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Plasmid Files

pMCSG43

Bacterial expression vector encoding E. coli DsbA plus OsmY followed by an N-terminal TVMV site and C-terminal TEV-6xHis.

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pMCSG43.dna
Map and Sequence File:    Download    Open   
Sequence Author:  Midwest Center for Structural Genomics
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ScaI (6078) PvuI (5968) BsaI (5659) AhdI (5598) PciI (4705) BspQI - SapI (4589) BstZ17I (4476) BsaAI (4457) PpuMI (3711) FspAI (3686) StyI (57) PaeR7I - PspXI - XhoI (158) EagI - NotI (166) HindIII (173) SalI (179) Eco53kI (188) SacI (190) EcoRI (192) BamHI (198) TspMI - XmaI (253) SmaI (255) BsrGI (298) DraIII (378) Acc65I (395) KpnI (399) BstBI (880) NdeI (906) XbaI (944) BspDI * - ClaI * (1013) SgrAI (1055) AgeI (1543) AscI (1554) BglII (1711) BfuAI - BspMI (1835) MfeI (1937) EcoNI (2139) PflMI (2186) BstAPI (2287) MluI (2604) BclI * (2618) PspOMI (2811) ApaI (2815) pMCSG43 6773 bp
ScaI  (6078)
1 site
A G T A C T T C A T G A
PvuI  (5968)
1 site
C G A T C G G C T A G C
BsaI  (5659)
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  (5598)
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  (4705)
1 site
A C A T G T T G T A C A

PciI is inhibited by nonionic detergents.
BspQI  (4589)
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  (4589)
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  (4476)
1 site
G T A T A C C A T A T G
BsaAI  (4457)
1 site
Y A C G T R R T G C A Y
PpuMI  (3711)
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  (3686)
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.
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.
TspMI  (253)
1 site
C C C G G G G G G C C C
XmaI  (253)
1 site
C C C G G G G G G C C C

Efficient cleavage requires at least two copies of the XmaI recognition sequence.
Full cleavage with XmaI may require a long incubation.
SmaI  (255)
1 site
C C C G G G G G G C C C

SmaI can be used at 37°C for brief incubations.
BsrGI  (298)
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  (378)
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.
Acc65I  (395)
1 site
G G T A C C C C A T G G
KpnI  (399)
1 site
G G T A C C C C A T G G
BstBI  (880)
1 site
T T C G A A A A G C T T
NdeI  (906)
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  (944)
1 site
T C T A G A A G A T C T
BspDI  (1013)
1 site
A T C G A T T A G C T A
* Blocked by Dam methylation.
ClaI  (1013)
1 site
A T C G A T T A G C T A
* Blocked by Dam methylation.
SgrAI  (1055)
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  (1543)
1 site
A C C G G T T G G C C A
AscI  (1554)
1 site
G G C G C G C C C C G C G C G G
BglII  (1711)
1 site
A G A T C T T C T A G A
BfuAI  (1835)
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  (1835)
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  (1937)
1 site
C A A T T G G T T A A C
EcoNI  (2139)
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  (2186)
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  (2287)
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  (2604)
1 site
A C G C G T T G C G C A
BclI  (2618)
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  (2811)
1 site
G G G C C C C C C G G G
ApaI  (2815)
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
2254 .. 3336  =  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
2254 .. 3336  =  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
5525 .. 6385  =  861 bp
286 amino acids  =  31.5 kDa
   Segment 2:  
   5525 .. 6316  =  792 bp
   263 amino acids  =  28.9 kDa
Product: β-lactamase
confers resistance to ampicillin, carbenicillin, and related antibiotics
AmpR
5525 .. 6385  =  861 bp
286 amino acids  =  31.5 kDa
   Segment 1:  signal sequence  
   6317 .. 6385  =  69 bp
   23 amino acids  =  2.6 kDa
Product: β-lactamase
confers resistance to ampicillin, carbenicillin, and related antibiotics
AmpR
5525 .. 6385  =  861 bp
286 amino acids  =  31.5 kDa
2 segments
Product: β-lactamase
confers resistance to ampicillin, carbenicillin, and related antibiotics
DsbA
1445 .. 2071  =  627 bp
208 amino acids  =  23.1 kDa
Product: bacterial periplasmic oxidoreductase
DsbA
1445 .. 2071  =  627 bp
208 amino acids  =  23.1 kDa
Product: bacterial periplasmic oxidoreductase
OsmY
305 .. 907  =  603 bp
201 amino acids  =  21.1 kDa
Product: osmotically inducible protein OsmY from E. coli
OsmY
305 .. 907  =  603 bp
201 amino acids  =  21.1 kDa
Product: osmotically inducible protein OsmY from E. coli
ori
4766 .. 5354  =  589 bp
high-copy-number ColE1/pMB1/pBR322/pUC origin of replication
ori
4766 .. 5354  =  589 bp
high-copy-number ColE1/pMB1/pBR322/pUC origin of replication
dsbA promoter
1213 .. 1444  =  232 bp
promoter for the E. coli dsbA gene (Belin and Boquet, 1994)
dsbA promoter
1213 .. 1444  =  232 bp
promoter for the E. coli dsbA gene (Belin and Boquet, 1994)
rop
4145 .. 4336  =  192 bp
63 amino acids  =  7.2 kDa
Product: Rop protein, which maintains plasmids at low copy number
rop
4145 .. 4336  =  192 bp
63 amino acids  =  7.2 kDa
Product: Rop protein, which maintains plasmids at low copy number
AmpR promoter
6386 .. 6489  =  104 bp
AmpR promoter
6386 .. 6489  =  104 bp
lacI promoter
2176 .. 2253  =  78 bp
lacI promoter
2176 .. 2253  =  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
952 .. 976  =  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
952 .. 976  =  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
232 .. 252  =  21 bp
7 amino acids  =  900.0 Da
Product: tobacco etch virus (TEV) protease recognition and cleavage site
TEV site
232 .. 252  =  21 bp
7 amino acids  =  900.0 Da
Product: tobacco etch virus (TEV) protease recognition and cleavage site
TVMV site
263 .. 283  =  21 bp
7 amino acids  =  866.0 Da
Product:
tobacco vein mottling virus (TVMV) NIa protease recognition and cleavage site
TVMV site
263 .. 283  =  21 bp
7 amino acids  =  866.0 Da
Product:
tobacco vein mottling virus (TVMV) NIa protease recognition and cleavage site
T7 promoter
977 .. 995  =  19 bp
promoter for bacteriophage T7 RNA polymerase
T7 promoter
977 .. 995  =  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
208 .. 225  =  18 bp
6 amino acids  =  840.9 Da
Product: 6xHis affinity tag
6xHis
208 .. 225  =  18 bp
6 amino acids  =  840.9 Da
Product: 6xHis affinity tag
RBS
916 .. 921  =  6 bp
ribosome binding site
RBS
916 .. 921  =  6 bp
ribosome binding site
ORF:  2377 .. 3336  =  960 bp
ORF:  319 amino acids  =  34.1 kDa
ORF:  440 .. 1237  =  798 bp
ORF:  265 amino acids  =  28.7 kDa
ORF:  1445 .. 2071  =  627 bp
ORF:  208 amino acids  =  23.1 kDa
ORF:  3392 .. 3748  =  357 bp
ORF:  118 amino acids  =  13.0 kDa
ORF:  4112 .. 4336  =  225 bp
ORF:  74 amino acids  =  8.5 kDa
ORF:  2139 .. 2504  =  366 bp
ORF:  121 amino acids  =  13.1 kDa
ORF:  3093 .. 3356  =  264 bp
ORF:  87 amino acids  =  8.9 kDa
ORF:  5655 .. 5921  =  267 bp
ORF:  88 amino acids  =  9.2 kDa
ORF:  137 .. 907  =  771 bp
ORF:  256 amino acids  =  27.1 kDa
ORF:  959 .. 1210  =  252 bp
ORF:  83 amino acids  =  8.6 kDa
ORF:  1811 .. 2215  =  405 bp
ORF:  134 amino acids  =  15.5 kDa
ORF:  3119 .. 3370  =  252 bp
ORF:  83 amino acids  =  9.1 kDa
ORF:  5525 .. 6385  =  861 bp
ORF:  286 amino acids  =  31.5 kDa
ORF:  3745 .. 4113  =  369 bp
ORF:  122 amino acids  =  14.2 kDa
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