProArgGlySerGlyMetLysGluThrAlaAlaAlaLysPheGluArgGlnHisMetAspSerProAspLeu
Kpn I

pET-30a(+)

Nco I

thrombin
EcoR V BamH I EcoR I Sac I
Sal I Hind III

Eag I
Not I

Xho I

HisTag

GGTACCGACGACGACGACAAGGCCATGGCTGATATCGGATCCGAATTCGAGCTCCGTCGACAAGCTTGCGGCCGCACTCGAGCACCACCACCACCACCACTGAGATCCGGCTGCTAA
GlyThrAspAspAspAspLysAlaMetAlaAspIleGlySerGluPheGluLeuArgArgGlnAlaCysGlyArgThrArgAlaProProProProProLeuArgSerGlyCysEnd
enterokinase
pET-30b(+)

...GCGATATCGGATCCGAATTCGAGCTCCGTCGACAAGCTTGCGGCCGCACTCGAGCACCACCACCACCACCACTGA...
AlaIleSerAspProAsnSerSerSerValAspLysLeuAlaAlaAlaLeuGluHisHisHisHisHisHisEnd

pET-30c(+)

...GGATATCTGTGGATCCGAATTCGAGCTCCGTCGACAAGCTTGCGGCCGCACTCGAGCACCACCACCACCACCACTGAGATCCGGCTGCTAA...
...GlyTyrLeuTrpIleArgIleArgAlaProSerThrSerLeuArgProHisSerSerThrThrThrThrThrThrGluIleArgLeuLeu...
Bpu1102 I

T7 terminator

CAAAGCCCGAAAGGAAGCTGAGTTGGCTGCTGCCACCGCTGAGCAATAACTAGCATAACCCCTTGGGGCCTCTAAACGGGTCTTGAGGGGTTTTTTG
T7 terminator primer #69337-3

pET-30a-c(+) cloning/expression region

Novagen ORDERING 800-526-7319

TECHNICAL SUPPORT

800-207-0144

pET-30a(+) Restriction Sites

Enzyme
AccI
AceIII

# Sites Locations
2
180
3047
7
943
1671
3229 5020
AciI
75
AflIII
2
1176 3277
AluI
22
AlwI
13
Alw21I
7
159
190
3095 3595
Alw44I
3
1156 3091
AlwNI
1
3693
ApaI
1
1387
ApaBI
1
860
ApoI
7
192
270
4982 4993
AvaI
2
158
4351
AvaII
5
1728 2104
BamHI
1
198
BanI
10
234
310
1096 1815
BanII
6
190
560
5255
BbsI
4
1322 1661
BbvI
25
BccI
14
Bce83I
6
21
1990
3907
BcefI
6
695
1036
5206
BcgI
8
160
194
2036 2854
BclI
1
1190
BfaI
6
70
385
5331
BglI
1
2240
BglII
1
241
BmgI
1
1385
BpmI
4
1014 1503
Bpu10I
2
2383 4496
Bpu1102I 1
80
BsaAI
2
3029 5180
BsaBI
3
449
459
BsaHI
5
499
520
BsaJI
10
57
212
2249 3437
BsaWI
7
2
1495
3630 4614
BsaXI
2
1835 5128
BsbI
2
2993 5087
BscGI
11
BsgI
3
1027 1227
BsiI
1
3450
BsiEI
5
169
1961
BslI
26
BsmI
2
4363 4440
BsmAI
6
873
1278
4495
BsmBI
3
1791 2918
BsmFI
4
637
2178
BsoFI
43
Bsp24I
10
466
498
1351 3770
Bsp1286I 12
BspEI
2
2
2466
BspGI
1
2803
BspLU11I 1
3277
BsrI
21
BsrBI
4
405
3210
BsrDI
2
1223 1589
BsrFI
7
486
495
4433 5281
BssHII
1
1587

2002

676

2786

1160

2927

2271

3591

1451

4092

4276

2192

2283

2562

498
1945
574

519
2071
1387

633
5217
4134

2035

2395

2160

3368

3666

1663

3779

4798

1468
2888

1502

2002

2291

3772

2137

2474
634
613
4350
1998

4079

2804

1133
619
4351
2466

1816
1811
4752
3483

2437
3193

3617

4479

1404

1791

2918

4495
2548

5395

1017
3802

1049
3948

4878

5324

862

2074

1319
3980

2234

TB095 12/98
Enzyme
Bst1107I
BstEII
BstXI
BstYI

# Sites
1
1
3
9

Cac8I
CjeI
CjePI
ClaI
CviJI
CviRI
DdeI
DpnI
DraIII
DrdI
DrdII
DsaI
EaeI
EagI
EarI
EciI
Eco47III
Eco57I
EcoNI
EcoO109I
EcoRI
EcoRII

40
24
18
2
85
31
11
23
1
3
2
3
4
1
3
3
3
1
2
3
1
9

EcoRV
FauI
FokI

1
17
9

FspI
GdiII
HaeI

1
4
7

HaeII
HaeIII
HgaI
HgiEII
HhaI
Hin4I
HincII
HindIII
HinfI
HpaI
HphI
KpnI
MaeII
MaeIII
MboII
MluI
MmeI

14
24
11
2
46
4
2
1
18
1
16
1
14
16
13
1
7

MnlI
MseI
MslI

25
25
6

MspI
MspA1I

29
9

MwoI
NarI
NciI
NcoI
NdeI
NgoAIV
NlaIII
NlaIV
NotI
NruI
NsiI

39
4
12
1
1
4
26
23
1
1
2

Locations
3048
1357
978
1107
132
198
2469 3918

453

5180
2970
899
212
166
166
794
953
581
3825
711
53
192
899
3424
206
1222
2822
2258
166
217
3755

1230
241
3929

740
4728

Enzyme
NspI
NspV
Pfl1108I
PflMI
PleI

# Sites
4
1
1
3
9

PshAI
Psp5II
Psp1406I
PvuI
PvuII
RcaI
RsaI
SacI
SalI
SapI
Sau96I
Sau3AI
ScrFI
SfaNI
SfcI
SgfI
SgrAI
SmaI
SphI
SspI
StyI
TaqI
TaqII

1
1
4
1
3
3
4
1
1
1
14
23
21
23
4
1
1
1
1
2
2
17
6

2636

TfiI

3303

ThaI
TseI
Tsp45I

36
25
7

Tsp509I
Tth111I
Tth111II

21
1
8

UbaJI
VspI
XbaI
XcmI
XhoI
XmnI

18
5
1
3
1
2

1952

4170

3385
5185
613
484

5135
2249
616

3161
3351
2082

4292
3497
2531

4391
609

2283

1214
3437

1754
4367

1850

1811
4724

1231
2963

2496
4117

2558
4723

484
904
4566

616
2225

1850
3292

774

3863

203
181
173

1075
1682

4165

3303

4707

1682
238

1176
3492
4686

3676
5157

4121

4315

4677

1228
2850

1516

1546

2264

2459

84
2868

283
2987

1206
3619

1776
3864

1869

499

520

634

1816

212
346
486

2074

2234

5281

166
4136
4329

4595

Locations
651
2622
268
2063
260
758
433
725
3656 4711
2021
2283
838
2206
4479
1776 1869
574
3997
236
1323
190
179
3161

2914

3281

4742
812
5115

1608
5123

2602

4965

2868
4872
3083

4314

3733

5399

418
4479
495
4353
651
4404
57

3542

1084
5084
1855
4390

1302

1975

3179

4733

2157
4446

2327
4618

2831
4709

3252

1357
4626

2185
5353

2716

2929

3024

3022
1015
3906

1708
4315

2738
4442

3867

3874

1861

1920

4678

4867

1548

1566

433
384
1032
158
2835

4972
212

4868

Enzymes that do not cut pET-30a(+):

AatII
AflII
AgeI
AscI
BaeI
BsaI
BseRI
BspMI
Bsu36I
DraI
Eam1105I FseI
MunI
NheI
PacI
PmeI
PstI
RleAI
RsrII
SacII
SexAI
SfiI
SnaBI
SpeI
Sse8387I StuI
SunI
SwaI

Novagen FAX 608-238-1388 E-MAIL novatech@novagen.com

3171

AvrII
BsrGI
MscI
PmlI
ScaI
SrfI