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pMCSG37

Bacterial vector with a 10xHis-MBP-6xHis-TEV leader for high-throughput purification of recombinant proteins.

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pMCSG37.dna
Map and Sequence File:    Download    Open   
Sequence Author:  Midwest Center for Structural Genomics
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AvaI - BsoBI - PaeR7I - PspXI - XhoI (158) ScaI (5731) PstI (5496) AhdI (5251) PciI (4358) BspQI - SapI (4242) BstZ17I (4129) PflFI - Tth111I (4103) Bpu10I (3464) PpuMI (3364) FspAI (3339) BmeT110I (159) EagI - NotI (166) HindIII (173) SalI (179) Eco53kI (188) SacI (190) EcoRI (192) BamHI (198) SspI (219) NcoI (408) BsmI (755) BmgBI (831) PsiI (1021) BsiWI (1079) Acc65I (1372) KpnI (1376) NdeI (1427) XbaI (1465) BspDI * - ClaI * (1534) SgrAI (1576) SphI (1732) EcoNI (1792) PflMI (1839) MluI (2257) BstEII (2438) PspOMI (2464) ApaI (2468) EcoRV (2707) HpaI (2763) PshAI (3102) pMCSG37 6426 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.
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  (5731)
1 site
A G T A C T T C A T G A
PstI  (5496)
1 site
C T G C A G G A C G T C
AhdI  (5251)
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.
PciI  (4358)
1 site
A C A T G T T G T A C A

PciI is inhibited by nonionic detergents.
BspQI  (4242)
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  (4242)
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  (4129)
1 site
G T A T A C C A T A T G
PflFI  (4103)
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  (4103)
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.
Bpu10I  (3464)
1 site
C C T N A G C G G A N T C G

Efficient cleavage requires at least two copies of the Bpu10I recognition sequence.
This recognition sequence is asymmetric, so ligating sticky ends generated by Bpu10I will not always regenerate a Bpu10I site.
Sticky ends from different Bpu10I sites may not be compatible.
PpuMI  (3364)
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  (3339)
1 site
R T G C G C A Y Y A C G C G T R
BmeT110I  (159)
1 site
C Y C G R G G R G C Y 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
HindIII  (173)
1 site
A A G C T T T T C G A A
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
EcoRI  (192)
1 site
G A A T T C C T T A 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.
SspI  (219)
1 site
A A T A T T T T A T A A
NcoI  (408)
1 site
C C A T G G G G T A C C
BsmI  (755)
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  (831)
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.
PsiI  (1021)
1 site
T T A T A A A A T A T T
BsiWI  (1079)
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.
Acc65I  (1372)
1 site
G G T A C C C C A T G G
KpnI  (1376)
1 site
G G T A C C C C A T G G
NdeI  (1427)
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  (1465)
1 site
T C T A G A A G A T C T
BspDI  (1534)
1 site
A T C G A T T A G C T A
* Blocked by Dam methylation.
ClaI  (1534)
1 site
A T C G A T T A G C T A
* Blocked by Dam methylation.
SgrAI  (1576)
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  (1732)
1 site
G C A T G C C G T A C G
EcoNI  (1792)
1 site
C C T N N N N N A G G G G A N N N N N T C C

The 1-base overhangs produced by EcoNI may be hard to ligate.
Sticky ends from different EcoNI sites may not be compatible.
PflMI  (1839)
1 site
C C A N N N N N T G G G G T N N N N N A C C

Sticky ends from different PflMI sites may not be compatible.
MluI  (2257)
1 site
A C G C G T T G C G C A
BstEII  (2438)
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.
PspOMI  (2464)
1 site
G G G C C C C C C G G G
ApaI  (2468)
1 site
G G G C C C C C C G G G

ApaI can be used between 25°C and 37°C.
EcoRV  (2707)
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.
HpaI  (2763)
1 site
G T T A A C C A A T T G
PshAI  (3102)
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.
MBP
274 .. 1371  =  1098 bp
366 amino acids  =  40.2 kDa
Product: maltose binding protein from E. coli
This version of the gene does not encode a signal sequence, so MBP will remain in the cytosol.
MBP
274 .. 1371  =  1098 bp
366 amino acids  =  40.2 kDa
Product: maltose binding protein from E. coli
This version of the gene does not encode a signal sequence, so MBP will remain in the cytosol.
lacI
1907 .. 2989  =  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
1907 .. 2989  =  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).
AmpR
5178 .. 6038  =  861 bp
286 amino acids  =  31.5 kDa
   Segment 2:  
   5178 .. 5969  =  792 bp
   263 amino acids  =  28.9 kDa
Product: β-lactamase
confers resistance to ampicillin, carbenicillin, and related antibiotics
AmpR
5178 ..&#