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Plasmid Files

pMCSG27 (linearized)

Linearized bacterial vector for ligation-independent cloning (LIC), with a C-terminal 10xHis-TVMV-MBP cassette.

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pMCSG27 (linearized) Sequence and MappMCSG27 (linearized).dna
Map and Sequence File   
Sequence Author:  Midwest Center for Structural Genomics
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 6xHis ScaI (4303) PstI (4068) AhdI (3823) PciI (2930) BspQI - SapI (2814) BstZ17I (2701) PflFI - Tth111I (2675) Bpu10I (2036) PpuMI (1936) FspAI (1911) PshAI (1674) AvaI - BsoBI - PaeR7I - PspXI - XhoI (5156) BmeT110I (5157) EagI - NotI (5164) HindIII (5171) SalI (5177) Eco53kI (5186) SacI (5188) EcoRI (5190) BamHI (5196) NcoI (5345) BsmI (5692) BmgBI (5768) BglII (5947) PsiI (5958) BsiWI (6016) TVMV site End (6351) Start (0) RBS XbaI (37) T7 promoter BspDI * - ClaI * (106) SgrAI (148) SphI (304) EcoNI (364) PflMI (411) MluI (829) BstEII (1010) PspOMI (1036) ApaI (1040) EcoRV (1279) HpaI (1335) pMCSG27 6351 bp
ScaI  (4303)
1 site
A G T A C T T C A T G A
PstI  (4068)
1 site
C T G C A G G A C G T C
AhdI  (3823)
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  (2930)
1 site
A C A T G T T G T A C A

PciI is inhibited by nonionic detergents.
BspQI  (2814)
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  (2814)
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  (2701)
1 site
G T A T A C C A T A T G
PflFI  (2675)
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  (2675)
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  (2036)
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  (1936)
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  (1911)
1 site
R T G C G C A Y Y A C G C G T R
PshAI  (1674)
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.
AvaI  (5156)
1 site
C Y C G R G G R G C Y C

Sticky ends from different AvaI sites may not be compatible.
BsoBI  (5156)
1 site
C Y C G R G G R G C Y C

Sticky ends from different BsoBI sites may not be compatible.
BsoBI is typically used at 37°C, but can be used at temperatures
up to 65°C.
PaeR7I  (5156)
1 site
C T C G A G G A G C T C

PaeR7I does not recognize the sequence CTCTCGAG.
PspXI  (5156)
1 site
V C T C G A G B B G A G C T C V
XhoI  (5156)
1 site
C T C G A G G A G C T C
BmeT110I  (5157)
1 site
C Y C G R G G R G C Y C
EagI  (5164)
1 site
C G G C C G G C C G G C
NotI  (5164)
1 site
G C G G C C G C C G C C G G C G
HindIII  (5171)
1 site
A A G C T T T T C G A A
SalI  (5177)
1 site
G T C G A C C A G C T G
Eco53kI  (5186)
1 site
G A G C T C C T C G A G
SacI  (5188)
1 site
G A G C T C C T C G A G
EcoRI  (5190)
1 site
G A A T T C C T T A A G
BamHI  (5196)
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  (5345)
1 site
C C A T G G G G T A C C
BsmI  (5692)
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  (5768)
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  (5947)
1 site
A G A T C T T C T A G A
PsiI  (5958)
1 site
T T A T A A A A T A T T
BsiWI  (6016)
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.
End  (6351)
0 sites
Start  (0)
0 sites
XbaI  (37)
1 site
T C T A G A A G A T C T
BspDI  (106)
1 site
A T C G A T T A G C T A
* Blocked by Dam methylation.
ClaI  (106)
1 site
A T C G A T T A G C T A
* Blocked by Dam methylation.
SgrAI  (148)
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  (304)
1 site
G C A T G C C G T A C G
EcoNI  (364)
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  (411)
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  (829)
1 site
A C G C G T T G C G C A
BstEII  (1010)
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  (1036)
1 site
G G G C C C C C C G G G
ApaI  (1040)
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  (1279)
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  (1335)
1 site
G T T A A C C A A T T G
MBP
5211 .. 6308  =  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
5211 .. 6308  =  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
479 .. 1561  =  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
479 .. 1561  =  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
3750 .. 4610  =  861 bp
286 amino acids  =  31.5 kDa
   Segment 2:  
   3750 .. 4541  =  792 bp
   263 amino acids  =  28.9 kDa
Product: β-lactamase
confers resistance to ampicillin, carbenicillin, and
related antibiotics
AmpR
3750 .. 4610  =  861 bp
286 amino acids  =  31.5 kDa
   Segment 1:  signal sequence  
   4542 .. 4610  =  69 bp
   23 amino acids  =  2.6 kDa
Product: β-lactamase
confers resistance to ampicillin, carbenicillin, and
related antibiotics
AmpR
3750 .. 4610  =  861 bp
286 amino acids  =  31.5 kDa
2 segments
Product: β-lactamase
confers resistance to ampicillin, carbenicillin, and
related antibiotics
ori
2991 .. 3579  =  589 bp
high-copy-number ColE1/pMB1/pBR322/pUC origin
of replication
ori
2991 .. 3579  =  589 bp
high-copy-number ColE1/pMB1/pBR322/pUC origin
of replication
rop
2370 .. 2561  =  192 bp
63 amino acids  =  7.2 kDa
Product: Rop protein, which maintains plasmids at
low copy number
rop
2370 .. 2561  =  192 bp
63 amino acids  =  7.2 kDa
Product: Rop protein, which maintains plasmids at
low copy number
AmpR promoter
4611 .. 4714  =  104 bp
AmpR promoter
4611 .. 4714  =  104 bp
lacI promoter
401 .. 478  =  78 bp
lacI promoter
401 .. 478  =  78 bp
T7 terminator
5024 .. 5071  =  48 bp
transcription terminator for bacteriophage T7 RNA
polymerase
T7 terminator
5024 .. 5071  =  48 bp
transcription terminator for bacteriophage T7 RNA
polymerase
10xHis
6330 .. 6359  =  30 bp
10 amino acids  =  1.4 kDa
Product: 10xHis affinity tag
10xHis
6330 .. 6359  =  30 bp
10 amino acids  =  1.4 kDa
Product: 10xHis affinity tag
lac operator
45 .. 69  =  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
45 .. 69  =  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
6309 .. 6329  =  21 bp
7 amino acids  =  866.0 Da
Product:
tobacco vein mottling virus (TVMV) NIa
protease recognition and cleavage site
TVMV site
6309 .. 6329  =  21 bp
7 amino acids  =  866.0 Da
Product:
tobacco vein mottling virus (TVMV) NIa
protease recognition and cleavage site
T7 promoter
70 .. 88  =  19 bp
promoter for bacteriophage T7 RNA polymerase
T7 promoter
70 .. 88  =  19 bp
promoter for bacteriophage T7 RNA polymerase
6xHis
5138 .. 5155  =  18 bp
6 amino acids  =  840.9 Da
Product: 6xHis affinity tag
6xHis
5138 .. 5155  =  18 bp
6 amino acids  =  840.9 Da
Product: 6xHis affinity tag
RBS
9 .. 14  =  6 bp
ribosome binding site
RBS
9 .. 14  =  6 bp
ribosome binding site
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