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Plasmid Files

pMCSG40

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

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pMCSG40.dna
Map and Sequence File:    Download    Open   
Sequence Author:  Midwest Center for Structural Genomics
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ScaI (6101) PvuI (5991) BsaI (5682) AhdI (5621) PciI (4728) BspQI - SapI (4612) BstZ17I (4499) BsaAI (4480) PpuMI (3734) FspAI (3709) StyI (57) AvaI - BsoBI - PaeR7I - PspXI - XhoI (158) BmeT110I (159) EagI - NotI (166) HindIII (173) SalI (179) Eco53kI (188) SacI (190) EcoRI (192) BamHI (198) SspI (219) BsrGI (321) DraIII (401) BstBI (903) NdeI (929) XbaI (967) 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
BmeT110I  (159)
1 site
C Y C G R G G R G C Y 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
   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
   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
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
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
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).
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
TVMV site
286 .. 306  =  21 bp
7 amino acids  =  866.0 Da
Product:
tobacco vein mottling virus (TVMV) NIa protease recognition and cleavage site
TVMV site
286 .. 306  =  21 bp
7 amino acids  =  866.0 Da
Product:
tobacco vein mottling virus (TVMV) NIa protease recognition and cleavage site
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
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
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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