pMCSG43 (linearized)

Linearized bacterial vector for ligation-independent cloning (LIC), encoding E. coli DsbA plus OsmY followed by an N-terminal TVMV site and C-terminal TEV-6xHis.

Sequence Author: Midwest Center for Structural Genomics

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T7 terminator ScaI (5823) PvuI (5713) BsaI (5404) AhdI (5343) PciI (4450) BspQI - SapI (4334) BstZ17I (4221) BsaAI (4202) PpuMI (3456) FspAI (3431) StyI (6575) AvaI - BsoBI - PaeR7I - PspXI - XhoI (6676) EagI - NotI (6684) HindIII (6691) SalI (6697) Eco53kI (6706) SacI (6708) EcoRI (6710) BamHI (6716) TEV site End (6763) Start (0) BsrGI (43) DraIII (123) Acc65I (140) KpnI (144) BstBI (625) NdeI (651) RBS XbaI (689) T7 promoter BspDI * - ClaI * (758) SgrAI (800) AgeI (1288) AscI (1299) BglII (1456) BfuAI - BspMI (1580) MfeI (1682) EcoNI (1884) PflMI (1931) BstAPI (2032) MluI (2349) BclI * (2363) PspOMI (2556) ApaI (2560) pMCSG43 6763 bp
ScaI  (5823)
1 site
A G T A C T T C A T G A
PvuI  (5713)
1 site
C G A T C G G C T A G C
BsaI  (5404)
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  (5343)
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  (4450)
1 site
A C A T G T T G T A C A

PciI is inhibited by nonionic detergents.
BspQI  (4334)
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  (4334)
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  (4221)
1 site
G T A T A C C A T A T G
BsaAI  (4202)
1 site
Y A C G T R R T G C A Y
PpuMI  (3456)
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  (3431)
1 site
R T G C G C A Y Y A C G C G T R
StyI  (6575)
1 site
C C W W G G G G W W C C

Sticky ends from different StyI sites may not be compatible.
AvaI  (6676)
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  (6676)
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  (6676)
1 site
C T C G A G G A G C T C

PaeR7I does not recognize the sequence CTCTCGAG.
PspXI  (6676)
1 site
V C T C G A G B B G A G C T C V
XhoI  (6676)
1 site
C T C G A G G A G C T C
EagI  (6684)
1 site
C G G C C G G C C G G C
NotI  (6684)
1 site
G C G G C C G C C G C C G G C G
HindIII  (6691)
1 site
A A G C T T T T C G A A
SalI  (6697)
1 site
G T C G A C C A G C T G
Eco53kI  (6706)
1 site
G A G C T C C T C G A G
SacI  (6708)
1 site
G A G C T C C T C G A G
EcoRI  (6710)
1 site
G A A T T C C T T A A G
BamHI  (6716)
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  (6763)
0 sites
Start  (0)
0 sites
BsrGI  (43)
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.
DraIII  (123)
1 site
C A C N N N G T G G T G N N N C A C

Sticky ends from different DraIII sites may not be compatible.
Acc65I  (140)
1 site
G G T A C C C C A T G G
KpnI  (144)
1 site
G G T A C C C C A T G G
BstBI  (625)
1 site
T T C G A A A A G C T T
NdeI  (651)
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  (689)
1 site
T C T A G A A G A T C T
BspDI  (758)
1 site
A T C G A T T A G C T A
* Blocked by Dam methylation.
ClaI  (758)
1 site
A T C G A T T A G C T A
* Blocked by Dam methylation.
SgrAI  (800)
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.
AgeI  (1288)
1 site
A C C G G T T G G C C A
AscI  (1299)
1 site
G G C G C G C C C C G C G C G G
BglII  (1456)
1 site
A G A T C T T C T A G A
BfuAI  (1580)
1 site
A C C T G C ( N ) 4 T G G A C G ( N ) 4 ( N ) 4

Efficient cleavage requires at least two copies of the BfuAI recognition sequence.
Sticky ends from different BfuAI sites may not be compatible.
BfuAI is typically used at 50°C, but is 50% active at 37°C.
BspMI  (1580)
1 site
A C C T G C ( N ) 4 T G G A C G ( N ) 4 ( N ) 4

Efficient cleavage requires at least two copies of the BspMI recognition sequence.
Sticky ends from different BspMI sites may not be compatible.
MfeI  (1682)
1 site
C A A T T G G T T A A C
EcoNI  (1884)
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  (1931)
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  (2032)
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  (2349)
1 site
A C G C G T T G C G C A
BclI  (2363)
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.
PspOMI  (2556)
1 site
G G G C C C C C C G G G
ApaI  (2560)
1 site
G G G C C C C C C G G G

ApaI can be used between 25°C and 37°C.
lacI
1999 .. 3081  =  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
1999 .. 3081  =  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
5270 .. 6130  =  861 bp
286 amino acids  =  31.5 kDa
2 segments
   Segment 2:  
   5270 .. 6061  =  792 bp
   263 amino acids  =  28.9 kDa
Product: β-lactamase
confers resistance to ampicillin, carbenicillin, and related antibiotics
AmpR
5270 .. 6130  =  861 bp
286 amino acids  =  31.5 kDa
2 segments
   Segment 1:  signal sequence  
   6062 .. 6130  =  69 bp
   23 amino acids  =  2.6 kDa
Product: β-lactamase
confers resistance to ampicillin, carbenicillin, and related antibiotics
AmpR
5270 .. 6130  =  861 bp
286 amino acids  =  31.5 kDa
2 segments
Product: β-lactamase
confers resistance to ampicillin, carbenicillin, and related antibiotics
TVMV site
8 .. 28  =  21 bp
7 amino acids  =  865.9 Da
Product:
tobacco vein mottling virus (TVMV) NIa protease recognition and cleavage site
TVMV site
8 .. 28  =  21 bp
7 amino acids  =  865.9 Da
Product:
tobacco vein mottling virus (TVMV) NIa protease recognition and cleavage site
OsmY
50 .. 652  =  603 bp
201 amino acids  =  21.1 kDa
Product: osmotically inducible protein OsmY from E. coli
OsmY
50 .. 652  =  603 bp
201 amino acids  =  21.1 kDa
Product: osmotically inducible protein OsmY from E. coli
DsbA
1190 .. 1816  =  627 bp
208 amino acids  =  23.1 kDa
Product: bacterial periplasmic oxidoreductase
DsbA
1190 .. 1816  =  627 bp
208 amino acids  =  23.1 kDa
Product: bacterial periplasmic oxidoreductase
ori
4511 .. 5099  =  589 bp
high-copy-number ColE1/pMB1/pBR322/pUC origin of replication
ori
4511 .. 5099  =  589 bp
high-copy-number ColE1/pMB1/pBR322/pUC origin of r