pMCSG24

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

Sequence Author: Midwest Center for Structural Genomics

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T7 promoter PfoI (4464) AclI (4372) BstAPI (4356) MluI (4032) ApaI (3829) PspOMI (3825) HpaI (3530) BsmBI - Esp3I (3417) PluTI (3397) SfoI (3395) NarI * (3394) KasI (3393) XbaI (3112) SgrAI (2439) BmtI (2316) NheI (2312) BspHI (2265) lac operator RBS NcoI (69) ATG 6xHis AscI (125) PstI - SbfI (135) AflII (163) BsrGI (190) T7 promoter RBS NdeI (298) ATG 6xHis BspQI - SapI (418) MscI (546) XmnI (732) BstBI (736) SwaI (766) ScaI (912) Acc65I (1014) KpnI (1018) SspI (1043) AvaI - BsoBI - PaeR7I - PspXI - XhoI (1100) PacI (1175) AvrII (1179) Bsu36I (1263) AgeI (1312) DrdI - PflFI - Tth111I (1372) BsaAI (1461) NgoMIV (1565) NaeI (1567) BglI (1589) PvuI (1839) pMCSG24 4527 bp
PfoI  (4464)
1 site
T C C N G G A A G G N C C T

Sticky ends from different PfoI sites may not be compatible.
AclI  (4372)
1 site
A A C G T T T T G C A A
BstAPI  (4356)
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.
MluI  (4032)
1 site
A C G C G T T G C G C A
ApaI  (3829)
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  (3825)
1 site
G G G C C C C C C G G G
HpaI  (3530)
1 site
G T T A A C C A A T T G
BsmBI  (3417)
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.
BsmBI-v2 is an improved version of BsmBI.
Esp3I  (3417)
1 site
C G T C T C N G C A G A G N ( N ) 4

Sticky ends from different Esp3I sites may not be compatible.
PluTI  (3397)
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  (3395)
1 site
G G C G C C C C G C G G
NarI  (3394)
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  (3393)
1 site
G G C G C C C C G C G G
XbaI  (3112)
1 site
T C T A G A A G A T C T
SgrAI  (2439)
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.
BmtI  (2316)
1 site
G C T A G C C G A T C G
NheI  (2312)
1 site
G C T A G C C G A T C G
BspHI  (2265)
1 site
T C A T G A A G T A C T
NcoI  (69)
1 site
C C A T G G G G T A C C
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.
BspQI  (418)
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  (418)
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.
MscI  (546)
1 site
T G G C C A A C C G G T
XmnI  (732)
1 site
G A A N N N N T T C C T T N N N N A A G
BstBI  (736)
1 site
T T C G A A A A G C T T
SwaI  (766)
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.
ScaI  (912)
1 site
A G T A C T T C A T G A
Acc65I  (1014)
1 site
G G T A C C C C A T G G
KpnI  (1018)
1 site
G G T A C C C C A T G G
SspI  (1043)
1 site
A A T A T T T T A T A A
AvaI  (1100)
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  (1100)
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  (1100)
1 site
C T C G A G G A G C T C

PaeR7I does not recognize the sequence CTCTCGAG.
PspXI  (1100)
1 site
V C T C G A G B B G A G C T C V
XhoI  (1100)
1 site
C T C G A G G A G C T C
PacI  (1175)
1 site
T T A A T T A A A A T T A A T T
AvrII  (1179)
1 site
C C T A G G G G A T C C
Bsu36I  (1263)
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  (1312)
1 site
A C C G G T T G G C C A
DrdI  (1372)
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  (1372)
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  (1372)
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.
BsaAI  (1461)
1 site
Y A C G T R R T G C A Y
NgoMIV  (1565)
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  (1567)
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  (1589)
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.
PvuI  (1839)
1 site
C G A T C G G C T A G C
lacI
3305 .. 4387  =  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
3305 .. 4387  =  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
1426 .. 2217  =  792 bp
263 amino acids  =  29.3 kDa
Product: aminoglycoside adenylyltransferase (Murphy, 1985)
confers resistance to spectinomycin and streptomycin
SmR
1426 .. 2217  =  792 bp
263 amino acids  =  29.3 kDa
Product: aminoglycoside adenylyltransferase (Murphy, 1985)
confers resistance to spectinomycin and streptomycin
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
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
GST
339 .. 992  =  654 bp
218 amino acids  =  25.5 kDa
Product: glutathione S-transferase from Schistosoma japonicum
GST
339 .. 992  =  654 bp
218 amino acids  =  25.5 kDa
Product: glutathione S-transferase from Schistosoma japonicum
TEV site
1020 .. 1040  =  21 bp
7 amino acids  =  900.0 Da
Product: tobacco etch virus (TEV) protease recognition and cleavage site
TEV site
1020 .. 1040  =  21 bp
7 amino acids  =  900.0 Da
Product: tobacco etch virus (TEV) protease recognition and cleavage site
CloDF13 ori
2357 .. 3095  =  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
2357 .. 3095  =  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
2218 .. 2309  =  92 bp
AmpR promoter
2218 .. 2309  =  92 bp
lacI promoter
4388 .. 4465  =  78 bp
lacI promoter
4388 .. 4465  =  78 bp
T7 terminator
1208 .. 1255  =  48 bp
transcription terminator for bacteriophage T7 RNA polymerase
T7 terminator
1208 .. 1255  =  48 bp
transcription terminator for bacteriophage T7 RNA polymerase
S-Tag
1112 .. 1156  =  45 bp
15 amino acids  =  1.7 kDa
Product: affinity and epitope tag derived from pancreatic ribonuclease A
S-Tag
1112 .. 1156  =  45 bp
15 amino acids  =  1.7 kDa
Product: affinity and epitope tag derived from pancreatic ribonuclease A
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
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
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).
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).
T7 promoter
4511 .. 2  =  19 bp
promoter for bacteriophage T7 RNA polymerase
T7 promoter
4511 .. 2  =  19 bp
promoter for bacteriophage T7 RNA polymerase
T7 promoter
214 .. 232  =  19 bp
promoter for bacteriophage T7 RNA polymerase
T7 promoter
214 .. 232  =  19 bp
promoter for bacteriophage T7 RNA polymerase
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
ORF:  1795 .. 2178  =  384 bp
ORF:  127 amino acids  =  14.6 kDa
ORF:  3271 .. 3522  =  252 bp
ORF:  83 amino acids  =  9.1 kDa
ORF:  1640 .. 1918  =  279 bp
ORF:  92 amino acids  =  9.4 kDa
ORF:  1928 .. 2185  =  258 bp
ORF:  85 amino acids  =  9.1 kDa
ORF:  300 .. 1058  =  759 bp
ORF:  252 amino acids  =  29.3 kDa
ORF:  892 .. 1149  =  258 bp
ORF:  85 amino acids  =  9.4 kDa
ORF:  1426 .. 2217  =  792 bp
ORF:  263 amino acids  =  29.3 kDa
ORF:  4137 .. 158  =  549 bp
ORF:  182 amino acids  =