pET-32a-c(+) Vectors

TB122 12/98
The pET-32 series is designed for cloning and high-level expression of peptide sequences fused
with the 109aa TrxTag thioredoxin protein (1). Cloning sites are available for producing fusion
proteins also containing cleavable HisTag and STag sequences for detection and purification.
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 circle 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.
69015-3
69016-3
69017-3

pET-32a DNA
pET-32b DNA
pET-32c DNA

1. LaVallie, E.R., DiBlasio, E.A., Kovacic, S., Grant, K.L., Schendel, P.F. and McCoy, J.M. (1993) Bio/Technology
11, 187193.
Ava I(158)
Xho I(158)
Eag I(166)
Not I(166)
Hind III(173)
Sal I(179)
Sac I(190)
EcoR I(192)
BamH I(198)
EcoR V(206)
Nco I(212)

pET-32a(+) sequence landmarks

764-780
763
366-692
327-344
249-293

Bpu1102 I(80)

158-217
140-157
26-72
1171-2250
3684
4445-5302
5434-5889

Bgl II(241)
Kpn I(238)
Nsp V(268)
Msc I(351)

Dra III(5658)

f1

-5889)
(5434
gin
ori

Sca I(4995)

Pst I(4760)
Bsa I(4576)

Xba I(729)
SgrA I(840)

Pvu I(4885)

Sph I(996)
EcoN I(1056)
ApaB I(1205)

Eam1105 I(4357)

lacI (1171
-22
50)

The maps for pET-32b(+) and pET-32c(+)

are the same as pET-32a(+) (shown) with
the following exceptions: pET-32b(+) is a
5899bp plasmid; subtract 1bp from each site
beyond BamH I at 198. pET-32c(+) is a
5901bp plasmid; add 1bp to each site
beyond BamH I at 198 except for EcoR V,
which cuts at 209.

Rsr II(589)

trxA
(36
669
2

Ap (4
445
-53
02
)

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

pET-32a(+)
(5900bp)

AlwN I(4038)

Mlu I(1521)
Bcl I(1535)
BstE II(1702)
Bmg I(1730)
Apa I(1732)

or

BssH II(1932)

i(

68

Hpa I(2027)

4)

BspLU11 I(3622)
Sap I(3506)
Bst1107 I(3393)
Tth111 I(3367)
BspG I(3148)
T7 promoter

lac operator

PshA I(2366)
Psp5 II(2628)

rbs

Xba I

TAATACGACTCACTATAGGGGAATTGTGAGCGGATAACAATTCCCCTCTAGAAATAATTTTGTTTAACTTTAAGAAGGAGA
TrxTag

HisTag

Msc I

TATACATATGAGC...315bp...CTGGCCGGTTCTGGTTCTGGCCATATGCACCATCATCATCATCATTCTTCTGGTCTGGTGCCACGCGGTTCT
MetSer 105aa...LeuAlaGlySerGlySerGlyHisMetHisHisHisHisHisHisSerSerGlyLeuValProArgGlySer
STag

STag primer #69945-3

Bgl II
Kpn I

Nsp V

thrombin

GGTATGAAAGAAACCGCTGCTGCTAAATTCGAACGCCAGCACATGGACAGCCCAGATCTGGGTACCGACGACGACGACAAG
GlyMetLysGluThrAlaAlaAlaLysPheGluArgGlnHisMetAspSerProAspLeuGlyThrAspAspAspAspLys
Nco I

pET-32a(+)
EcoR V BamH I EcoR I Sac I

Sal I

Hind III

Eag I
Not I

Ava I
Xho I

enterokinase

HisTag

GCCATGGCTGATATCGGATCCGAATTCGAGCTCCGTCGACAAGCTTGCGGCCGCACTCGAGCACCACCACCACCACCACTGAGATCCGGCTGCTAA
AlaMetAlaAspIleGlySerGluPheGluLeuArgArgGlnAlaCysGlyArgThrArgAlaProProProProProLeuArgSerGlyCysEnd
GCCATGGCGATATCGGATCCGAATTCGAGCTCCGTCGACAAGCTTGCGGCCGCACTCGAGCACCACCACCACCACCACTGAGATCCGGCTGCTAA
AlaMetAlaIleSerAspProAsnSerSerSerValAspLysLeuAlaAlaAlaLeuGluHisHisHisHisHisHisEnd

pET-32b(+)

GCCATGGGATATCTGTGGATCCGAATTCGAGCTCCGTCGACAAGCTTGCGGCCGCACTCGAGCACCACCACCACCACCACTGAGATCCGGCTGCTAA pET-32c(+)
AlaMetGlyTyrLeuTrpIleArgIleArgAlaProSerThrSerLeuArgProHisSerSerThrThrThrThrThrThrGluIleArgLeuLeuThr
T7 terminator

Bpu1102 I

CAAAGCCCGAAAGGAAGCTGAGTTGGCTGCTGCCACCGCTGAGCAATAACTAGCATAACCCCTTGGGGCCTCTAAACGGGTCTTGAGGGGTTTTTTG
LysProGluArgLysLeuSerTrpLeuLeuProProLeuSerAsnAsnEnd
T7 terminator primer #69337-3

pET-32a-c(+) cloning/expression region

Novagen ORDERING 800-526-7319

TECHNICAL SUPPORT

800-207-0144

pET-32a(+) Restriction Sites

Enzyme
AccI
AceIII

# Sites Locations
2
180
3392
8
1288 2016
3574 4814
AciI
85
AflIII
2
1521 3622
AluI
25
AlwI
17
Alw21I
10
159
190
2616 3440
Alw44I
5
410
1501
AlwNI
1
4038
ApaI
1
1732
ApaBI
1
1205
ApoI
5
192
270
AvaI
1
158
AvaII
8
589
2073
2907 4653
BamHI
1
198
BanI
11
BanII
5
190
905
BbsI
4
1667 2006
BbvI
29
BccI
14
Bce83I
7
21
2335
4252 5120
BcefI
5
1040 1381
BcgI
14
BclI
1
1535
BfaI
7
70
730
4705 5809
BglI
2
2585 4635
BglII
1
241
BmgI
1
1730
BpmI
5
1359 1848
Bpu10I
1
2728
Bpu1102I 1
80
BsaI
1
4576
BsaAI
2
3374 5658
BsaBI
3
794
804
BsaHI
6
844
865
5052
BsaJI
9
57
212
964
2156
BsaWI
7
2
1840
3975 4806
BsaXI
2
2180 5606
BsbI
2
3338 5565
BscGI
14
BsgI
3
1372 1572
BsiI
2
3795 5179
BsiEI
6
169
2306
5034
BslI
25
BsmAI
7
1218 1623
4576 5352
BsmBI
2
2136 3263
BsmFI
4
982
2523
BsoFI
50
Bsp24I
10
811
843
1696 4115
Bsp1286I 14
BspEI
2
2
2811
BspGI
1
3148
BspLU11I 1
3622
BsrI
25
BsrBI
4
750
3555
BsrDI
4
1568 1934
BsrFI
8
363
831
2579 4595
BssHII
1
1932
Bst1107I 1
3393
BstEII
1
1702

2347
5498

414
3940
3436

3131

1021
5101
3936

3272

1505
5186
5182

1796

5460

5471

2449
4875

2537

2628

919
2380

1732
2740

5733

2505

3713

4011

2008

4124

5684

2636

2482

4117

3149

4370

1478

2161

469
2594
2343

496
3782
2811

958
3828

2782

1749

3962

2136

2893

5873

1362
4147

1394
4293

5356
4576
840
5759

5802
4750
1207

Enzyme
BstXI
BstYI
Cac8I
CjeI
CjePI
ClaI
CviJI
CviRI
DdeI
DpnI
DraI
DraIII
DrdI
DrdII
DsaI
EaeI

# Sites
3
12
39
26
18
2
90
28
11
32
3
1
3
3
4
7

EagI
Eam1105I
EarI
EciI
Eco47III
Eco57I
EcoNI
EcoO109I
EcoRI
EcoRII

1
1
3
4
3
2
1
3
1
9

EcoRV
FauI
FokI
FspI
GdiII

1
17
13
2
6

HaeI

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

14
27
13
3
46
4
2
1
16
1
18
1
15
18
15
1
3
28
1
28
9

MspI
MspA1I

32
10

MwoI
NarI
NciI
NcoI
NdeI
NgoAIV
NlaIII
NlaIV
NotI
NspI
NspV

41
4
12
1
2
4
26
27
1
4
1

4585

2819
979

3538

TB122 12/98

4885

3263

1664
4325

2419

Locations
1323 1452

508

4381
5658
3315
357
212
166
2195
166
4515
1139
1298
926
4170
1056
53
192
367
2156
206

2603
166
4903
217
3648

Enzyme
Pfl1108I
PflMI
PleI

1575

PshAI
1
Psp5II
1
Psp1406I 6

798

4400

5092

3730
1244
469
349
4903

5613
5663
958
365

3506
3696
2427
5182

5310
3842
2876

954

2628

495
3648

1244
3769

4737
365
351
4100

2594
829

961

4670

1559
3782

2099

829

961

2195

1249

2570

3637

419

1119

4208

203
181
173

1420
2027

4514

4588

2027

4021

5635

1573
3195

1861
4767

1891
4926

2609
5285

2804

84
3213

283
3332

1551
3964

2121
4209

2214
5150

844

865

979

2161

212
346
831

691
2419

2579

5759

2967

3259

3626

351

166
996
268

PstI
PvuI
PvuII
RcaI
RsaI
RsrII
SacI
SalI
SapI
Sau96I
Sau3AI
ScaI
ScrFI
SfaNI
SfcI
SgrAI
SphI
SspI
StyI
TaqI
TaqII

1
1
3
3
5
1
1
1
1
19
32
1
21
21
5
1
1
2
2
18
10

TfiI

ThaI
TseI
Tsp45I

35
29
8

Tsp509I
Tth111I
Tth111II

18
1
6

UbaJI
VspI
XbaI
XcmI
XhoI
XmnI

21
4
1
3
1
3

Locations
2408 4533
260
1103
466
778
3516 4001
2366
2628
1183 2551
5443
4760
4885
2121 2214
919
4342
236
642
589
190
179
3506

1070
4504

1157
5593

1953
5601

2947

4741

5114

3213
5350
1668

3428

4995

4078

4756

5877

4995

763
840
996
5319
57

3887

429
4863
566
3597

1429
5048
2200

1647
5201
2502

2320
5218
2672

3524
5562
3176

1702
4771

2530
4982

3061
5831

3274

3369

3367
1360
4251

2053

3083

4212

4219

2206

2265

4687

1893

1911

3180

5114

778
729
1377
158
388

5450
212

Enzymes that do not cut pET-32a(+):

AatII
AflII
AgeI
AscI
BaeI
BseRI
BsmI
BspMI
Bsu36I
FseI
MunI
NheI
NsiI
PacI
PmeI
PmlI
SacII
SexAI
SfiI
SgfI
SnaBI
SpeI
SrfI
Sse8387I
SunI
SwaI

238

1521
3837

# Sites
2
2
10

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

AvrII
BsrGI
NruI
RleAI
SmaI
StuI