pMCSG30

Bacterial expression vector with a 6xHis-TEV-MBP leader and a SacB negative selection marker, for high-throughput purification of recombinant proteins.

Sequence Author: Midwest Center for Structural Genomics

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AvaI - BmeT110I - BsoBI - PaeR7I - PspXI - XhoI (158) ScaI (7619) PstI (7384) AhdI (7139) BspQI - SapI (6130) PflFI - Tth111I (5991) PpuMI (5252) FspAI (5227) PshAI (4990) EcoRV (4595) ApaI (4356) PspOMI (4352) BstEII (4326) EagI - NotI (166) SalI (179) Eco53kI (188) SacI (190) BamHI (198) SfiI (249) SacII (598) SnaBI (926) BsrGI (991) AanI (1035) MscI (1412) StuI * (1434) SfiI (2133) NcoI (2281) BsmI (2628) BmgBI (2704) AanI (2894) BsiWI (2952) TEV site 6xHis ATG NdeI (3315) RBS XbaI (3353) T7 promoter SgrAI (3464) SphI (3620) EcoNI (3680) PflMI (3727) MluI (4145) pMCSG30 8314 bp
AvaI  (158)
1 site
C Y C G R G G R G C Y C

Sticky ends from different AvaI sites may not be compatible.
BmeT110I  (158)
1 site
C Y C G R G G R G C Y C
BsoBI  (158)
1 site
C Y C G R G G R G C Y C

Sticky ends from different BsoBI sites may not be compatible.
BsoBI is typically used at 37°C, but can be used at temperatures up to 65°C.
PaeR7I  (158)
1 site
C T C G A G G A G C T C

PaeR7I does not recognize the sequence CTCTCGAG.
PspXI  (158)
1 site
V C T C G A G B B G A G C T C V
XhoI  (158)
1 site
C T C G A G G A G C T C
ScaI  (7619)
1 site
A G T A C T T C A T G A
PstI  (7384)
1 site
C T G C A G G A C G T C
AhdI  (7139)
1 site
G A C N N N N N G T C C T G N N N N N C A G

The 1-base overhangs produced by AhdI may be hard to ligate.
Sticky ends from different AhdI sites may not be compatible.
BspQI  (6130)
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  (6130)
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.
PflFI  (5991)
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  (5991)
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.
PpuMI  (5252)
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.
FspAI  (5227)
1 site
R T G C G C A Y Y A C G C G T R
PshAI  (4990)
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.
EcoRV  (4595)
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.
ApaI  (4356)
1 site
G G G C C C C C C G G G

ApaI can be used between 25°C and 37°C.
PspOMI  (4352)
1 site
G G G C C C C C C G G G
BstEII  (4326)
1 site
G G T N A C C C C A N T G G

Sticky ends from different BstEII sites may not be compatible.
BstEII is typically used at 60°C, but is 50% active at 37°C.
EagI  (166)
1 site
C G G C C G G C C G G C
NotI  (166)
1 site
G C G G C C G C C G C C G G C G
SalI  (179)
1 site
G T C G A C C A G C T G
Eco53kI  (188)
1 site
G A G C T C C T C G A G
SacI  (190)
1 site
G A G C T C C T C G A G
BamHI  (198)
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.
SfiI  (249)
2 sites
G G C C N N N N N G G C C C C G G N N N N N C C G G

Efficient cleavage requires at least two copies of the SfiI recognition sequence.
Sticky ends from different SfiI sites may not be compatible.
SacII  (598)
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.
SnaBI  (926)
1 site
T A C G T A A T G C A T
BsrGI  (991)
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.
AanI  (1035)
2 sites
T T A T A A A A T A T T
MscI  (1412)
1 site
T G G C C A A C C G G T
StuI  (1434)
1 site
A G G C C T T C C G G A
* Blocked by Dcm methylation.
SfiI  (2133)
2 sites
G G C C N N N N N G G C C C C G G N N N N N C C G G

Efficient cleavage requires at least two copies of the SfiI recognition sequence.
Sticky ends from different SfiI sites may not be compatible.
NcoI  (2281)
1 site
C C A T G G G G T A C C
BsmI  (2628)
1 site
G A A T G C N C T T A C G N

Sticky ends from different BsmI sites may not be compatible.
BmgBI  (2704)
1 site
C A C G T C G T G C A G

This recognition sequence is asymmetric, so ligating blunt ends generated by BmgBI will not always regenerate a BmgBI site.
AanI  (2894)
2 sites
T T A T A A A A T A T T
BsiWI  (2952)
1 site
C G T A C G G C A T G C

BsiWI is typically used at 55°C, but is 50% active at 37°C.
NdeI  (3315)
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  (3353)
1 site
T C T A G A A G A T C T
SgrAI  (3464)
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  (3620)
1 site
G C A T G C C G T A C G
EcoNI  (3680)
1 site
C C T