pMCSG24 (linearized)

Linearized bacterial coexpression vector for ligation-independent cloning (LIC), with a 6xHis-GST-TEV leader and a CloDF13 origin.

Sequence Author: Midwest Center for Structural Genomics

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T7 promoter PfoI (3421) AclI (3329) BstAPI (3313) MluI (2989) ApaI (2786) PspOMI (2782) HpaI (2487) BsmBI - Esp3I (2374) PluTI (2354) SfoI (2352) NarI * (2351) KasI (2350) XbaI (2069) SgrAI (1396) BmtI (1273) NheI (1269) BspHI (1222) RBS NcoI (3553) ATG 6xHis AscI (3609) PstI - SbfI (3619) AflII (3647) BsrGI (3674) T7 promoter RBS NdeI (3782) ATG 6xHis BspQI - SapI (3902) MscI (4030) XmnI (4216) BstBI (4220) SwaI (4250) ScaI (4396) Acc65I (4498) KpnI (4502) End (4512) Start (0) AvaI - BsoBI - PaeR7I - PspXI - XhoI (57) PacI (132) AvrII (136) Bsu36I (220) AgeI (269) DrdI - PflFI - Tth111I (329) BsaAI (418) NgoMIV (522) NaeI (524) BglI (546) PvuI (796) pMCSG24 4512 bp
PfoI  (3421)
1 site
T C C N G G A A G G N C C T

Sticky ends from different PfoI sites may not be compatible.
AclI  (3329)
1 site
A A C G T T T T G C A A
BstAPI  (3313)
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  (2989)
1 site
A C G C G T T G C G C A
ApaI  (2786)
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  (2782)
1 site
G G G C C C C C C G G G
HpaI  (2487)
1 site
G T T A A C C A A T T G
BsmBI  (2374)
1 site
C G T C T C N G C A G A G N ( N ) 4

Sticky ends from different BsmBI sites may not be compatible.
BsmBI-v2 is an improved version of BsmBI.
Esp3I  (2374)
1 site
C G T C T C N G C A G A G N ( N ) 4

Sticky ends from different Esp3I sites may not be compatible.
PluTI  (2354)
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  (2352)
1 site
G G C G C C C C G C G G
NarI  (2351)
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  (2350)
1 site
G G C G C C C C G C G G
XbaI  (2069)
1 site
T C T A G A A G A T C T
SgrAI  (1396)
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.
BmtI  (1273)
1 site
G C T A G C C G A T C G
NheI  (1269)
1 site
G C T A G C C G A T C G
BspHI  (1222)
1 site
T C A T G A A G T A C T
NcoI  (3553)
1 site
C C A T G G G G T A C C
AscI  (3609)
1 site
G G C G C G C C C C G C G C G G
PstI  (3619)
1 site
C T G C A G G A C G T C
SbfI  (3619)
1 site
C C T G C A G G G G A C G T C C
AflII  (3647)
1 site
C T T A A G G A A T T C
BsrGI  (3674)
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  (3782)
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  (3902)
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  (3902)
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.
MscI  (4030)
1 site
T G G C C A A C C G G T
XmnI  (4216)
1 site
G A A N N N N T T C C T T N N N N A A G
BstBI  (4220)
1 site
T T C G A A A A G C T T
SwaI  (4250)
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.
ScaI  (4396)
1 site
A G T A C T T C A T G A
Acc65I  (4498)
1 site
G G T A C C C C A T G G
KpnI  (4502)
1 site
G G T A C C C C A T G G
End  (4512)
0 sites
Start  (0)
0 sites
AvaI  (57)
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  (57)
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  (57)
1 site
C T C G A G G A G C T C

PaeR7I does not recognize the sequence CTCTCGAG.
PspXI  (57)
1 site
V C T C G A G B B G A G C T C V
XhoI  (57)
1 site
C T C G A G G A G C T C
PacI  (132)
1 site
T T A A T T A A A A T T A A T T
AvrII  (136)
1 site
C C T A G G G G A T C C
Bsu36I  (220)
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.
AgeI  (269)
1 site
A C C G G T T G G C C A
DrdI  (329)
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  (329)
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  (329)
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.
BsaAI  (418)
1 site
Y A C G T R R T G C A Y
NgoMIV  (522)
1 site
G C C G G C C G G C C G

Efficient cleavage requires at least two copies of the NgoMIV recognition sequence.
NaeI  (524)
1 site
G C C G G C C G G C C G

Efficient cleavage requires at least two copies of the NaeI recognition sequence.
BglI  (546)
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.
PvuI  (796)
1 site
C G A T C G G C T A G C