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pMCSG14

Bacterial coexpression vector with a 6xHis-GST-TEV leader and a p15A origin, for high-throughput purification of recombinant proteins.

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pMCSG14.dna
Map and Sequence File:    Download    Open   
Sequence Author:  Midwest Center for Structural Genomics
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NcoI (69) BstAPI (4583) ApaLI (4279) MluI (4259) BstEII (4077) ApaI (4056) PspOMI (4052) BciVI (3807) HpaI (3757) PluTI (3624) SfoI (3622) NarI * (3621) KasI (3620) AcuI (3399) XbaI (3339) BssS α I (2874) SacII (2750) SgrAI (2584) BstZ17I (2511) BmtI (2502) NheI (2498) AfeI (2497) BfuAI - BspMI (124) AscI (125) PstI - SbfI (135) AflII (163) BsrGI (190) NdeI (298) BspQI - SapI (418) PciI (577) SwaI (766) Acc65I (1014) KpnI (1018) SspI (1043) AvaI - BsoBI - PaeR7I - PspXI - XhoI (1100) BmeT110I (1101) PacI (1175) AvrII (1179) BlpI (1197) Bsu36I (1263) DrdI - PflFI - Tth111I (1372) BspEI (1920) Bpu10I (2144) BsaAI (2227) pMCSG14 4754 bp
NcoI  (69)
1 site
C C A T G G G G T A C C
BstAPI  (4583)
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.
ApaLI  (4279)
1 site
G T G C A C C A C G T G
MluI  (4259)
1 site
A C G C G T T G C G C A
BstEII  (4077)
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.
ApaI  (4056)
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  (4052)
1 site
G G G C C C C C C G G G
BciVI  (3807)
1 site
G T A T C C ( N ) 5 N C A T A G G ( N ) 5

The 1-base overhangs produced by BciVI may be hard to ligate.
Sticky ends from different BciVI sites may not be compatible.
HpaI  (3757)
1 site
G T T A A C C A A T T G
PluTI  (3624)
1 site
G G C G C C C C G C G G

Efficient cleavage requires at least two copies of the PluTI recognition sequence.
SfoI  (3622)
1 site
G G C G C C C C G C G G
NarI  (3621)
1 site
G G C G C C C C G C G G
* Blocked by Dcm methylation.
Efficient cleavage requires at least two copies of the NarI recognition sequence.
KasI  (3620)
1 site
G G C G C C C C G C G G
AcuI  (3399)
1 site
C T G A A G ( N ) 14 N N G A C T T C ( N ) 14

Efficient cleavage requires at least two copies of the AcuI recognition sequence.
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).
XbaI  (3339)
1 site
T C T A G A A G A T C T
BssSαI  (2874)
1 site
C A C G A G G T G C T C
SacII  (2750)
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.
SgrAI  (2584)
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.
BstZ17I  (2511)
1 site
G T A T A C C A T A T G
BmtI  (2502)
1 site
G C T A G C C G A T C G
NheI  (2498)
1 site
G C T A G C C G A T C G
AfeI  (2497)
1 site
A G C G C T T C G C G A
BfuAI  (124)
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  (124)
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.
AscI  (125)
1 site
G G C G C G C C C C G C G C G G
PstI  (135)
1 site
C T G C A G G A C G T C
SbfI  (135)
1 site
C C T G C A G G G G A C G T C C
AflII  (163)
1 site
C T T A A G G A A T T C
BsrGI  (190)
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  (298)
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.
BspQI  (418)
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  (418)
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.
PciI  (577)
1 site
A C A T G T T G T A C A

PciI is inhibited by nonionic detergents.
SwaI  (766)
1 site
A T T T A A A T T A A A T T T A

SwaI is typically used at 25°C, but is 50% active at 37°C.
Acc65I  (1014)
1 site
G G T A C C C C A T G G
KpnI  (1018)
1 site
G G T A C C C C A T G G
SspI  (1043)
1 site
A A T A T T T T A T A A
AvaI  (1100)
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  (1100)
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  (1100)
1 site
C T C G A G G A G C T C

PaeR7I does not recognize the sequence CTCTCGAG.
PspXI  (1100)
1 site
V C T C G A G B B G A G C T C V
XhoI  (1100)
1 site
C T C G A G G A G C T C
BmeT110I  (1101)
1 site
C Y C G R G G R G C Y C
PacI  (1175)
1 site
T T A A T T A A A A T T A A T T
AvrII  (1179)
1 site
C C T A G G G G A T C C
BlpI  (1197)
1 site
G C T N A G C C G A N T C G

Sticky ends from different BlpI sites may not be compatible.
Bsu36I  (1263)
1 site
C C T N A G G G G A N T C C

Sticky ends from different Bsu36I sites may not be compatible.
DrdI  (1372)
1 site
G A C N N N N N N G T C C T G N N N N N N C A G

Sticky ends from different DrdI sites may not be compatible.
PflFI  (1372)
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  (1372)
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.
BspEI  (1920)
1 site
T C C G G A A G G C C T
Bpu10I  (2144)
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.
BsaAI  (2227)
1 site
Y A C G T R R T G C A Y
lacI
3532 .. 4614  =  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
3532 .. 4614  =  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).
CmR
1475 .. 2134  =  660 bp
219 amino acids  =  25.7 kDa
Product: chloramphenicol acetyltransferase
confers resistance to chloramphenicol
CmR
1475 .. 2134  =  660 bp
219 amino acids  =  25.7 kDa
Product: chloramphenicol acetyltransferase
confers resistance to chloramphenicol
GST
339 .. 992  =  654 bp
218 amino acids  =  25.5 kDa
Product: glutathione S-transferase from Schistosoma japonicum
GST
339 .. 992  =  654 bp
218 amino acids  =  25.5 kDa
Product: glutathione S-transferase from Schistosoma japonicum
p15A ori
2763 .. 3307  =  545 bp