pMCSG11 (linearized)

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

Sequence Author: Midwest Center for Structural Genomics

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T7 promoter EcoNI (3684) BstAPI (3540) ApaLI (3236) AflIII - MluI (3216) BclI * (3202) BstEII (3034) ApaI (3013) PspOMI (3009) BciVI (2764) HpaI (2714) PluTI (2581) SfoI (2579) NarI * (2578) KasI (2577) AcuI (2356) XbaI (2296) NspI (2033) BssSI - BssSαI (1831) SacII (1707) SgrAI (1541) RBS NcoI (3780) ATG 6xHis BfuAI - BspMI (3835) AscI (3836) PstI - SbfI (3846) AflII (3874) BsrGI (3901) T7 promoter lac operator RBS NdeI (4009) ATG 6xHis BglII (4043) Acc65I (4050) KpnI (4054) End (4064) Start (0) AvaI - BsoBI - PaeR7I - PspXI - XhoI (57) PacI (132) AvrII (136) BlpI (154) EcoO109I (181) Bsu36I (220) DrdI - PflFI - Tth111I (329) ScaI (460) MscI (610) BspEI (877) Bpu10I (1101) BsaAI (1184) AfeI (1454) NheI (1455) BmtI (1459) BstZ17I (1468) XmnI (1511) pMCSG11 4064 bp
EcoNI  (3684)
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.
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
AflIII  (3216)
1 site
A C R Y G T T G Y R C A

Sticky ends from different AflIII sites may not be compatible.
MluI  (3216)
1 site
A C G C G T T G C G C A
BclI  (3202)
1 site
T G A T C A A C T A G T
* Blocked by Dam methylation.
BclI is typically used at 50-55°C, but is 50% active at 37°C.
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
NspI  (2033)
1 site
R C A T G Y Y G T A C R
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.
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.
BglII  (4043)
1 site
A G A T C T T C T A G A
Acc65I  (4050)
1 site
G G T A C C C C A T G G
KpnI  (4054)
1 site
G G T A C C C C A T G G
End  (4064)
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.
EcoO109I  (181)
1 site
R G G N C C Y Y C C N G G R

Sticky ends from different EcoO109I 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 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.
ScaI  (460)
1 site
A G T A C T T C A T G A
MscI  (610)
1 site
T G G C C A A C C G G T
BspEI  (877)
1 site
T C C G G A A G G C C T
Bpu10I  (1101)
1 site
C C T N A G C G G A N T C G

Cleavage may be enhanced when more than one copy of the Bpu10I recognition sequence is present.
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  (1184)
1 site
Y A C G T R R T G C A Y
AfeI  (1454)
1 site
A G C G C T T C G C G A
NheI  (1455)
1 site
G C T A G C C G A T C G
BmtI  (1459)
1 site
G C T A G C C G A T C G
BstZ17I  (1468)
1 site
G T A T A C C A T A T G
XmnI  (1511)
1 site
G A A N N N N T T C C T T N N N N A A G
lacI
2489 .. 3571  =  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
2489 .. 3571  =  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
432 .. 1091  =  660 bp
219 amino acids  =  25.7 kDa
Product: chloramphenicol acetyltransferase
confers resistance to chloramphenicol
CmR
432 .. 1091  =  660 bp
219 amino acids  =  25.7 kDa
Product: chloramphenicol acetyltransferase
confers resistance to chloramphenicol
p15A ori
1720 .. 2264  =  545 bp
Plasmids containing the medium-copy-number p15A origin of replication can be propagated in E. coli cells that contain a second plasmid with the ColE1 origin.
p15A ori
1720 .. 2264  =  545 bp
Plasmids containing the medium-copy-number p15A origin of replication can be propagated in E. coli cells that contain a second plasmid with the ColE1 origin.
cat promoter
1092 .. 1194  =  103 bp
promoter of the E. coli cat gene
cat promoter
1092 .. 1194  =  103 bp
promoter of the E. coli cat gene
lacI promoter
3572 .. 3649  =  78 bp
lacI promoter
3572 .. 3649  =  78 bp
ATG
4011 .. 4013  =  3 bp
1 amino acid  =  149.2 Da
Product: start codon
ATG
4011 .. 4013  =  3 bp
1 amino acid  =  149.2 Da
Product: start codon
6xHis
4014 .. 4031  =  18 bp
6 amino acids  =  840.9 Da