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Plasmid Files

pMCSG28

Bacterial vector encoding a C-terminal TEV-6xHis cassette, for high-throughput purification of recombinant proteins.

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pMCSG28 Sequence and MappMCSG28.dna
Map and Sequence File   
Sequence Author:  Midwest Center for Structural Genomics
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 T7 terminator ScaI (4558) PvuI (4448) PstI (4323) BsaI (4139) AhdI (4078) AlwNI (3601) PciI (3185) BspQI - SapI (3069) BstZ17I (2956) BsaAI (2937) PflFI - Tth111I (2930) StyI (57) BlpI (80) 6xHis PaeR7I - PspXI - XhoI (158) EagI - NotI (166) HindIII (173) SalI (179) Eco53kI (188) SacI (190) EcoRI (192) BamHI (198) 6xHis TEV site TspMI - XmaI (253) SmaI (255) RBS XbaI (292) lac operator T7 promoter BspDI * - ClaI * (361) SgrAI (403) SphI (559) EcoNI (619) PflMI (666) BstAPI (767) MluI (1084) BclI * (1098) BstEII (1265) PspOMI (1291) ApaI (1295) BssHII (1495) EcoRV (1534) HpaI (1590) PshAI (1929) FspAI (2166) PpuMI (2191) Bpu10I (2291) pMCSG28 5253 bp
ScaI  (4558)
1 site
A G T A C T T C A T G A
PvuI  (4448)
1 site
C G A T C G G C T A G C
PstI  (4323)
1 site
C T G C A G G A C G T C
BsaI  (4139)
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  (4078)
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.
AlwNI  (3601)
1 site
C A G N N N C T G G T C N N N G A C

Sticky ends from different AlwNI sites may not be compatible.
PciI  (3185)
1 site
A C A T G T T G T A C A

PciI is inhibited by nonionic detergents.
BspQI  (3069)
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  (3069)
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  (2956)
1 site
G T A T A C C A T A T G
BsaAI  (2937)
1 site
Y A C G T R R T G C A Y
PflFI  (2930)
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  (2930)
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.
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.
BlpI  (80)
1 site
G C T N A G C C G A N T C G

Sticky ends from different BlpI 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.
XbaI  (292)
1 site
T C T A G A A G A T C T
BspDI  (361)
1 site
A T C G A T T A G C T A
* Blocked by Dam methylation.
ClaI  (361)
1 site
A T C G A T T A G C T A
* Blocked by Dam methylation.
SgrAI  (403)
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  (559)
1 site
G C A T G C C G T A C G
EcoNI  (619)
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  (666)
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  (767)
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  (1084)
1 site
A C G C G T T G C G C A
BclI  (1098)
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.
BstEII  (1265)
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  (1291)
1 site
G G G C C C C C C G G G
ApaI  (1295)
1 site
G G G C C C C C C G G G

ApaI can be used between 25°C and 37°C.
BssHII  (1495)
1 site
G C G C G C C G C G C G

BssHII is typically used at 50°C, but is 75% active at 37°C.
EcoRV  (1534)
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  (1590)
1 site
G T T A A C C A A T T G
PshAI  (1929)
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  (2166)
1 site
R T G C G C A Y Y A C G C G T R
PpuMI  (2191)
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.
Bpu10I  (2291)
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.
lacI
734 .. 1816  =  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
734 .. 1816  =  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
4005 .. 4865  =  861 bp
286 amino acids  =  31.5 kDa
   Segment 2:  
   4005 .. 4796  =  792 bp
   263 amino acids  =  28.9 kDa
Product: β-lactamase
confers resistance to ampicillin, carbenicillin, and
related antibiotics
AmpR
4005 .. 4865  =  861 bp
286 amino acids  =  31.5 kDa
   Segment 1:  signal sequence  
   4797 .. 4865  =  69 bp
   23 amino acids  =  2.6 kDa
Product: β-lactamase
confers resistance to ampicillin, carbenicillin, and
related antibiotics
AmpR
4005 .. 4865  =  861 bp
286 amino acids  =  31.5 kDa
2 segments
Product: β-lactamase
confers resistance to ampicillin, carbenicillin, and
related antibiotics
ori
3246 .. 3834  =  589 bp
high-copy-number ColE1/pMB1/pBR322/pUC origin
of replication
ori
3246 .. 3834  =  589 bp
high-copy-number ColE1/pMB1/pBR322/pUC origin
of replication
rop
2625 .. 2816  =  192 bp
63 amino acids  =  7.2 kDa
Product: Rop protein, which maintains plasmids at
low copy number
rop
2625 .. 2816  =  192 bp
63 amino acids  =  7.2 kDa
Product: Rop protein, which maintains plasmids at
low copy number
AmpR promoter
4866 .. 4969  =  104 bp
AmpR promoter
4866 .. 4969  =  104 bp
lacI promoter
656 .. 733  =  78 bp
lacI promoter
656 .. 733  =  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
300 .. 324  =  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
300 .. 324  =  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
T7 promoter
325 .. 343  =  19 bp
promoter for bacteriophage T7 RNA polymerase
T7 promoter
325 .. 343  =  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
264 .. 269  =  6 bp
ribosome binding site
RBS
264 .. 269  =  6 bp
ribosome binding site
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