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Plasmid Files

pMCSG32

Bacterial vector encoding N-terminal MBP-TVMV and C-terminal TEV-6xHis, for high-throughput purification of recombinant proteins.

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pMCSG32.dna
Map and Sequence File:    Download    Open   
Sequence Author:  Midwest Center for Structural Genomics
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PaeR7I - PspXI - XhoI (158) ScaI (5723) PstI (5488) AhdI (5243) PciI (4350) BspQI - SapI (4234) BstZ17I (4121) PflFI - Tth111I (4095) Bpu10I (3456) PpuMI (3356) FspAI (3331) EagI - NotI (166) HindIII (173) SalI (179) Eco53kI (188) SacI (190) EcoRI (192) BamHI (198) TspMI - XmaI (253) SmaI (255) BsrGI (298) NcoI (454) BsmI (801) BmgBI (877) BglII (1056) PsiI (1067) BsiWI (1125) NdeI (1419) XbaI (1457) BspDI * - ClaI * (1526) SgrAI (1568) SphI (1724) EcoNI (1784) PflMI (1831) MluI (2249) BstEII (2430) PspOMI (2456) ApaI (2460) EcoRV (2699) HpaI (2755) PshAI (3094) pMCSG32 6418 bp
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
ScaI  (5723)
1 site
A G T A C T T C A T G A
PstI  (5488)
1 site
C T G C A G G A C G T C
AhdI  (5243)
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  (4350)
1 site
A C A T G T T G T A C A

PciI is inhibited by nonionic detergents.
BspQI  (4234)
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  (4234)
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  (4121)
1 site
G T A T A C C A T A T G
PflFI  (4095)
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  (4095)
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.
Bpu10I  (3456)
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.
PpuMI  (3356)
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  (3331)
1 site
R T G C G C A Y Y A C G C G T R
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.
NcoI  (454)
1 site
C C A T G G G G T A C C
BsmI  (801)
1 site
G A A T G C N C T T A C G N

Sticky ends from different BsmI sites may not be compatible.
BmgBI  (877)
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.
BglII  (1056)
1 site
A G A T C T T C T A G A
PsiI  (1067)
1 site
T T A T A A A A T A T T
BsiWI  (1125)
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.
NdeI  (1419)
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  (1457)
1 site
T C T A G A A G A T C T
BspDI  (1526)
1 site
A T C G A T T A G C T A
* Blocked by Dam methylation.
ClaI  (1526)
1 site
A T C G A T T A G C T A
* Blocked by Dam methylation.
SgrAI  (1568)
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.
SphI  (1724)
1 site
G C A T G C C G T A C G
EcoNI  (1784)
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  (1831)
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.
MluI  (2249)
1 site
A C G C G T T G C G C A
BstEII  (2430)
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.
PspOMI  (2456)
1 site
G G G C C C C C C G G G
ApaI  (2460)
1 site
G G G C C C C C C G G G

ApaI can be used between 25°C and 37°C.
EcoRV  (2699)
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.
HpaI  (2755)
1 site
G T T A A C C A A T T G
PshAI  (3094)
1 site
G A C N N N N G T C C T G N N N N C A G

PshAI quickly loses activity at 37°C, but can be used at 25°C for long incubations.
MBP
320 .. 1420  =  1101 bp
367 amino acids  =  40.3 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
320 .. 1420  =  1101 bp
367 amino acids  =  40.3 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
1899 .. 2981  =  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
1899 .. 2981  =  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
5170 .. 6030  =  861 bp
286 amino acids  =  31.5 kDa
   Segment 2:  
   5170 .. 5961  =  792 bp
   263 amino acids  =  28.9 kDa
Product: β-lactamase
confers resistance to ampicillin, carbenicillin, and related antibiotics
AmpR
5170 .. 6030  =  861 bp
286 amino acids  =  31.5 kDa
   Segment 1:  signal sequence  
   5962 .. 6030  =  69 bp
   23 amino acids  =  2.6 kDa
Product: β-lactamase
confers resistance to ampicillin, carbenicillin, and related antibiotics
AmpR
5170 .. 6030  =  861 bp
286 amino acids  =  31.5 kDa
2 segments
Product: β-lactamase
confers resistance to ampicillin, carbenicillin, and related antibiotics
ori
4411 .. 4999  =  589 bp
high-copy-number ColE1/pMB1/pBR322/pUC origin of replication
ori
4411 .. 4999  =  589 bp
high-copy-number ColE1/pMB1/pBR322/pUC origin of replication
rop
3790 .. 3981  =  192 bp
63 amino acids  =  7.2 kDa
Product: Rop protein, which maintains plasmids at low copy number
rop
3790 .. 3981  =  192 bp
63 amino acids  =  7.2 kDa
Product: Rop protein, which maintains plasmids at low copy number
AmpR promoter
6031 .. 6134  =  104 bp
AmpR promoter
6031 .. 6134  =  104 bp
lacI promoter
1821 .. 1898  =  78 bp
lacI promoter
1821 .. 1898  =  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
1465 .. 1489  =  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
1465 .. 1489  =  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
1490 .. 1508  =  19 bp
promoter for bacteriophage T7 RNA polymerase
T7 promoter
1490 .. 1508  =  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
1429 .. 1434  =  6 bp
ribosome binding site
RBS
1429 .. 1434  =  6 bp
ribosome binding site
ORF:  3037 .. 3393  =  357 bp
ORF:  118 amino acids  =  13.0 kDa
ORF:  3757 .. 3981  =  225 bp
ORF:  74 amino acids  =  8.5 kDa
ORF:  1301 .. 2149  =  849 bp
ORF:  282 amino acids  =  30.1 kDa
ORF:  2738 .. 3001  =  264 bp
ORF:  87 amino acids  =  8.9 kDa
ORF:  5300 .. 5566  =  267 bp
ORF:  88 amino acids  =  9.2 kDa
ORF:  2022 .. 2981  =  960 bp
ORF:  319 amino acids  =  34.1 kDa
ORF:  137 .. 1420  =  1284 bp
ORF:  427 amino acids  =  46.9 kDa
ORF:  1472 .. 1723  =  252 bp
ORF:  83 amino acids  =  8.6 kDa
ORF:  1597 .. 1860  =  264 bp
ORF:  87 amino acids  =  9.5 kDa
ORF:  2764 .. 3015  =  252 bp
ORF:  83 amino acids  =  9.1 kDa
ORF:  5170 .. 6030  =  861 bp
ORF:  286 amino acids  =  31.5 kDa
ORF:  3390 .. 3758  =  369 bp
ORF:  122 amino acids  =  14.2 kDa
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