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Plasmid Files

pMCSG11

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

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pMCSG11 Sequence and MappMCSG11.dna
Map and Sequence File   
Sequence Author:  Midwest Center for Structural Genomics
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 T7 promoter EcoNI (4052) BstAPI (3908) ApaLI (3604) AflIII - MluI (3584) BclI * (3570) BstEII (3402) ApaI (3381) PspOMI (3377) BciVI (3132) HpaI (3082) PluTI (2949) SfoI (2947) NarI * (2946) KasI (2945) AcuI (2724) XbaI (2664) NspI (2401) BssS α I (2199) SacII (2075) RBS NcoI (69) ATG 6xHis BfuAI - BspMI (124) AscI (125) PstI - SbfI (135) AflII (163) BsrGI (190) T7 promoter lac operator RBS NdeI (298) ATG 6xHis BglII (332) Acc65I (339) KpnI (343) SspI (368) AvaI - BsoBI - PaeR7I - PspXI - XhoI (425) BmeT110I (426) PacI (500) AvrII (504) BlpI (522) EcoO109I (549) Bsu36I (588) DrdI - PflFI - Tth111I (697) ScaI (828) MscI (978) BspEI (1245) Bpu10I (1469) BsaAI (1552) AfeI (1822) NheI (1823) BmtI (1827) BstZ17I (1836) XmnI (1879) SgrAI (1909) pMCSG11 4079 bp
EcoNI  (4052)
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  (3908)
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  (3604)
1 site
G T G C A C C A C G T G
AflIII  (3584)
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  (3584)
1 site
A C G C G T T G C G C A
BclI  (3570)
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  (3402)
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  (3381)
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  (3377)
1 site
G G G C C C C C C G G G
BciVI  (3132)
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  (3082)
1 site
G T T A A C C A A T T G
PluTI  (2949)
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  (2947)
1 site
G G C G C C C C G C G G
NarI  (2946)
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  (2945)
1 site
G G C G C C C C G C G G
AcuI  (2724)
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  (2664)
1 site
T C T A G A A G A T C T
NspI  (2401)
1 site
R C A T G Y Y G T A C R
BssSαI  (2199)
1 site
C A C G A G G T G C T C
SacII  (2075)
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.
NcoI  (69)
1 site
C C A T G G G G T A C C
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

The sticky ends produced by AflII are hard to ligate.
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.
BglII  (332)
1 site
A G A T C T T C T A G A
Acc65I  (339)
1 site
G G T A C C C C A T G G
KpnI  (343)
1 site
G G T A C C C C A T G G
SspI  (368)
1 site
A A T A T T T T A T A A
AvaI  (425)
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  (425)
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  (425)
1 site
C T C G A G G A G C T C

PaeR7I does not recognize the sequence CTCTCGAG.
PspXI  (425)
1 site
V C T C G A G B B G A G C T C V
XhoI  (425)
1 site
C T C G A G G A G C T C
BmeT110I  (426)
1 site
C Y C G R G G R G C Y C
PacI  (500)
1 site
T T A A T T A A A A T T A A T T
AvrII  (504)
1 site
C C T A G G G G A T C C
BlpI  (522)
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  (549)
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  (588)
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  (697)
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  (697)
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  (697)
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  (828)
1 site
A G T A C T T C A T G A
MscI  (978)
1 site
T G G C C A A C C G G T
BspEI  (1245)
1 site
T C C G G A A G G C C T
Bpu10I  (1469)
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  (1552)
1 site
Y A C G T R R T G C A Y
AfeI  (1822)
1 site
A G C G C T T C G C G A
NheI  (1823)
1 site
G C T A G C C G A T C G
BmtI  (1827)
1 site
G C T A G C C G A T C G
BstZ17I  (1836)
1 site
G T A T A C C A T A T G
XmnI  (1879)
1 site
G A A N N N N T T C C T T N N N N A A G
SgrAI  (1909)
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.
lacI
2857 .. 3939  =  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
2857 .. 3939  =  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
800 .. 1459  =  660 bp
219 amino acids  =  25.7 kDa
Product: chloramphenicol acetyltransferase
confers resistance to chloramphenicol
CmR
800 .. 1459  =  660 bp
219 amino acids  =  25.7 kDa
Product: chloramphenicol acetyltransferase
confers resistance to chloramphenicol
p15A ori
2088 .. 2632  =  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
2088 .. 2632  =  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
1460 .. 1562  =  103 bp
promoter of the E. coli cat gene
cat promoter
1460 .. 1562  =  103 bp
promoter of the E. coli cat gene
lacI promoter
3940 .. 4017  =  78 bp
lacI promoter
3940 .. 4017  =  78 bp
T7 terminator
533 .. 580  =  48 bp
transcription terminator for bacteriophage T7 RNA
polymerase
T7 terminator
533 .. 580  =  48 bp
transcription terminator for bacteriophage T7 RNA
polymerase
S-Tag
437 .. 481  =  45 bp
15 amino acids  =  1.7 kDa
Product: affinity and epitope tag derived from
pancreatic ribonuclease A
S-Tag
437 .. 481  =  45 bp
15 amino acids  =  1.7 kDa
Product: affinity and epitope tag derived from
pancreatic ribonuclease A
lac operator
3 .. 27  =  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
3 .. 27  =  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
233 .. 257  =  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
233 .. 257  =  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
345 .. 365  =  21 bp
7 amino acids  =  900.0 Da
Product: tobacco etch virus (TEV) protease
recognition and cleavage site
TEV site
345 .. 365  =  21 bp
7 amino acids  =  900.0 Da
Product: tobacco etch virus (TEV) protease
recognition and cleavage site
T7 promoter
214 .. 232  =  19 bp
promoter for bacteriophage T7 RNA polymerase
T7 promoter
214 .. 232  =  19 bp
promoter for bacteriophage T7 RNA polymerase
T7 promoter
4063 .. 2  =  19 bp
promoter for bacteriophage T7 RNA polymerase
T7 promoter
4063 .. 2  =  19 bp
promoter for bacteriophage T7 RNA polymerase
6xHis
83 .. 100  =  18 bp
6 amino acids  =  840.9 Da
Product: 6xHis affinity tag
6xHis
83 .. 100  =  18 bp
6 amino acids  =  840.9 Da
Product: 6xHis affinity tag
6xHis
303 .. 320  =  18 bp
6 amino acids  =  840.9 Da
Product: 6xHis affinity tag
6xHis
303 .. 320  =  18 bp
6 amino acids  =  840.9 Da
Product: 6xHis affinity tag
RBS
58 .. 63  =  6 bp
ribosome binding site
RBS
58 .. 63  =  6 bp
ribosome binding site
RBS
286 .. 291  =  6 bp
ribosome binding site
RBS
286 .. 291  =  6 bp
ribosome binding site
ATG
71 .. 73  =  3 bp
1 amino acid  =  149.2 Da
Product: start codon
ATG
71 .. 73  =  3 bp
1 amino acid  =  149.2 Da
Product: start codon
ATG
300 .. 302  =  3 bp
1 amino acid  =  149.2 Da
Product: start codon
ATG
300 .. 302  =  3 bp
1 amino acid  =  149.2 Da
Product: start codon
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