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Plasmid Files

pMCSG27

Bacterial vector encoding a C-terminal 10xHis-TVMV-MBP cassette, for high-throughput purification of recombinant proteins.

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pMCSG27 Sequence and MappMCSG27.dna
Map and Sequence File   
Sequence Author:  Midwest Center for Structural Genomics
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 6xHis ScaI (5667) PstI (5432) AhdI (5187) PciI (4294) BspQI - SapI (4178) BstZ17I (4065) PflFI - Tth111I (4039) Bpu10I (3400) PpuMI (3300) PaeR7I - PspXI - XhoI (158) EagI - NotI (166) HindIII (173) SalI (179) Eco53kI (188) SacI (190) EcoRI (192) BamHI (198) NcoI (347) BsmI (694) BmgBI (770) BglII (949) PsiI (960) BsiWI (1018) TVMV site TspMI - XmaI (1362) SmaI (1364) RBS XbaI (1401) T7 promoter BspDI * - ClaI * (1470) SgrAI (1512) SphI (1668) EcoNI (1728) PflMI (1775) MluI (2193) BstEII (2374) PspOMI (2400) ApaI (2404) EcoRV (2643) HpaI (2699) PshAI (3038) FspAI (3275) pMCSG27 6362 bp
ScaI  (5667)
1 site
A G T A C T T C A T G A
PstI  (5432)
1 site
C T G C A G G A C G T C
AhdI  (5187)
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  (4294)
1 site
A C A T G T T G T A C A

PciI is inhibited by nonionic detergents.
BspQI  (4178)
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  (4178)
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  (4065)
1 site
G T A T A C C A T A T G
PflFI  (4039)
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  (4039)
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  (3400)
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  (3300)
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.
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.
NcoI  (347)
1 site
C C A T G G G G T A C C
BsmI  (694)
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  (770)
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  (949)
1 site
A G A T C T T C T A G A
PsiI  (960)
1 site
T T A T A A A A T A T T
BsiWI  (1018)
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.
TspMI  (1362)
1 site
C C C G G G G G G C C C
XmaI  (1362)
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  (1364)
1 site
C C C G G G G G G C C C

SmaI can be used at 37°C for brief incubations.
XbaI  (1401)
1 site
T C T A G A A G A T C T
BspDI  (1470)
1 site
A T C G A T T A G C T A
* Blocked by Dam methylation.
ClaI  (1470)
1 site
A T C G A T T A G C T A
* Blocked by Dam methylation.
SgrAI  (1512)
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  (1668)
1 site
G C A T G C C G T A C G
EcoNI  (1728)
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  (1775)
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  (2193)
1 site
A C G C G T T G C G C A
BstEII  (2374)
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  (2400)
1 site
G G G C C C C C C G G G
ApaI  (2404)
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  (2643)
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  (2699)
1 site
G T T A A C C A A T T G
PshAI  (3038)
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.
FspAI  (3275)
1 site
R T G C G C A Y Y A C G C G T R
MBP
213 .. 1310  =  1098 bp
366 amino acids  =  40.2 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
213 .. 1310  =  1098 bp
366 amino acids  =  40.2 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
1843 .. 2925  =  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
1843 .. 2925  =  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
5114 .. 5974  =  861 bp
286 amino acids  =  31.5 kDa
   Segment 2:  
   5114 .. 5905  =  792 bp
   263 amino acids  =  28.9 kDa
Product: β-lactamase
confers resistance to ampicillin, carbenicillin, and
related antibiotics
AmpR
5114 .. 5974  =  861 bp
286 amino acids  =  31.5 kDa
   Segment 1:  signal sequence  
   5906 .. 5974  =  69 bp
   23 amino acids  =  2.6 kDa
Product: β-lactamase
confers resistance to ampicillin, carbenicillin, and
related antibiotics
AmpR
5114 .. 5974  =  861 bp
286 amino acids  =  31.5 kDa
2 segments
Product: β-lactamase
confers resistance to ampicillin, carbenicillin, and
related antibiotics
ori
4355 .. 4943  =  589 bp
high-copy-number ColE1/pMB1/pBR322/pUC origin
of replication
ori
4355 .. 4943  =  589 bp
high-copy-number ColE1/pMB1/pBR322/pUC origin
of replication
rop
3734 .. 3925  =  192 bp
63 amino acids  =  7.2 kDa
Product: Rop protein, which maintains plasmids at
low copy number
rop
3734 .. 3925  =  192 bp
63 amino acids  =  7.2 kDa
Product: Rop protein, which maintains plasmids at
low copy number
AmpR promoter
5975 .. 6078  =  104 bp
AmpR promoter
5975 .. 6078  =  104 bp
lacI promoter
1765 .. 1842  =  78 bp
lacI promoter
1765 .. 1842  =  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
10xHis
1332 .. 1361  =  30 bp
10 amino acids  =  1.4 kDa
Product: 10xHis affinity tag
10xHis
1332 .. 1361  =  30 bp
10 amino acids  =  1.4 kDa
Product: 10xHis affinity tag
lac operator
1409 .. 1433  =  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
1409 .. 1433  =  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).
TVMV site
1311 .. 1331  =  21 bp
7 amino acids  =  866.0 Da
Product:
tobacco vein mottling virus (TVMV) NIa
protease recognition and cleavage site
TVMV site
1311 .. 1331  =  21 bp
7 amino acids  =  866.0 Da
Product:
tobacco vein mottling virus (TVMV) NIa
protease recognition and cleavage site
T7 promoter
1434 .. 1452  =  19 bp
promoter for bacteriophage T7 RNA polymerase
T7 promoter
1434 .. 1452  =  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
1373 .. 1378  =  6 bp
ribosome binding site
RBS
1373 .. 1378  =  6 bp
ribosome binding site
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