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Plasmid Files

pMCSG14 (linearized)

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

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pMCSG14 (linearized) Sequence and MappMCSG14 (linearized).dna
Map and Sequence File   
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) BssS α I (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) BmeT110I (58) 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

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  (2296)
1 site
T C T A G A A G A T C T
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

The sticky ends produced by AflII are hard to ligate.
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
BmeT110I  (58)
1 site
C Y C G R G G R G C Y 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 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.
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

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  (1184)
1 site
Y A C G T R R T G C A Y
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
GST
4050 .. 4703  =  654 bp
218 amino acids  =  25.5 kDa
Product: glutathione S-transferase from
Schistosoma japonicum
GST
4050 .. 4703  =  654 bp
218 amino acids  =  25.5 kDa
Product: glutathione S-transferase from
Schistosoma japonicum
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
T7 terminator
165 .. 212  =  48 bp
transcription terminator for bacteriophage T7 RNA
polymerase
T7 terminator
165 .. 212  =  48 bp
transcription terminator for bacteriophage T7 RNA
polymerase
S-Tag
69 .. 113  =  45 bp
15 amino acids  =  1.7 kDa
Product: affinity and epitope tag derived from
pancreatic ribonuclease A
S-Tag
69 .. 113  =  45 bp
15 amino acids  =  1.7 kDa
Product: affinity and epitope tag derived from
pancreatic ribonuclease A
lac operator
3714 .. 3738  =  25 bp
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).
lac operator
3714 .. 3738  =  25 bp
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).
lac operator
3944 .. 3968  =  25 bp
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).
lac operator
3944 .. 3968  =  25 bp
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).
TEV site
4731 .. 4751  =  21 bp
7 amino acids  =  900.0 Da
Product: tobacco etch virus (TEV) protease
recognition and cleavage site
TEV site
4731 .. 4751  =  21 bp
7 amino acids  =  900.0 Da
Product: tobacco etch virus (TEV) protease
recognition and cleavage site
T7 promoter
3695 .. 3713  =  19 bp
promoter for bacteriophage T7 RNA polymerase
T7 promoter
3695 .. 3713  =  19 bp
promoter for bacteriophage T7 RNA polymerase
T7 promoter
3925 .. 3943  =  19 bp
promoter for bacteriophage T7 RNA polymerase
T7 promoter
3925 .. 3943  =  19 bp
promoter for bacteriophage T7 RNA polymerase
6xHis
3794 .. 3811  =  18 bp
6 amino acids  =  840.9 Da
Product: 6xHis affinity tag
6xHis
3794 .. 3811  =  18 bp
6 amino acids  =  840.9 Da
Product: 6xHis affinity tag
6xHis
4014 .. 4031  =  18 bp
6 amino acids  =  840.9 Da
Product: 6xHis affinity tag
6xHis
4014 .. 4031  =  18 bp
6 amino acids  =  840.9 Da
Product: 6xHis affinity tag
RBS
3769 .. 3774  =  6 bp
ribosome binding site
RBS
3769 .. 3774  =  6 bp
ribosome binding site
RBS
3997 .. 4002  =  6 bp
ribosome binding site
RBS
3997 .. 4002  =  6 bp
ribosome binding site
ATG
3782 .. 3784  =  3 bp
1 amino acid  =  149.2 Da
Product: start codon
ATG
3782 .. 3784  =  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
ATG
4011 .. 4013  =  3 bp
1 amino acid  =  149.2 Da
Product: start codon
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