pMCSG40

Bacterial expression vector encoding E. coli DsbA plus OsmY fused to a TVMV-6xHis-TEV leader.

Sequence Author: Midwest Center for Structural Genomics

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T7 terminator ScaI (6101) PvuI (5991) BsaI (5682) AhdI (5621) PciI (4728) BspQI - SapI (4612) BstZ17I (4499) BsaAI (4480) PpuMI (3734) FspAI (3709) StyI (57) 6xHis AvaI - BsoBI - PaeR7I - PspXI - XhoI (158) EagI - NotI (166) HindIII (173) SalI (179) Eco53kI (188) SacI (190) EcoRI (192) BamHI (198) SspI (219) TEV site 6xHis TVMV site BsrGI (321) DraIII (401) BstBI (903) NdeI (929) RBS XbaI (967) lac operator T7 promoter BspDI * - ClaI * (1036) SgrAI (1078) AgeI (1566) AscI (1577) BglII (1734) BfuAI - BspMI (1858) MfeI (1960) EcoNI (2162) PflMI (2209) BstAPI (2310) MluI (2627) BclI * (2641) PspOMI (2834) ApaI (2838) pMCSG40 6796 bp
ScaI  (6101)
1 site
A G T A C T T C A T G A
PvuI  (5991)
1 site
C G A T C G G C T A G C
BsaI  (5682)
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.
AhdI  (5621)
1 site
G A C N N N N N G T C C T G N N N N N C A G

The 1-base overhangs produced by AhdI may be hard to ligate.
Sticky ends from different AhdI sites may not be compatible.
PciI  (4728)
1 site
A C A T G T T G T A C A

PciI is inhibited by nonionic detergents.
BspQI  (4612)
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  (4612)
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.
BstZ17I  (4499)
1 site
G T A T A C C A T A T G
BsaAI  (4480)
1 site
Y A C G T R R T G C A Y
PpuMI  (3734)
1 site
R G G W C C Y Y C C W G G R

Sticky ends from different PpuMI sites may not be compatible.
FspAI  (3709)
1 site
R T G C G C A Y Y A C G C G T R
StyI  (57)
1 site
C C W W G G G G W W C C

Sticky ends from different StyI sites may not be compatible.
AvaI  (158)
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  (158)
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  (158)
1 site
C T C G A G G A G C T C

PaeR7I does not recognize the sequence CTCTCGAG.
PspXI  (158)
1 site
V C T C G A G B B G A G C T C V
XhoI  (158)
1 site
C T C G A G G A G C T C
EagI  (166)
1 site
C G G C C G G C C G G C
NotI  (166)
1 site
G C G G C C G C C G C C G G C G
HindIII  (173)
1 site
A A G C T T T T C G A A
SalI  (179)
1 site
G T C G A C C A G C T G
Eco53kI  (188)
1 site
G A G C T C C T C G A G
SacI  (190)
1 site
G A G C T C C T C G A G
EcoRI  (192)
1 site
G A A T T C C T T A A G
BamHI  (198)
1 site
G G A T C C C C T A G G

After cleavage, BamHI-HF® (but not the original BamHI) can remain bound to DNA and alter its electrophoretic mobility.
SspI  (219)
1 site
A A T A T T T T A T A A
BsrGI  (321)
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.
DraIII  (401)
1 site
C A C N N N G T G G T G N N N C A C

Sticky ends from different DraIII sites may not be compatible.
BstBI  (903)
1 site
T T C G A A A A G C T T
NdeI  (929)
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.
XbaI  (967)
1 site
T C T A G A A G A T C T
BspDI  (1036)
1 site
A T C G A T T A G C T A
* Blocked by Dam methylation.
ClaI  (1036)
1 site
A T C G A T T A G C T A
* Blocked by Dam methylation.
SgrAI  (1078)
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.
AgeI  (1566)
1 site
A C C G G T T G G C C A
AscI  (1577)
1 site
G G C G C G C C C C G C G C G G
BglII  (1734)
1 site
A G A T C T T C T A G A
BfuAI  (1858)
1 site
A C C T G C ( N ) 4 T G G A C G ( N ) 4 ( N ) 4

Efficient cleavage requires at least two copies of the BfuAI recognition sequence.
Sticky ends from different BfuAI sites may not be compatible.
BfuAI is typically used at 50°C, but is 50% active at 37°C.
BspMI  (1858)
1 site
A C C T G C ( N ) 4 T G G A C G ( N ) 4 ( N ) 4

Efficient cleavage requires at least two copies of the BspMI recognition sequence.
Sticky ends from different BspMI sites may not be compatible.
MfeI  (1960)
1 site
C A A T T G G T T A A C
EcoNI  (2162)
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.
PflMI  (2209)
1 site
C C A N N N N N T G G G G T N N N N N A C C

Sticky ends from different PflMI sites may not be compatible.
BstAPI  (2310)
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  (2627)
1 site
A C G C G T T G C G C A
BclI  (2641)
1 site
T G A T C A A C T A G T
* Blocked by Dam methylation.
BclI is typically used at 50-55°C, but is 50% active at 37°C.
PspOMI  (2834)
1 site
G G G C C C C C C G G G
ApaI  (2838)
1 site
G G G C C C C C C G G G

ApaI can be used between 25°C and 37°C.
lacI
2277 .. 3359  =  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
2277 .. 3359  =  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).
AmpR
5548 .. 6408  =  861 bp
286 amino acids  =  31.5 kDa
2 segments
   Segment 2:  
   5548 .. 6339  =  792 bp
   263 amino acids  =  28.9 kDa
Product: β-lactamase
confers resistance to ampicillin, carbenicillin, and related antibiotics
AmpR
5548 .. 6408  =  861 bp
286 amino acids  =  31.5 kDa
2 segments
   Segment 1:  signal sequence  
   6340 .. 6408  =  69 bp
   23 amino acids  =  2.6 kDa
Product: β-lactamase
confers resistance to ampicillin, carbenicillin, and related antibiotics
AmpR
5548 .. 6408  =  861 bp
286 amino acids  =  31.5 kDa
2 segments
Product: β-lactamase
confers resistance to ampicillin, carbenicillin, and related antibiotics
TEV site
223 .. 243  =  21 bp
7 amino acids  =  900.0 Da
Product: tobacco etch virus (TEV) protease recognition and cleavage site
TEV site
223 .. 243  =  21 bp
7 amino acids  =  900.0 Da
Product: tobacco etch virus (TEV) protease recognition and cleavage site
6xHis
268 .. 285  =  18 bp
6 amino acids  =  840.9 Da
Product: 6xHis affinity tag
6xHis
268 .. 285  =  18 bp
6 amino acids  =  840.9 Da
Product: 6xHis affinity tag
TVMV site
286 .. 306  =  21 bp
7 amino acids  =  865.9 Da
Product:
tobacco vein mottling virus (TVMV) NIa protease recognition and cleavage site
TVMV site
286 .. 306  =  21 bp
7 amino acids  =  865.9 Da
Product:
tobacco vein mottling virus (TVMV) NIa protease recognition and cleavage site
OsmY
328 .. 930  =  603 bp
201 amino acids  =  21.1 kDa
Product: osmotically inducible protein OsmY from E. coli
OsmY
328 .. 930  =  603 bp
201 amino acids  =  21.1 kDa
Product: osmotically inducible protein OsmY from E. coli
DsbA
1468 .. 2094  =  627 bp
208 amino acids  =  23.1 kDa
Product: bacterial periplasmic oxidoreductase
DsbA
1468 .. 2094  =  627 bp
208 amino acids  =  23.1 kDa
Product: bacterial periplasmic oxidoreductase
ori
4789 .. 5377  =  589 bp
high-copy-number ColE1/pMB1/pBR322/pUC origin of replication
ori
4789 .. 5377  =  589 bp
high-copy-number ColE1/pMB1/pBR322/pUC origin of replication
dsbA promoter
1236 .. 1467  =  232 bp
promoter for the E. coli dsbA gene (Belin and Boquet, 1994)
dsbA promoter
1236 .. 1467  =  232 bp
promoter for the E. coli dsbA gene (Belin and Boquet, 1994)
rop
4168 .. 4359  =  192 bp
63 amino acids  =  7.2 kDa
Product: Rop protein, which maintains plasmids at low copy number
rop
4168 .. 4359  =  192 bp
63 amino acids  =  7.2 kDa
Product: Rop protein, which maintains plasmids at low copy number
AmpR promoter
6409 .. 6512  =  104 bp
AmpR promoter
6409 .. 6512  =  104 bp
lacI promoter
2199 .. 2276  =  78 bp
lacI promoter
2199 .. 2276  =  78 bp
T7 terminator
26 .. 73  =  48 bp
transcription terminator for bacteriophage T7 RNA polymerase
T7 terminator
26 .. 73  =  48 bp
transcription terminator for bacteriophage T7 RNA polymerase
lac operator
975 .. 999  =  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
975 .. 999  =  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
1000 .. 1018  =  19 bp
promoter for bacteriophage T7 RNA polymerase
T7 promoter
1000 .. 1018  =  19 bp
promoter for bacteriophage T7 RNA polymerase
6xHis
140 .. 157  =  18 bp
6 amino acids  =  840.9 Da
Product: 6xHis affinity tag
6xHis
140 .. 157  =  18 bp
6 amino acids  =  840.9 Da
Product: 6xHis affinity tag
RBS
939 .. 944  =  6 bp
ribosome binding site
RBS
939 .. 944  =  6 bp
ribosome binding site
ORF:  271 .. 1260  =  990 bp
ORF:  329 amino acids  =  36.0 kDa
ORF:  1468 .. 2094  =  627 bp
ORF:  208 amino acids  =  23.1 kDa
ORF:  3415 .. 3771  =  357 bp
ORF:  118 amino acids  =  13.0 kDa
ORF:  4135 .. 4359  =  225 bp
ORF:  74 amino acids  =  8.5 kDa
ORF:  2162 .. 2527  =  366 bp
ORF:  121 amino acids  =  13.1 kDa
ORF:  3116 .. 3379  =  264 bp
ORF:  87 amino acids  =  8.9 kDa
ORF:  5678 .. 5944  =  267 bp
ORF:  88 amino acids  =  9.2 kDa
ORF:  2400 .. 3359  =  960 bp
ORF:  319 amino acids  =  34.1 kDa
ORF:  205 .. 930  =  726 bp
ORF:  241 amino acids  =  25.5 kDa
ORF:  982 .. 1233  =  252 bp
ORF:  83 amino acids  =  8.6 kDa
ORF:  1834 .. 2238  =  405 bp
ORF:  134 amino acids  =  15.5 kDa
ORF:  3142 .. 3393  =  252 bp
ORF:  83 amino acids  =  9.1 kDa
ORF:  5548 .. 6408  =  861 bp
ORF:  286 amino acids  =  31.5 kDa
ORF:  3768 .. 4136  =  369 bp
ORF:  122 amino acids  =  14.2 kDa
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