pMCSG22

Bacterial coexpression vector with a 6xHis-S- loop-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 EcoNI (3879) PfoI (3843) AclI (3751) BstAPI (3735) MluI (3411) ApaI (3208) PspOMI (3204) HpaI (2909) BsmBI - Esp3I (2796) PluTI (2776) SfoI (2774) NarI * (2773) KasI (2772) XbaI (2491) RBS NcoI (69) ATG 6xHis AscI (125) PstI - SbfI (135) AflII (163) BsrGI (190) T7 promoter lac operator RBS NdeI (298) ATG 6xHis BglII (332) Acc65I (393) KpnI (397) TEV site SspI (422) AvaI - BsoBI - PaeR7I - PspXI - XhoI (479) PacI (554) AvrII (558) EcoO109I (603) Bsu36I (642) DrdI - PflFI - Tth111I (751) BsaAI (840) NgoMIV (944) NaeI (946) BglI (968) PvuI (1218) BspHI (1644) NheI (1691) BmtI (1695) PciI (1791) NspI (1795) SgrAI (1818) pMCSG22 3906 bp
EcoNI  (3879)
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.
PfoI  (3843)
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  (3751)
1 site
A A C G T T T T G C A A
BstAPI  (3735)
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  (3411)
1 site
A C G C G T T G C G C A
ApaI  (3208)
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  (3204)
1 site
G G G C C C C C C G G G
HpaI  (2909)
1 site
G T T A A C C A A T T G
BsmBI  (2796)
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  (2796)
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  (2776)
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  (2774)
1 site
G G C G C C C C G C G G
NarI  (2773)
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  (2772)
1 site
G G C G C C C C G C G G
XbaI  (2491)
1 site
T C T A G A A G A T 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.
BglII  (332)
1 site
A G A T C T T C T A G A
Acc65I  (393)
1 site
G G T A C C C C A T G G
KpnI  (397)
1 site
G G T A C C C C A T G G
SspI  (422)
1 site
A A T A T T T T A T A A
AvaI  (479)
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  (479)
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  (479)
1 site
C T C G A G G A G C T C

PaeR7I does not recognize the sequence CTCTCGAG.
PspXI  (479)
1 site
V C T C G A G B B G A G C T C V
XhoI  (479)
1 site
C T C G A G G A G C T C
PacI  (554)
1 site
T T A A T T A A A A T T A A T T
AvrII  (558)
1 site
C C T A G G G G A T C C
EcoO109I  (603)
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  (642)
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  (751)
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  (751)
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  (751)
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  (840)
1 site
Y A C G T R R T G C A Y
NgoMIV  (944)
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  (946)
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  (968)
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  (1218)
1 site
C G A T C G G C T A G C
BspHI  (1644)
1 site
T C A T G A A G T A C T
NheI  (1691)
1 site
G C T A G C C G A T C G
BmtI  (1695)
1 site
G C T A G C C G A T C G
PciI  (1791)
1 site
A C A T G T T G T A C A

PciI is inhibited by nonionic detergents.
NspI  (1795)
1 site
R C A T G Y Y G T A C R
SgrAI  (1818)
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
2684 .. 3766  =  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
2684 .. 3766  =  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
805 .. 1596  =  792 bp
263 amino acids  =  29.3 kDa
Product: aminoglycoside adenylyltransferase (Murphy, 1985)
confers resistance to spectinomycin and streptomycin
SmR
805 .. 1596  =  792 bp
263 amino acids  =  29.3 kDa
Product: aminoglycoside adenylyltransferase (Murphy, 1985)
confers resistance to spectinomycin and streptomycin
CloDF13 ori
1736 .. 2474  =  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
1736 .. 2474  =  739 bp
Plasmids containing the CloDF13 (CDF) origin of replication can be propagated in E. coli cells that contain additional plasmids with compatible origins.
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
S loop
339 .. 392  =  54 bp
18 amino acids  =  1.7 kDa
Product: GroES chaperone mobile loop that interacts with GroEL
S loop
339 .. 392  =  54 bp
18 amino acids  =  1.7 kDa
Product: GroES chaperone mobile loop that interacts with GroEL
TEV site
399 .. 419  =  21 bp
7 amino acids  =  900.0 Da
Product: tobacco etch virus (TEV) protease recognition and cleavage site
TEV site
399 .. 419  =  21 bp
7 amino acids  =  900.0 Da
Product: tobacco etch virus (TEV) protease recognition and cleavage site
AmpR promoter
1597 .. 1688  =  92 bp
AmpR promoter
1597 .. 1688  =  92 bp
lacI promoter
3767 .. 3844  =  78 bp
lacI promoter
3767 .. 3844  =  78 bp
T7 terminator
587 .. 634  =  48 bp
transcription terminator for bacteriophage T7 RNA polymerase
T7 terminator
587 .. 634  =  48 bp
transcription terminator for bacteriophage T7 RNA polymerase
S-Tag
491 .. 535  =  45 bp
15 amino acids  =  1.7 kDa
Product: affinity and epitope tag derived from pancreatic ribonuclease A
S-Tag
491 .. 535  =  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
3890 .. 2  =  19 bp
promoter for bacteriophage T7 RNA polymerase
T7 promoter
3890 .. 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:  1174 .. 1557  =  384 bp
ORF:  127 amino acids  =  14.6 kDa
ORF:  2650 .. 2901  =  252 bp
ORF:  83 amino acids  =  9.1 kDa
ORF:  1019 .. 1297  =  279 bp
ORF:  92 amino acids  =  9.4 kDa
ORF:  1307 .. 1564  =  258 bp
ORF:  85 amino acids  =  9.1 kDa
ORF:  271 .. 528  =  258 bp
ORF:  85 amino acids  =  9.8 kDa
ORF:  805 .. 1596  =  792 bp
ORF:  263 amino acids  =  29.3 kDa
ORF:  3516 .. 158  =  549 bp
ORF:  182 amino acids  =  19.6 kDa
ORF:  2664 .. 2927  =  264 bp
ORF:  87 amino acids  =  8.9 kDa
ORF:  2684 .. 3643  =  960 bp
ORF:  319 amino acids  =  34.1 kDa
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Download pMCSG22.dna file

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