pET-40b(+)

Bacterial vector for expressing periplasmic DsbC fusion proteins.

Sequence Author: MilliporeSigma (Novagen)

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PaeR7I - PspXI - XhoI (174) 8xHis AvrII (131) T7 terminator DraIII (5948) AsiSI - PvuI (5247) NruI (4904) AcuI (4593) BssSI - BssSαI (4218) PciI (4045) BspQI - SapI (3929) BstZ17I (3816) PflFI - Tth111I (3790) EagI - NotI (182) HindIII (189) SalI (195) Eco53kI (204) SacI (206) EcoRI (208) BamHI (214) EcoRV (222) NcoI (227) SrfI (246) BseRI (250) enterokinase site Acc65I (267) KpnI (271) BglII (274) BstBI (301) MfeI (332) ScaI (341) thrombin site SacII (365) 6xHis SpeI (396) BfuAI - BspMI (600) BsrGI (941) NdeI (1114) RBS XbaI (1152) T7 promoter SgrAI (1263) SphI (1419) BstAPI (1627) MluI (1944) BclI * (1958) NmeAIII (2150) PspOMI (2151) ApaI (2155) BssHII (2355) HpaI (2450) PshAI (2789) BglI (3008) FspI - FspAI (3026) PpuMI (3051) pET-40b(+) 6190 bp
PaeR7I  (174)
1 site
C T C G A G G A G C T C

PaeR7I does not recognize the sequence CTCTCGAG.
PspXI  (174)
1 site
V C T C G A G B B G A G C T C V
XhoI  (174)
1 site
C T C G A G G A G C T C
AvrII  (131)
1 site
C C T A G G G G A T C C
DraIII  (5948)
1 site
C A C N N N G T G G T G N N N C A C

Sticky ends from different DraIII sites may not be compatible.
AsiSI  (5247)
1 site
G C G A T C G C C G C T A G C G
PvuI  (5247)
1 site
C G A T C G G C T A G C
NruI  (4904)
1 site
T C G C G A A G C G C T
AcuI  (4593)
1 site
C T G A A G ( N ) 14 N N G A C T T C ( N ) 14

Cleavage may be enhanced when more than one copy of the AcuI recognition sequence is present.
Sticky ends from different AcuI sites may not be compatible.
After cleavage, AcuI can remain bound to DNA and alter its electrophoretic mobility.
For full activity, add fresh S-adenosylmethionine (SAM).
BssSI  (4218)
1 site
C A C G A G G T G C T C
BssSαI  (4218)
1 site
C A C G A G G T G C T C
PciI  (4045)
1 site
A C A T G T T G T A C A

PciI is inhibited by nonionic detergents.
BspQI  (3929)
1 site
G C T C T T C N C G A G A A G N N N N

Sticky ends from different BspQI sites may not be compatible.
SapI  (3929)
1 site
G C T C T T C N C G A G A A G N N N N

Sticky ends from different SapI sites may not be compatible.
SapI gradually settles in solution, so a tube of SapI should be mixed before removing an aliquot.
BstZ17I  (3816)
1 site
G T A T A C C A T A T G
PflFI  (3790)
1 site
G A C N N N G T C C T G N N N C A G

The 1-base overhangs produced by PflFI may be hard to ligate.
Sticky ends from different PflFI sites may not be compatible.
Tth111I  (3790)
1 site
G A C N N N G T C C T G N N N C A G

The 1-base overhangs produced by Tth111I may be hard to ligate.
Sticky ends from different Tth111I sites may not be compatible.
EagI  (182)
1 site
C G G C C G G C C G G C
NotI  (182)
1 site
G C G G C C G C C G C C G G C G
HindIII  (189)
1 site
A A G C T T T T C G A A
SalI  (195)
1 site
G T C G A C C A G C T G
Eco53kI  (204)
1 site
G A G C T C C T C G A G
SacI  (206)
1 site
G A G C T C C T C G A G
EcoRI  (208)
1 site
G A A T T C C T T A A G
BamHI  (214)
1 site
G G A T C C C C T A G G

After cleavage, BamHI-HF® (but not the original BamHI) can remain bound to DNA and alter its electrophoretic mobility.
EcoRV  (222)
1 site
G A T A T C C T A T A G

EcoRV is reportedly more prone than its isoschizomer Eco32I to delete a base after cleavage.
NcoI  (227)
1 site
C C A T G G G G T A C C
SrfI  (246)
1 site
G C C C G G G C C G G G C C C G
BseRI  (250)
1 site
G A G G A G ( N ) 8 N N C T C C T C ( N ) 8

Sticky ends from different BseRI sites may not be compatible.
BseRI quickly loses activity at 37°C.
Prolonged incubation with BseRI may lead to degradation of the DNA.
Acc65I  (267)
1 site
G G T A C C C C A T G G
KpnI  (271)
1 site
G G T A C C C C A T G G
BglII  (274)
1 site
A G A T C T T C T A G A
BstBI  (301)
1 site
T T C G A A A A G C T T
MfeI  (332)
1 site
C A A T T G G T T A A C
ScaI  (341)
1 site
A G T A C T T C A T G A
SacII  (365)
1 site
C C G C G G G G C G C C

Efficient cleavage requires at least two copies of the SacII recognition sequence.
SpeI  (396)
1 site
A C T A G T T G A T C A
BfuAI  (600)
1 site
A C C T G C ( N ) 4 T G G A C G ( N ) 4 ( N ) 4

Efficient cleavage requires at least two copies of the BfuAI recognition sequence.
Sticky ends from different BfuAI sites may not be compatible.
BfuAI is typically used at 50°C, but is 50% active at 37°C.
BspMI  (600)
1 site
A C C T G C ( N ) 4 T G G A C G ( N ) 4 ( N ) 4

Efficient cleavage requires at least two copies of the BspMI recognition sequence.
Sticky ends from different BspMI sites may not be compatible.
BsrGI  (941)
1 site
T G T A C A A C A T G T

BsrGI is typically used at 37°C, but is even more active at 60°C.
NdeI  (1114)
1 site
C A T A T G G T A T A C

Prolonged incubation with NdeI may lead to removal of additional nucleotides.
XbaI  (1152)
1 site
T C T A G A A G A T C T
SgrAI  (1263)
1 site
C R C C G G Y G G Y G G C C R C

Efficient cleavage requires at least two copies of the SgrAI recognition sequence.
SphI  (1419)
1 site
G C A T G C C G T A C G
BstAPI  (1627)
1 site
G C A N N N N N T G C C G T N N N N N A C G

Sticky ends from different BstAPI sites may not be compatible.
MluI  (1944)
1 site
A C G C G T T G C G C A
BclI  (1958)
1 site
T G A T C A A C T A G T
* Blocked by Dam methylation.
BclI is typically used at 50-55°C, but is 50% active at 37°C.
NmeAIII  (2150)
1 site
G C C G A G ( N ) 18-19 N N C G G C T C ( N ) 18-19

Efficient cleavage requires at least two copies of the NmeAIII recognition sequence.
Sticky ends from different NmeAIII sites may not be compatible.
For full activity, add fresh S-adenosylmethionine (SAM).
PspOMI  (2151)
1 site
G G G C C C C C C G G G
ApaI  (2155)
1 site
G G G C C C C C C G G G

ApaI can be used between 25°C and 37°C.
BssHII  (2355)
1 site
G C G C G C C G C G C G

BssHII is typically used at 50°C, but is 75% active at 37°C.
HpaI  (2450)
1 site
G T T A A C C A A T T G
PshAI  (2789)
1 site
G A C N N N N G T C C T G N N N N C A G

PshAI quickly loses activity at 37°C, but can be used at 25°C for long incubations.
BglI  (3008)
1 site
G C C N N N N N G G C C G G N N N N N C C G

Sticky ends from different BglI sites may not be compatible.
FspI  (3026)
1 site
T G C G C A A C G C G T
FspAI  (3026)
1 site
R T G C G C A Y Y A C G C G T R
PpuMI  (3051)
1 site
R G G W C C Y Y C C W G G R

Sticky ends from different PpuMI sites may not be compatible.
lacI
1594 .. 2676  =  1083 bp
360 amino acids  =  38.6 kDa
Product: lac repressor
The lac repressor binds to the lac operator to inhibit transcription in E. coli. This inhibition can be relieved by adding lactose or isopropyl-β-D-thiogalactopyranoside (IPTG).
lacI
1594 .. 2676  =  1083 bp
360 amino acids  =  38.6 kDa
Product: lac repressor
The lac repressor binds to the lac operator to inhibit transcription in E. coli. This inhibition can be relieved by adding lactose or isopropyl-β-D-thiogalactopyranoside (IPTG).
8xHis
150 .. 173  =  24 bp
8 amino acids  =  1.1 kDa
Product: 8xHis affinity tag
8xHis
150 .. 173  =  24 bp
8 amino acids  =  1.1 kDa
Product: 8xHis affinity tag
enterokinase site
252 .. 266  =  15 bp
5 amino acids  =  606.5 Da
Product: enterokinase recognition and cleavage site
enterokinase site
252 .. 266  =  15 bp
5 amino acids  =  606.5 Da
Product: enterokinase recognition and cleavage site
S-Tag
282 .. 326  =  45 bp
15 amino acids  =  1.7 kDa
Product: affinity and epitope tag derived from pancreatic ribonuclease A
S-Tag
282 .. 326  =  45 bp
15 amino acids  =  1.7 kDa
Product: affinity and epitope tag derived from pancreatic ribonuclease A
thrombin site
342 .. 359  =  18 bp
6 amino acids  =  627.7 Da
Product: thrombin recognition and cleavage site
thrombin site
342 .. 359  =  18 bp
6 amino acids  =  627.7 Da
Product: thrombin recognition and cleavage site
6xHis
369 .. 386  =  18 bp
6 amino acids  =  840.9 Da
Product: 6xHis affinity tag
6xHis
369 .. 386  =  18 bp
6 amino acids  =  840.9 Da
Product: 6xHis affinity tag
DsbC
408 .. 1115  =  708 bp
236 amino acids  =  25.6 kDa
Product: bacterial periplasmic protein disulfide isomerase
DsbC
408 .. 1115  =  708 bp
236 amino acids  =  25.6 kDa
Product: bacterial periplasmic protein disulfide isomerase
KanR
4816 .. 5631  =  816 bp
271 amino acids  =  31.0 kDa
Product: aminoglycoside phosphotransferase
confers resistance to kanamycin
KanR
4816 .. 5631  =  816 bp
271 amino acids  =  31.0 kDa
Product: aminoglycoside phosphotransferase
confers resistance to kanamycin
ori
4106 .. 4694  =  589 bp
high-copy-number ColE1/pMB1/pBR322/pUC origin of replication
ori
4106 .. 4694  =  589 bp
high-copy-number ColE1/pMB1/pBR322/pUC origin of replication
f1 ori
5724 .. 6179  =  456 bp
f1 bacteriophage origin of replication; arrow indicates direction of (+) strand synthesis
f1 ori
5724 .. 6179  =  456 bp
f1 bacteriophage origin of replication; arrow indicates direction of (+) strand synthesis
rop
3485 .. 3676  =  192 bp
63 amino acids  =  7.2 kDa
Product: Rop protein
rop
3485 .. 3676  =  192 bp
63 amino acids  =  7.2 kDa
Product: Rop protein
lacI promoter
1516 .. 1593  =  78 bp
lacI promoter
1516 .. 1593  =  78 bp
MCS
174 .. 250  =  77 bp
multiple cloning site
MCS
174 .. 250  =  77 bp
multiple cloning site
T7 terminator
26 .. 73  =  48 bp
transcription terminator for bacteriophage T7 RNA polymerase
T7 terminator
26 .. 73  =  48 bp
transcription terminator for bacteriophage T7 RNA polymerase
lac operator
1160 .. 1184  =  25 bp
The lac repressor binds to the lac operator to inhibit transcription in E. coli. This inhibition can be relieved by adding lactose or isopropyl-β-D-thiogalactopyranoside (IPTG).
lac operator
1160 .. 1184  =  25 bp
The lac repressor binds to the lac operator to inhibit transcription in E. coli. This inhibition can be relieved by adding lactose or isopropyl-β-D-thiogalactopyranoside (IPTG).
T7 promoter
1185 .. 1203  =  19 bp
promoter for bacteriophage T7 RNA polymerase
T7 promoter
1185 .. 1203  =  19 bp
promoter for bacteriophage T7 RNA polymerase
RBS
1124 .. 1129  =  6 bp
ribosome binding site
RBS
1124 .. 1129  =  6 bp
ribosome binding site
ORF:  1717 .. 2676  =  960 bp
ORF:  319 amino acids  =  34.1 kDa
ORF:  4816 .. 5631  =  816 bp
ORF:  271 amino acids  =  31.0 kDa
ORF:  2732 .. 3088  =  357 bp
ORF:  118 amino acids  =  13.0 kDa
ORF:  3452 .. 3676  =  225 bp
ORF:  74 amino acids  =  8.5 kDa
ORF:  546 .. 854  =  309 bp
ORF:  102 amino acids  =  11.9 kDa
ORF:  1116 .. 1844  =  729 bp
ORF:  242 amino acids  =  25.5 kDa
ORF:  2433 .. 2696  =  264 bp
ORF:  87 amino acids  =  8.9 kDa
ORF:  1292 .. 1555  =  264 bp
ORF:  87 amino acids  =  9.5 kDa
ORF:  2459 .. 2710  =  252 bp
ORF:  83 amino acids  =  9.1 kDa
ORF:  3085 .. 3453  =  369 bp
ORF:  122 amino acids  =  14.2 kDa
ORF:  147 .. 1115  =  969 bp
ORF:  322 amino acids  =  34.7 kDa
ORF:  1167 .. 1418  =  252 bp
ORF:  83 amino acids  =  8.6 kDa
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