pMCSG13

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

Sequence Author: Midwest Center for Structural Genomics

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T7 promoter EcoNI (5171) ApaLI (4723) AflIII - MluI (4703) BstEII (4521) ApaI (4500) PspOMI (4496) HpaI (4201) PluTI (4068) SfoI (4066) NarI * (4065) KasI (4064) XbaI (3783) NspI (3520) BssSI - BssSαI (3318) SacII (3194) SgrAI (3028) XmnI (2998) BstZ17I (2955) BmtI (2946) NheI (2942) RBS ATG 6xHis AscI (125) PstI - SbfI (135) AflII (163) BsrGI (190) T7 promoter RBS NdeI (298) ATG 6xHis BsaI (405) BsiWI (632) PvuI (667) PsiI (694) BmgBI (884) BsaBI (1017) EcoRV (1451) TEV site SspI (1487) AvaI - BsoBI - PaeR7I - PspXI - XhoI (1544) PacI (1619) AvrII (1623) EcoO109I (1668) Bsu36I (1707) DrdI - PflFI - Tth111I (1816) ScaI (1947) MscI (2097) Bpu10I (2588) pMCSG13 5198 bp
EcoNI  (5171)
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.
ApaLI  (4723)
1 site
G T G C A C C A C G T G
AflIII  (4703)
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  (4703)
1 site
A C G C G T T G C G C A
BstEII  (4521)
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  (4500)
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  (4496)
1 site
G G G C C C C C C G G G
HpaI  (4201)
1 site
G T T A A C C A A T T G
PluTI  (4068)
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  (4066)
1 site
G G C G C C C C G C G G
NarI  (4065)
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  (4064)
1 site
G G C G C C C C G C G G
XbaI  (3783)
1 site
T C T A G A A G A T C T
NspI  (3520)
1 site
R C A T G Y Y G T A C R
BssSI  (3318)
1 site
C A C G A G G T G C T C
BssSαI  (3318)
1 site
C A C G A G G T G C T C
SacII  (3194)
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  (3028)
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.
XmnI  (2998)
1 site
G A A N N N N T T C C T T N N N N A A G
BstZ17I  (2955)
1 site
G T A T A C C A T A T G
BmtI  (2946)
1 site
G C T A G C C G A T C G
NheI  (2942)
1 site
G C T A G C C G A T C G
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.
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.
PvuI  (667)
1 site
C G A T C G G C T A G 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.
BsaBI  (1017)
1 site
G A T N N N N A T C C T A N N N N T A G
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
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.
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.
ScaI  (1947)
1 site
A G T A C T T C A T G A
MscI  (2097)
1 site
T G G C C A A C C G G T
Bpu10I  (2588)
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.
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
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.
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
lacI
3976 .. 5058  =  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
3976 .. 5058  =  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
1919 .. 2578  =  660 bp
219 amino acids  =  25.7 kDa
Product: chloramphenicol acetyltransferase
confers resistance to chloramphenicol
CmR
1919 .. 2578  =  660 bp
219 amino acids  =  25.7 kDa
Product: chloramphenicol acetyltransferase
confers resistance to chloramphenicol
p15A ori
3207 .. 3751  =  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
3207 .. 3751  =  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
2579 .. 2681  =  103 bp
promoter of the E. coli cat gene
cat promoter
2579 .. 2681  =  103 bp
promoter of the E. coli cat gene
lacI promoter
5059 .. 5136  =  78 bp
lacI promoter
5059 .. 5136  =  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
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
5182 .. 2  =  19 bp
promoter for bacteriophage T7 RNA polymerase
T7 promoter
5182 .. 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:  3122 .. 3400  =  279 bp
ORF:  92 amino acids  =  10.4 kDa
ORF:  300 .. 1502  =  1203 bp
ORF:  400 amino acids  =  44.0 kDa
ORF:  3942 .. 4193  =  252 bp
ORF:  83 amino acids  =  9.1 kDa
ORF:  4808 .. 158  =  549 bp
ORF:  182 amino acids  =  19.6 kDa
ORF:  213 .. 461  =  249 bp
ORF:  82 amino acids  =  9.8 kDa
ORF:  1919 .. 2578  =  660 bp
ORF:  219 amino acids  =  25.7 kDa
ORF:  3089 .. 3361  =  273 bp
ORF:  90 amino acids  =  9.7 kDa
ORF:  3956 .. 4219  =  264 bp
ORF:  87 amino acids  =  8.9 kDa
ORF:  3976 .. 4935  =  960 bp
ORF:  319 amino acids  =  34.1 kDa
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