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pMCSG13

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

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pMCSG13.dna
Map and Sequence File:    Download    Open   
Sequence Author:  Midwest Center for Structural Genomics
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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) BssS α I (3318) SacII (3194) SgrAI (3028) XmnI (2998) BstZ17I (2955) BmtI (2946) NheI (2942) AscI (125) PstI - SbfI (135) AflII (163) BsrGI (190) NdeI (298) BsaI (405) BsiWI (632) PvuI (667) PsiI (694) BmgBI (884) BsaBI (1017) EcoRV (1451) SspI (1487) AvaI - BsoBI - PaeR7I - PspXI - XhoI (1544) BmeT110I (1545) 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
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
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.
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

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.
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
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
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
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
5182 .. 2  =  19 bp
promoter for bacteriophage T7 RNA polymerase
T7 promoter
5182 .. 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
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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