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pMCSG23

Bacterial coexpression vector with a 6xHis-MBP-TEV leader and a CloDF13 origin, for high-throughput purification of recombinant proteins.

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pMCSG23.dna
Map and Sequence File:    Download    Open   
Sequence Author:  Midwest Center for Structural Genomics
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EcoNI (4944) AclI (4816) MluI (4476) ApaI (4273) PspOMI (4269) HpaI (3974) BsmBI (3861) PluTI (3841) SfoI (3839) NarI * (3838) KasI (3837) XbaI (3556) SgrAI (2883) NspI (2860) PciI (2856) BmtI (2760) NheI (2756) BspHI (2709) AscI (125) PstI - SbfI (135) AflII (163) BsrGI (190) NdeI (298) BsaI (405) BspEI (461) BsiWI (632) PsiI (694) BmgBI (884) BsmI (962) AlwNI (987) BsaBI (1017) AfeI (1252) EcoRV (1451) SspI (1487) AvaI - BsoBI - PaeR7I - PspXI - XhoI (1544) BmeT110I (1545) PacI (1619) AvrII (1623) EcoO109I (1668) Bsu36I (1707) AgeI (1756) DrdI - PflFI - Tth111I (1816) NgoMIV (2009) NaeI (2011) BglI (2033) pMCSG23 4971 bp
EcoNI  (4944)
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.
AclI  (4816)
1 site
A A C G T T T T G C A A
MluI  (4476)
1 site
A C G C G T T G C G C A
ApaI  (4273)
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  (4269)
1 site
G G G C C C C C C G G G
HpaI  (3974)
1 site
G T T A A C C A A T T G
BsmBI  (3861)
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.
PluTI  (3841)
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  (3839)
1 site
G G C G C C C C G C G G
NarI  (3838)
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  (3837)
1 site
G G C G C C C C G C G G
XbaI  (3556)
1 site
T C T A G A A G A T C T
SgrAI  (2883)
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.
NspI  (2860)
1 site
R C A T G Y Y G T A C R
PciI  (2856)
1 site
A C A T G T T G T A C A

PciI is inhibited by nonionic detergents.
BmtI  (2760)
1 site
G C T A G C C G A T C G
NheI  (2756)
1 site
G C T A G C C G A T C G
BspHI  (2709)
1 site
T C A T G A A G T A C T
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
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.
BsaI  (405)
1 site
G G T C T C N C C A G A G N ( N ) 4

Sticky ends from different BsaI sites may not be compatible.
BsaI can be used between 37°C and 50°C.
BspEI  (461)
1 site
T C C G G A A G G C C T
BsiWI  (632)
1 site
C G T A C G G C A T G C

BsiWI is typically used at 55°C, but is 50% active at 37°C.
PsiI  (694)
1 site
T T A T A A A A T A T T
BmgBI  (884)
1 site
C A C G T C G T G C A G

This recognition sequence is asymmetric, so ligating blunt ends generated by BmgBI will not always regenerate a BmgBI site.
BsmI  (962)
1 site
G A A T G C N C T T A C G N

Sticky ends from different BsmI sites may not be compatible.
AlwNI  (987)
1 site
C A G N N N C T G G T C N N N G A C

Sticky ends from different AlwNI sites may not be compatible.
BsaBI  (1017)
1 site
G A T N N N N A T C C T A N N N N T A G
AfeI  (1252)
1 site
A G C G C T T C G C G A
EcoRV  (1451)
1 site
G A T A T C C T A T A G

EcoRV is reportedly more prone than its isoschizomer Eco32I to delete a base after cleavage.
SspI  (1487)
1 site
A A T A T T T T A T A A
AvaI  (1544)
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  (1544)
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  (1544)
1 site
C T C G A G G A G C T C

PaeR7I does not recognize the sequence CTCTCGAG.
PspXI  (1544)
1 site
V C T C G A G B B G A G C T C V
XhoI  (1544)
1 site
C T C G A G G A G C T C
BmeT110I  (1545)
1 site
C Y C G R G G R G C Y C
PacI  (1619)
1 site
T T A A T T A A A A T T A A T T
AvrII  (1623)
1 site
C C T A G G G G A T C C
EcoO109I  (1668)
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  (1707)
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  (1756)
1 site
A C C G G T T G G C C A
DrdI  (1816)
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  (1816)
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  (1816)
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.
NgoMIV  (2009)
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  (2011)
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  (2033)
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.
MBP
345 .. 1442  =  1098 bp
366 amino acids  =  40.2 kDa
Product: maltose binding protein from E. coli
This version of the gene does not encode a signal sequence, so MBP will remain in the cytosol.
MBP
345 .. 1442  =  1098 bp
366 amino acids  =  40.2 kDa
Product: maltose binding protein from E. coli
This version of the gene does not encode a signal sequence, so MBP will remain in the cytosol.
lacI
3749 .. 4831  =  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
3749 .. 4831  =  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).
SmR
1870 .. 2661  =  792 bp
263 amino acids  =  29.3 kDa
Product: aminoglycoside adenylyltransferase (Murphy, 1985)
confers resistance to spectinomycin and streptomycin
SmR
1870 .. 2661  =  792 bp
263 amino acids  =  29.3 kDa
Product: aminoglycoside adenylyltransferase (Murphy, 1985)
confers resistance to spectinomycin and streptomycin
CloDF13 ori
2801 .. 3539  =  739 bp
Plasmids containing the CloDF13 (CDF) origin of replication can be propagated in E. coli cells that contain additional plasmids with compatible origins.
CloDF13 ori
2801 .. 3539  =  739 bp
Plasmids containing the CloDF13 (CDF) origin of replication can be propagated in E. coli cells that contain additional plasmids with compatible origins.
AmpR promoter
2662 .. 2753  =  92 bp
AmpR promoter
2662 .. 2753  =  92 bp
lacI promoter
4832 .. 4909  =  78 bp
lacI promoter
4832 .. 4909  =  78 bp
T7 terminator
1652 .. 1699  =  48 bp
transcription terminator for bacteriophage T7 RNA polymerase
T7 terminator
1652 .. 1699  =  48 bp
transcription terminator for bacteriophage T7 RNA polymerase
S-Tag
1556 .. 1600  =  45 bp
15 amino acids  =  1.7 kDa
Product: affinity and epitope tag derived from pancreatic ribonuclease A
S-Tag
1556 .. 1600  =  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
1464 .. 1484  =  21 bp
7 amino acids  =  900.0 Da
Product: tobacco etch virus (TEV) protease recognition and cleavage site
TEV site
1464 .. 1484  =  21 bp
7 amino acids  =  900.0 Da
Product: tobacco etch virus (TEV) protease recognition and cleavage site
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T7 promoter
214 .. 232  =  19 bp
promoter for bacteriophage T7 RNA polymerase