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Plasmid Files

pMCSG71

Compact bacterial vector encoding N-terminal MBP-TVMV and C-terminal TEV-6xHis, plus tRNA genes for rare Arg and Ile codons.

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pMCSG71 Sequence and MappMCSG71.dna
Map and Sequence File   
Sequence Author:  Midwest Center for Structural Genomics
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 ScaI (5245) FspI (4987) BglI (4885) AhdI (4765) DrdI (3980) PciI (3872) BspQI - SapI (3756) BstZ17I (3643) PshAI (3617) BsmBI (3387) HpaI (3278) EcoRV (3222) ApaI (2983) PspOMI (2979) T7 terminator 6xHis PaeR7I - PspXI - XhoI (158) EagI - NotI (166) HindIII (173) SalI (179) Eco53kI (188) SacI (190) EcoRI (192) BamHI (198) 6xHis TspMI - XmaI (253) SmaI (255) TVMV site BsrGI (298) BsmI (801) BmgBI (877) BglII (1056) BsiWI (1125) NdeI (1419) RBS XbaI (1457) T7 promoter BspDI * - ClaI * (1526) AgeI (1568) AscI (1785) SbfI (1796) BstBI (1801) MreI (1933) EcoNI (2307) PflMI (2354) MluI (2772) BstEII (2953) pMCSG71 5940 bp
ScaI  (5245)
1 site
A G T A C T T C A T G A
FspI  (4987)
1 site
T G C G C A A C G C G T
BglI  (4885)
1 site
G C C N N N N N G G C C G G N N N N N C C G

Sticky ends from different BglI sites may not be compatible.
AhdI  (4765)
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.
DrdI  (3980)
1 site
G A C N N N N N N G T C C T G N N N N N N C A G

Sticky ends from different DrdI sites may not be compatible.
PciI  (3872)
1 site
A C A T G T T G T A C A

PciI is inhibited by nonionic detergents.
BspQI  (3756)
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  (3756)
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  (3643)
1 site
G T A T A C C A T A T G
PshAI  (3617)
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.
BsmBI  (3387)
1 site
C G T C T C N G C A G A G N ( N ) 4

Sticky ends from different BsmBI sites may not be compatible.
HpaI  (3278)
1 site
G T T A A C C A A T T G
EcoRV  (3222)
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.
ApaI  (2983)
1 site
G G G C C C C C C G G G

ApaI can be used between 25°C and 37°C.
PspOMI  (2979)
1 site
G G G C C C C C C G G G
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.
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
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.
AgeI  (1568)
1 site
A C C G G T T G G C C A

AgeI quickly loses activity at 37°C, but can be used at 25°C for
long incubations.
AscI  (1785)
1 site
G G C G C G C C C C G C G C G G
SbfI  (1796)
1 site
C C T G C A G G G G A C G T C C
BstBI  (1801)
1 site
T T C G A A A A G C T T
MreI  (1933)
1 site
C G C C G G C G G C G G C C G C
EcoNI  (2307)
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  (2354)
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  (2772)
1 site
A C G C G T T G C G C A
BstEII  (2953)
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.
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
2422 .. 3504  =  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
2422 .. 3504  =  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
4692 .. 5552  =  861 bp
286 amino acids  =  31.5 kDa
   Segment 2:  
   4692 .. 5483  =  792 bp
   263 amino acids  =  28.9 kDa
Product: β-lactamase
confers resistance to ampicillin, carbenicillin, and
related antibiotics
AmpR
4692 .. 5552  =  861 bp
286 amino acids  =  31.5 kDa
   Segment 1:  signal sequence  
   5484 .. 5552  =  69 bp
   23 amino acids  =  2.6 kDa
Product: β-lactamase
confers resistance to ampicillin, carbenicillin, and
related antibiotics
AmpR
4692 .. 5552  =  861 bp
286 amino acids  =  31.5 kDa
2 segments
Product: β-lactamase
confers resistance to ampicillin, carbenicillin, and
related antibiotics
ori
3933 .. 4521  =  589 bp
high-copy-number ColE1/pMB1/pBR322/pUC origin
of replication
ori
3933 .. 4521  =  589 bp
high-copy-number ColE1/pMB1/pBR322/pUC origin
of replication
AmpR promoter
5553 .. 5656  =  104 bp
AmpR promoter
5553 .. 5656  =  104 bp
lacI promoter
2344 .. 2421  =  78 bp
lacI promoter
2344 .. 2421  =  78 bp
argU
1783 .. 1859  =  77 bp
Product: E. coli tRNA-Arg recognizing AGA and AGG
argU
1783 .. 1859  =  77 bp
Product: E. coli tRNA-Arg recognizing AGA and AGG
ileX
2129 .. 2204  =  76 bp
Product: E. coli tRNA-Ile recognizing AUA
ileX
2129 .. 2204  =  76 bp
Product: E. coli tRNA-Ile recognizing AUA
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
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