pMCSG14 (linearized)

Linearized bacterial coexpression vector for ligation-independent cloning (LIC), with a 6xHis-GST-TEV leader and a p15A origin.

Sequence Author: Midwest Center for Structural Genomics

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T7 promoter BstAPI (3540) ApaLI (3236) MluI (3216) BstEII (3034) ApaI (3013) PspOMI (3009) BciVI (2764) HpaI (2714) PluTI (2581) SfoI (2579) NarI * (2578) KasI (2577) AcuI (2356) XbaI (2296) BssSI - BssSαI (1831) SacII (1707) SgrAI (1541) BstZ17I (1468) BmtI (1459) NheI (1455) AfeI (1454) RBS NcoI (3780) ATG 6xHis BfuAI - BspMI (3835) AscI (3836) PstI - SbfI (3846) AflII (3874) BsrGI (3901) T7 promoter RBS NdeI (4009) ATG 6xHis BspQI - SapI (4129) PciI (4288) SwaI (4477) Acc65I (4725) KpnI (4729) End (4739) Start (0) AvaI - BsoBI - PaeR7I - PspXI - XhoI (57) PacI (132) AvrII (136) BlpI (154) Bsu36I (220) DrdI - PflFI - Tth111I (329) BspEI (877) Bpu10I (1101) BsaAI (1184) pMCSG14 4739 bp
BstAPI  (3540)
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  (3236)
1 site
G T G C A C C A C G T G
MluI  (3216)
1 site
A C G C G T T G C G C A
BstEII  (3034)
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  (3013)
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  (3009)
1 site
G G G C C C C C C G G G
BciVI  (2764)
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  (2714)
1 site
G T T A A C C A A T T G
PluTI  (2581)
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  (2579)
1 site
G G C G C C C C G C G G
NarI  (2578)
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  (2577)
1 site
G G C G C C C C G C G G
AcuI  (2356)
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).
XbaI  (2296)
1 site
T C T A G A A G A T C T
BssSI  (1831)
1 site
C A C G A G G T G C T C
BssSαI  (1831)
1 site
C A C G A G G T G C T C
SacII  (1707)
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  (1541)
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  (1468)
1 site
G T A T A C C A T A T G
BmtI  (1459)
1 site
G C T A G C C G A T C G
NheI  (1455)
1 site
G C T A G C C G A T C G
AfeI  (1454)
1 site
A G C G C T T C G C G A
NcoI  (3780)
1 site
C C A T G G G G T A C C
BfuAI  (3835)
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  (3835)
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  (3836)
1 site
G G C G C G C C C C G C G C G G
PstI  (3846)
1 site
C T G C A G G A C G T C
SbfI  (3846)
1 site
C C T G C A G G G G A C G T C C
AflII  (3874)
1 site
C T T A A G G A A T T C
BsrGI  (3901)
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  (4009)
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  (4129)
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  (4129)
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  (4288)
1 site
A C A T G T T G T A C A

PciI is inhibited by nonionic detergents.
SwaI  (4477)
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  (4725)
1 site
G G T A C C C C A T G G
KpnI  (4729)
1 site
G G T A C C C C A T G G
End  (4739)
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
BlpI  (154)
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  (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.
DrdI  (329)
1 site
G A C N N N