2024年3月10日发(作者：仝名)
pET-32a-c(+) Vectors
pET-32a DNA
pET-32b DNA
pET-32c DNA
Cat. No.
69015-3
69016-3
69017-3
TB122 12/98
The pET-32 series is designed for cloning and high-level expression of peptide sequences fused
with the 109aa Trx•Tag™ thioredoxin protein (1). Cloning sites are available for producing fusion
proteins also containing cleavable His•Tag
®
and S•Tag™ sequences for detection and purification.
Unique sites are shown on the circle map. Note that the sequence is numbered by the pBR322 con-
vention, 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 corre-
sponds to the coding strand. Therefore, single-stranded sequencing should be performed using the
T7 terminator primer (Cat. No. 69337-3).
ie, E.R., DiBlasio, E.A., Kovacic, S., Grant, K.L., Schendel, P.F. and McCoy, J.M. (1993) Bio/Technology
11, 187–193.
pET-32a(+) sequence landmarks
A
p
(
4
4
4
5
-
5
3
0
2
)
T7 promoter
T7 transcription start
Trx•Tag coding sequence
His•Tag coding sequence
S•Tag coding sequence
Multiple cloning sites
(NcoI - XhoI)
His•Tag coding sequence
T7 terminator
lacIcoding sequence
pBR322 origin
blacoding sequence
f1 origin
764-780
763
366-692
327-344
249-293
158-217
140-157
26-72
1171-2250
3684
4445-5302
5434-5889
Bpu1102 I
(80)
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)
Bgl II
(241)
Kpn I
(238)
Nsp V
(268)
Msc I
(351)
Rsr II
(589)
Dra III
(5658)
Sca I
(4995)
Pvu I
(4885)
4
-
5
8
8
9
)
(
5
4
3
n
i
g
o
r
i
f
1
t
r
x
A
(
3
6
6
-
6
9
2
)
Xba I
(729)
SgrA I
(840)
Sph I
(996)
EcoN I
(1056)
ApaB I
(1205)
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.
Pst I
(4760)
Bsa I
(4576)
Eam1105 I
(4357)–
l
a
c
I
(
1
1
7
1
-
2
2
5
0
)
pET-32a(+)
(5900bp)
Mlu I
(1521)
Bcl I
(1535)
BstE II
(1702)
Bmg I
(1730)
Apa I
(1732)
BssH II
(1932)
AlwN I
(4038)
BspLU11 I
(3622)
Sap I
(3506)
Bst1107 I
(3393)
Tth111 I
(3367)
BspG I
(3148)
lac operator
rbs
Xba
I
TAATACGACTCACTATAGGGGAATTGTGAGCGGATAACAATTCCCCTCTAGAAATAATTTTGTTTAACTTTAAGAAGGAGA
Trx•Tag
His•Tag
Msc
I
T7 promoter
CTGGCCGGTTCTGGTTCTGGCCATATGCACCATCATCATCATCATTCTTCTGGTCTGGTGCCACGCGGTTCT
MetSer LeuAlaGlySerGlySerGlyHisMetHisHisHisHisHisHisSerSerGlyLeuValProArgGlySer
S•Tag primer #69945-3
thrombin
S•Tag
Nsp
V
Bgl
II
Kpn
I
GGTATGAAAGAAACCGCTGCTGCTAAATTCGAACGCCAGCACATGGACAGCCCAGATCTGGGTACCGACGACGACGACAAG
GlyMetLysGluThrAlaAlaAlaLysPheGluArgGlnHisMetAspSerProAspLeuGlyThrAspAspAspAspLys
enterokinase
pET-32a(+)
Eag
I
Ava
I
His•Tag
Nco
I
Eco
R V
Bam
H I
Eco
R I
Sac
I
Sal
I
Hin
d III
Not
I
Xho
I
GCCATGGCTGATATCGGATCCGAATTCGAGCTCCGTCGACAAGCTTGCGGCCGCACTCGAGCACCACCACCACCACCACTGAGATCCGGCTGCTAA
AlaMetAlaAspIleGlySerGluPheGluLeuArgArgGlnAlaCysGlyArgThrArgAlaProProProProProLeuArgSerGlyCysEnd
GCCATGGCGATATCGGATCCGAATTCGAGCTCCGTCGACAAGCTTGCGGCCGCACTCGAGCACCACCACCACCACCACTGAGATCCGGCTGCTAA pET-32b(+)
AlaMetAlaIleSerAspProAsnSerSerSerValAspLysLeuAlaAlaAlaLeuGluHisHisHisHisHisHisEnd
GCCATGGGATATCTGTGGATCCGAATTCGAGCTCCGTCGACAAGCTTGCGGCCGCACTCGAGCACCACCACCACCACCACTGAGATCCGGCTGCTAA pET-32c(+)
AlaMetGlyTyrLeuTrpIleArgIleArgAlaProSerThrSerLeuArgProHisSerSerThrThrThrThrThrThrGluIleArgLeuLeuThr
T7 terminator
Bpu
1102 I
CAAAGCCCGAAAGGAAGCTGAGTTGGCTGCTGCCACCGCTGAGCAATAACTAGCATAACCCCTTGGGGCCTCTAAACGGGTCTTGAGGGGTTTTTTG
LysProGluArgLysLeuSerTrpLeuLeuProProLeuSerAsnAsnEnd
pET-32a-c(+) cloning/expression region
Novagen
•
ORDERING
800-526-7319
•
TECHNICALSUPPORT
T7 terminator primer #69337-3
o
r
i
(
6
8
4
)
Hpa I
(2027)
3
PshA I
(2366)
Psp5 II
(2628)
800-207-0144
pET-32a(+) Restriction Sites
Enzyme
AccI
AceIII
# SitesLocations
21803392
812882016
35744814
AciI85
AflIII215213622
AluI25
AlwI17
Alw21I10159190
26163440
Alw44I54101501
AlwNI14038
ApaI11732
ApaBI11205
ApoI5192270
AvaI1158
AvaII85892073
29074653
BamHI1198
BanI11
BanII5190905
BbsI416672006
BbvI29
BccI14
Bce83I7212335
42525120
BcefI510401381
BcgI14
BclI11535
BfaI770730
47055809
BglI225854635
BglII1241
BmgI11730
BpmI513591848
Bpu10I12728
Bpu1102I180
BsaI14576
BsaAI233745658
BsaBI3794804
BsaHI6844865
5052
BsaJI957212
9642156
BsaWI721840
39754806
BsaXI221805606
BsbI233385565
BscGI14
BsgI313721572
BsiI237955179
BsiEI61692306
5034
BslI25
BsmAI712181623
45765352
BsmBI221363263
BsmFI49822523
BsoFI50
Bsp24I10811843
16964115
Bsp1286I14
BspEI222811
BspGI13148
BspLU11I13622
BsrI25
BsrBI47503555
BsrDI415681934
BsrFI8363831
25794595
BssHII11932
Bst1107I13393
BstEII11702
2347
5498
31313272
Enzyme
BstXI
BstYI
Cac8I
CjeI
CjePI
ClaI
CviJI
CviRI
DdeI
DpnI
DraI
DraIII
DrdI
DrdII
DsaI
EaeI
EagI
Eam1105I
EarI
EciI
Eco47III
Eco57I
EcoNI
EcoO109I
EcoRI
EcoRII
EcoRV
FauI
FokI
FspI
GdiII
HaeI
HaeII
HaeIII
HgaI
HgiEII
HhaI
Hin4I
HincII
HindIII
HinfI
HpaI
HphI
KpnI
MaeII
MaeIII
MboII
MluI
MmeI
MnlI
MscI
MseI
MslI
MspI
MspA1I
MwoI
NarI
NciI
NcoI
NdeI
NgoAIV
NlaIII
NlaIV
NotI
NspI
NspV
# Sites
3
12
39
26
18
2
90
28
11
32
3
1
3
3
4
7
1
1
3
4
3
2
1
3
1
9
1
17
13
2
6
7
14
27
13
3
46
4
2
1
16
1
18
1
15
18
15
1
3
28
1
28
9
32
10
41
4
12
1
2
4
26
27
1
4
1
Locations
5
Enzyme
Pfl1108I
PflMI
PleI
# Sites
2
2
10
Locations
24084533
2601103
466778
35164001
2366
2628
11832551
5443
4760
4885
21212214
9194342
236642
589
190
179
3506
1070
4504
1157
5593
TB122 12/98
1953
5601
508798
PshAI1
Psp5II1
Psp1406I6
PstI
PvuI
PvuII
RcaI
RsaI
RsrII
SacI
SalI
SapI
Sau96I
Sau3AI
ScaI
ScrFI
SfaNI
SfcI
SgrAI
SphI
SspI
StyI
TaqI
TaqII
TfiI
1
1
3
3
5
1
1
1
1
19
32
1
21
21
5
1
1
2
2
18
10
6
35
29
8
18
1
6
21
4
1
3
1
3
294747415114
414
3940
3436
1021
5101
3936
1505
5186
5182
1796
2449
4875
5460
2537
5471
2628
919
2380
1732
2740
5733
2505
2008
3713
4124
4011
5684
263641174370
4381
5658
3315
357
212
166
2195
166
4515
1139
1298
926
4170
1056
53
192
367
2156
206
4400
3730
1244
469
349
4903
5092
5613
5663
958
365
3213
5350
5
2594
829961
3506
3696
2427
5182
954
495
3648
5310
3842
2876
4995
4670
2628
1244
3769
1559
3782
2099
763
840
996
5319
57
429
4863
566
3597
38874
5450
212
1429
5048
2200
1647
5201
2502
2320
5218
2672
3524
5562
3176
248231494585
2603
166
4903
217
3648
4737
365
351
4100
829
1249
961
2570
2195
3637
ThaI
TseI
Tsp45I
Tsp509I
Tth111I
Tth111II
1702
4771
3367
1360
4251
778
729
1377
158
388
2530
4982
3061
5831
32743369
2819
979
469
2594
2343
2219
1478
496
3782
2811
2161
958
3828
419
203
181
173
2027
238
1119
1420
2027
4208
45144588
UbaJI
VspI
XbaI
XcmI
XhoI
XmnI
2206
1893
3180
2265
1911
5114
4687
2782
353839624885
1521
3837
351
1573
3195
84
3213
844
212
346
831
40215635
3
Enzymes that do not cut pET-32a(+):
AatIIAflIIAgeIAscI
BaeIBseRIBsmIBspMI
Bsu36IFseIMunINheI
NsiIPacIPmeIPmlI
SacIISexAISfiISgfI
SnaBISpeISrfISse8387I
SunISwaI
2609
5285
2121
4209
2161
2804
AvrII
BsrGI
NruI
RleAI
SmaI
StuI
2893
1362
4147
5873
1394
4293
1664
4325
1861
4767
283
3332
865
1891
4926
1551
3964
979
2214
5150
5356
4576
840
5759
5802
4750
1207
691
241925795759
2419
166
996
268
296732593626
Novagen
•
FAX
608-238-1388
•
E
-
novatech@
2024年3月10日发(作者：仝名)
pET-32a-c(+) Vectors
pET-32a DNA
pET-32b DNA
pET-32c DNA
Cat. No.
69015-3
69016-3
69017-3
TB122 12/98
The pET-32 series is designed for cloning and high-level expression of peptide sequences fused
with the 109aa Trx•Tag™ thioredoxin protein (1). Cloning sites are available for producing fusion
proteins also containing cleavable His•Tag
®
and S•Tag™ sequences for detection and purification.
Unique sites are shown on the circle map. Note that the sequence is numbered by the pBR322 con-
vention, 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 corre-
sponds to the coding strand. Therefore, single-stranded sequencing should be performed using the
T7 terminator primer (Cat. No. 69337-3).
ie, E.R., DiBlasio, E.A., Kovacic, S., Grant, K.L., Schendel, P.F. and McCoy, J.M. (1993) Bio/Technology
11, 187–193.
pET-32a(+) sequence landmarks
A
p
(
4
4
4
5
-
5
3
0
2
)
T7 promoter
T7 transcription start
Trx•Tag coding sequence
His•Tag coding sequence
S•Tag coding sequence
Multiple cloning sites
(NcoI - XhoI)
His•Tag coding sequence
T7 terminator
lacIcoding sequence
pBR322 origin
blacoding sequence
f1 origin
764-780
763
366-692
327-344
249-293
158-217
140-157
26-72
1171-2250
3684
4445-5302
5434-5889
Bpu1102 I
(80)
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)
Bgl II
(241)
Kpn I
(238)
Nsp V
(268)
Msc I
(351)
Rsr II
(589)
Dra III
(5658)
Sca I
(4995)
Pvu I
(4885)
4
-
5
8
8
9
)
(
5
4
3
n
i
g
o
r
i
f
1
t
r
x
A
(
3
6
6
-
6
9
2
)
Xba I
(729)
SgrA I
(840)
Sph I
(996)
EcoN I
(1056)
ApaB I
(1205)
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.
Pst I
(4760)
Bsa I
(4576)
Eam1105 I
(4357)–
l
a
c
I
(
1
1
7
1
-
2
2
5
0
)
pET-32a(+)
(5900bp)
Mlu I
(1521)
Bcl I
(1535)
BstE II
(1702)
Bmg I
(1730)
Apa I
(1732)
BssH II
(1932)
AlwN I
(4038)
BspLU11 I
(3622)
Sap I
(3506)
Bst1107 I
(3393)
Tth111 I
(3367)
BspG I
(3148)
lac operator
rbs
Xba
I
TAATACGACTCACTATAGGGGAATTGTGAGCGGATAACAATTCCCCTCTAGAAATAATTTTGTTTAACTTTAAGAAGGAGA
Trx•Tag
His•Tag
Msc
I
T7 promoter
CTGGCCGGTTCTGGTTCTGGCCATATGCACCATCATCATCATCATTCTTCTGGTCTGGTGCCACGCGGTTCT
MetSer LeuAlaGlySerGlySerGlyHisMetHisHisHisHisHisHisSerSerGlyLeuValProArgGlySer
S•Tag primer #69945-3
thrombin
S•Tag
Nsp
V
Bgl
II
Kpn
I
GGTATGAAAGAAACCGCTGCTGCTAAATTCGAACGCCAGCACATGGACAGCCCAGATCTGGGTACCGACGACGACGACAAG
GlyMetLysGluThrAlaAlaAlaLysPheGluArgGlnHisMetAspSerProAspLeuGlyThrAspAspAspAspLys
enterokinase
pET-32a(+)
Eag
I
Ava
I
His•Tag
Nco
I
Eco
R V
Bam
H I
Eco
R I
Sac
I
Sal
I
Hin
d III
Not
I
Xho
I
GCCATGGCTGATATCGGATCCGAATTCGAGCTCCGTCGACAAGCTTGCGGCCGCACTCGAGCACCACCACCACCACCACTGAGATCCGGCTGCTAA
AlaMetAlaAspIleGlySerGluPheGluLeuArgArgGlnAlaCysGlyArgThrArgAlaProProProProProLeuArgSerGlyCysEnd
GCCATGGCGATATCGGATCCGAATTCGAGCTCCGTCGACAAGCTTGCGGCCGCACTCGAGCACCACCACCACCACCACTGAGATCCGGCTGCTAA pET-32b(+)
AlaMetAlaIleSerAspProAsnSerSerSerValAspLysLeuAlaAlaAlaLeuGluHisHisHisHisHisHisEnd
GCCATGGGATATCTGTGGATCCGAATTCGAGCTCCGTCGACAAGCTTGCGGCCGCACTCGAGCACCACCACCACCACCACTGAGATCCGGCTGCTAA pET-32c(+)
AlaMetGlyTyrLeuTrpIleArgIleArgAlaProSerThrSerLeuArgProHisSerSerThrThrThrThrThrThrGluIleArgLeuLeuThr
T7 terminator
Bpu
1102 I
CAAAGCCCGAAAGGAAGCTGAGTTGGCTGCTGCCACCGCTGAGCAATAACTAGCATAACCCCTTGGGGCCTCTAAACGGGTCTTGAGGGGTTTTTTG
LysProGluArgLysLeuSerTrpLeuLeuProProLeuSerAsnAsnEnd
pET-32a-c(+) cloning/expression region
Novagen
•
ORDERING
800-526-7319
•
TECHNICALSUPPORT
T7 terminator primer #69337-3
o
r
i
(
6
8
4
)
Hpa I
(2027)
3
PshA I
(2366)
Psp5 II
(2628)
800-207-0144
pET-32a(+) Restriction Sites
Enzyme
AccI
AceIII
# SitesLocations
21803392
812882016
35744814
AciI85
AflIII215213622
AluI25
AlwI17
Alw21I10159190
26163440
Alw44I54101501
AlwNI14038
ApaI11732
ApaBI11205
ApoI5192270
AvaI1158
AvaII85892073
29074653
BamHI1198
BanI11
BanII5190905
BbsI416672006
BbvI29
BccI14
Bce83I7212335
42525120
BcefI510401381
BcgI14
BclI11535
BfaI770730
47055809
BglI225854635
BglII1241
BmgI11730
BpmI513591848
Bpu10I12728
Bpu1102I180
BsaI14576
BsaAI233745658
BsaBI3794804
BsaHI6844865
5052
BsaJI957212
9642156
BsaWI721840
39754806
BsaXI221805606
BsbI233385565
BscGI14
BsgI313721572
BsiI237955179
BsiEI61692306
5034
BslI25
BsmAI712181623
45765352
BsmBI221363263
BsmFI49822523
BsoFI50
Bsp24I10811843
16964115
Bsp1286I14
BspEI222811
BspGI13148
BspLU11I13622
BsrI25
BsrBI47503555
BsrDI415681934
BsrFI8363831
25794595
BssHII11932
Bst1107I13393
BstEII11702
2347
5498
31313272
Enzyme
BstXI
BstYI
Cac8I
CjeI
CjePI
ClaI
CviJI
CviRI
DdeI
DpnI
DraI
DraIII
DrdI
DrdII
DsaI
EaeI
EagI
Eam1105I
EarI
EciI
Eco47III
Eco57I
EcoNI
EcoO109I
EcoRI
EcoRII
EcoRV
FauI
FokI
FspI
GdiII
HaeI
HaeII
HaeIII
HgaI
HgiEII
HhaI
Hin4I
HincII
HindIII
HinfI
HpaI
HphI
KpnI
MaeII
MaeIII
MboII
MluI
MmeI
MnlI
MscI
MseI
MslI
MspI
MspA1I
MwoI
NarI
NciI
NcoI
NdeI
NgoAIV
NlaIII
NlaIV
NotI
NspI
NspV
# Sites
3
12
39
26
18
2
90
28
11
32
3
1
3
3
4
7
1
1
3
4
3
2
1
3
1
9
1
17
13
2
6
7
14
27
13
3
46
4
2
1
16
1
18
1
15
18
15
1
3
28
1
28
9
32
10
41
4
12
1
2
4
26
27
1
4
1
Locations
5
Enzyme
Pfl1108I
PflMI
PleI
# Sites
2
2
10
Locations
24084533
2601103
466778
35164001
2366
2628
11832551
5443
4760
4885
21212214
9194342
236642
589
190
179
3506
1070
4504
1157
5593
TB122 12/98
1953
5601
508798
PshAI1
Psp5II1
Psp1406I6
PstI
PvuI
PvuII
RcaI
RsaI
RsrII
SacI
SalI
SapI
Sau96I
Sau3AI
ScaI
ScrFI
SfaNI
SfcI
SgrAI
SphI
SspI
StyI
TaqI
TaqII
TfiI
1
1
3
3
5
1
1
1
1
19
32
1
21
21
5
1
1
2
2
18
10
6
35
29
8
18
1
6
21
4
1
3
1
3
294747415114
414
3940
3436
1021
5101
3936
1505
5186
5182
1796
2449
4875
5460
2537
5471
2628
919
2380
1732
2740
5733
2505
2008
3713
4124
4011
5684
263641174370
4381
5658
3315
357
212
166
2195
166
4515
1139
1298
926
4170
1056
53
192
367
2156
206
4400
3730
1244
469
349
4903
5092
5613
5663
958
365
3213
5350
5
2594
829961
3506
3696
2427
5182
954
495
3648
5310
3842
2876
4995
4670
2628
1244
3769
1559
3782
2099
763
840
996
5319
57
429
4863
566
3597
38874
5450
212
1429
5048
2200
1647
5201
2502
2320
5218
2672
3524
5562
3176
248231494585
2603
166
4903
217
3648
4737
365
351
4100
829
1249
961
2570
2195
3637
ThaI
TseI
Tsp45I
Tsp509I
Tth111I
Tth111II
1702
4771
3367
1360
4251
778
729
1377
158
388
2530
4982
3061
5831
32743369
2819
979
469
2594
2343
2219
1478
496
3782
2811
2161
958
3828
419
203
181
173
2027
238
1119
1420
2027
4208
45144588
UbaJI
VspI
XbaI
XcmI
XhoI
XmnI
2206
1893
3180
2265
1911
5114
4687
2782
353839624885
1521
3837
351
1573
3195
84
3213
844
212
346
831
40215635
3
Enzymes that do not cut pET-32a(+):
AatIIAflIIAgeIAscI
BaeIBseRIBsmIBspMI
Bsu36IFseIMunINheI
NsiIPacIPmeIPmlI
SacIISexAISfiISgfI
SnaBISpeISrfISse8387I
SunISwaI
2609
5285
2121
4209
2161
2804
AvrII
BsrGI
NruI
RleAI
SmaI
StuI
2893
1362
4147
5873
1394
4293
1664
4325
1861
4767
283
3332
865
1891
4926
1551
3964
979
2214
5150
5356
4576
840
5759
5802
4750
1207
691
241925795759
2419
166
996
268
296732593626
Novagen
•
FAX
608-238-1388
•
E
-
novatech@