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Plasmid Files

pMCSG10 (linearized)

Linearized bacterial vector for ligation-independent cloning (LIC), with a 6xHis-GST-TEV leader.

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pMCSG10 (linearized) Sequence and MappMCSG10 (linearized).dna
Map and Sequence File   
Sequence Author:  Midwest Center for Structural Genomics
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 T7 terminator PvuI (4937) PstI (4812) BsaI (4628) AhdI (4567) AlwNI (4090) BstZ17I (3445) BsaAI (3426) PflFI - Tth111I (3419) Bpu10I (2780) PpuMI (2680) FspAI (2655) StyI (5799) BlpI (5822) AvaI - BsoBI - PaeR7I - PspXI - XhoI (5900) BmeT110I (5901) EagI - NotI (5908) HindIII (5915) SalI (5921) Eco53kI (5930) SacI (5932) EcoRI (5934) BamHI (5940) End (5946) Start (0) Acc65I (25) KpnI (29) SwaI (277) BstBI (305) MscI (497) 6xHis Feature 14 NdeI (743) RBS XbaI (781) T7 promoter BspDI * - ClaI * (850) SgrAI (892) SphI (1048) PflMI (1155) BstAPI (1256) MluI (1573) BstEII (1754) PspOMI (1780) ApaI (1784) BssHII (1984) EcoRV (2023) HpaI (2079) PshAI (2418) pMCSG10 5946 bp
PvuI  (4937)
1 site
C G A T C G G C T A G C
PstI  (4812)
1 site
C T G C A G G A C G T C
BsaI  (4628)
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  (4567)
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  (4090)
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.
BstZ17I  (3445)
1 site
G T A T A C C A T A T G
BsaAI  (3426)
1 site
Y A C G T R R T G C A Y
PflFI  (3419)
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  (3419)
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  (2780)
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  (2680)
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  (2655)
1 site
R T G C G C A Y Y A C G C G T R
StyI  (5799)
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  (5822)
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.
AvaI  (5900)
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  (5900)
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  (5900)
1 site
C T C G A G G A G C T C

PaeR7I does not recognize the sequence CTCTCGAG.
PspXI  (5900)
1 site
V C T C G A G B B G A G C T C V
XhoI  (5900)
1 site
C T C G A G G A G C T C
BmeT110I  (5901)
1 site
C Y C G R G G R G C Y C
EagI  (5908)
1 site
C G G C C G G C C G G C
NotI  (5908)
1 site
G C G G C C G C C G C C G G C G
HindIII  (5915)
1 site
A A G C T T T T C G A A
SalI  (5921)
1 site
G T C G A C C A G C T G
Eco53kI  (5930)
1 site
G A G C T C C T C G A G
SacI  (5932)
1 site
G A G C T C C T C G A G
EcoRI  (5934)
1 site
G A A T T C C T T A A G
BamHI  (5940)
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.
End  (5946)
0 sites
Start  (0)
0 sites
Acc65I  (25)
1 site
G G T A C C C C A T G G
KpnI  (29)
1 site
G G T A C C C C A T G G
SwaI  (277)
1 site
A T T T A A A T T A A A T T T A

SwaI is typically used at 25°C, but is 50% active at 37°C.
BstBI  (305)
1 site
T T C G A A A A G C T T
MscI  (497)
1 site
T G G C C A A C C G G T
NdeI  (743)
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  (781)
1 site
T C T A G A A G A T C T
BspDI  (850)
1 site
A T C G A T T A G C T A
* Blocked by Dam methylation.
ClaI  (850)
1 site
A T C G A T T A G C T A
* Blocked by Dam methylation.
SgrAI  (892)
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  (1048)
1 site
G C A T G C C G T A C G
PflMI  (1155)
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  (1256)
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  (1573)
1 site
A C G C G T T G C G C A
BstEII  (1754)
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  (1780)
1 site
G G G C C C C C C G G G
ApaI  (1784)
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  (1984)
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  (2023)
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  (2079)
1 site
G T T A A C C A A T T G
PshAI  (2418)
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.
lacI
1223 .. 2305  =  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
1223 .. 2305  =  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
4494 .. 5354  =  861 bp
286 amino acids  =  31.5 kDa
   Segment 2:  
   4494 .. 5285  =  792 bp
   263 amino acids  =  28.9 kDa
Product: β-lactamase
confers resistance to ampicillin, carbenicillin, and
related antibiotics
AmpR
4494 .. 5354  =  861 bp
286 amino acids  =  31.5 kDa
   Segment 1:  signal sequence  
   5286 .. 5354  =  69 bp
   23 amino acids  =  2.6 kDa
Product: β-lactamase
confers resistance to ampicillin, carbenicillin, and
related antibiotics
AmpR
4494 .. 5354  =  861 bp
286 amino acids  =  31.5 kDa
2 segments
Product: β-lactamase
confers resistance to ampicillin, carbenicillin, and
related antibiotics
GST
52 .. 705  =  654 bp
218 amino acids  =  25.5 kDa
Product: glutathione S-transferase from
Schistosoma japonicum
GST
52 .. 705  =  654 bp
218 amino acids  =  25.5 kDa
Product: glutathione S-transferase from
Schistosoma japonicum
ori
3735 .. 4323  =  589 bp
high-copy-number ColE1/pMB1/pBR322/pUC origin
of replication
ori
3735 .. 4323  =  589 bp
high-copy-number ColE1/pMB1/pBR322/pUC origin
of replication
rop
3114 .. 3305  =  192 bp
63 amino acids  =  7.2 kDa
Product: Rop protein, which maintains plasmids at
low copy number
rop
3114 .. 3305  =  192 bp
63 amino acids  =  7.2 kDa
Product: Rop protein, which maintains plasmids at
low copy number
AmpR promoter
5355 .. 5458  =  104 bp
AmpR promoter
5355 .. 5458  =  104 bp
lacI promoter
1145 .. 1222  =  78 bp
lacI promoter
1145 .. 1222  =  78 bp
T7 terminator
5768 .. 5815  =  48 bp
transcription terminator for bacteriophage T7 RNA
polymerase
T7 terminator
5768 .. 5815  =  48 bp
transcription terminator for bacteriophage T7 RNA
polymerase
lac operator
789 .. 813  =  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
789 .. 813  =  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
4 .. 24  =  21 bp
7 amino acids  =  900.0 Da
Product: tobacco etch virus (TEV) protease
recognition and cleavage site
TEV site
4 .. 24  =  21 bp
7 amino acids  =  900.0 Da
Product: tobacco etch virus (TEV) protease
recognition and cleavage site
T7 promoter
814 .. 832  =  19 bp
promoter for bacteriophage T7 RNA polymerase
T7 promoter
814 .. 832  =  19 bp
promoter for bacteriophage T7 RNA polymerase
6xHis
724 .. 741  =  18 bp
6 amino acids  =  840.9 Da
Product: 6xHis affinity tag
6xHis
724 .. 741  =  18 bp
6 amino acids  =  840.9 Da
Product: 6xHis affinity tag
6xHis
5882 .. 5899  =  18 bp
6 amino acids  =  840.9 Da
Product: 6xHis affinity tag
6xHis
5882 .. 5899  =  18 bp
6 amino acids  =  840.9 Da
Product: 6xHis affinity tag
RBS
753 .. 758  =  6 bp
ribosome binding site
RBS
753 .. 758  =  6 bp
ribosome binding site
Feature 14
742 .. 744  =  3 bp
1 amino acid  =  149.2 Da
Product: start codon
Feature 14
742 .. 744  =  3 bp
1 amino acid  =  149.2 Da
Product: start codon
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