pMCSG75 (linearized)

Linearized bacterial vector for ligation-independent cloning (LIC), with a C-terminal TEV-AviTag™-6xHis cassette, the BirA biotin ligase, and tRNA genes for rare Arg and Ile codons.

Sequence Author: Midwest Center for Structural Genomics

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BspEI (4522) ScaI (3825) FspI (3567) BglI (3465) BsaI (3406) AhdI (3345) DrdI (2560) PciI (2452) BspQI - SapI (2336) BstZ17I (2223) PshAI (2197) BsmBI - Esp3I (1967) HpaI (1858) EcoRV (1802) ApaI (1563) PspOMI (1559) BstEII (1533) BlpI (4600) AvaI - BsoBI - PaeR7I - PspXI - XhoI (4678) EagI - NotI (4686) HindIII (4693) SalI (4699) Eco53kI (4708) SacI (4710) EcoRI (4712) BsmI (5171) DraIII (5599) RBS 6xHis TEV site End (5866) Start (0) RBS XbaI (37) T7 promoter BspDI * - ClaI * (106) AgeI (148) AscI (365) SbfI (376) BfuAI - BspMI (394) MreI (513) AfeI (599) NcoI (783) PsiI (798) EcoNI (887) PfoI (919) PflMI (934) BstAPI (1035) MluI (1352) BclI * (1366) pMCSG75 5866 bp
BspEI  (4522)
1 site
T C C G G A A G G C C T
ScaI  (3825)
1 site
A G T A C T T C A T G A
FspI  (3567)
1 site
T G C G C A A C G C G T
BglI  (3465)
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.
BsaI  (3406)
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  (3345)
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  (2560)
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  (2452)
1 site
A C A T G T T G T A C A

PciI is inhibited by nonionic detergents.
BspQI  (2336)
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  (2336)
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  (2223)
1 site
G T A T A C C A T A T G
PshAI  (2197)
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  (1967)
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.
BsmBI-v2 is an improved version of BsmBI.
Esp3I  (1967)
1 site
C G T C T C N G C A G A G N ( N ) 4

Sticky ends from different Esp3I sites may not be compatible.
HpaI  (1858)
1 site
G T T A A C C A A T T G
EcoRV  (1802)
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  (1563)
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  (1559)
1 site
G G G C C C C C C G G G
BstEII  (1533)
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.
BlpI  (4600)
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  (4678)
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  (4678)
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  (4678)
1 site
C T C G A G G A G C T C

PaeR7I does not recognize the sequence CTCTCGAG.
PspXI  (4678)
1 site
V C T C G A G B B G A G C T C V
XhoI  (4678)
1 site
C T C G A G G A G C T C
EagI  (4686)
1 site
C G G C C G G C C G G C
NotI  (4686)
1 site
G C G G C C G C C G C C G G C G
HindIII  (4693)
1 site
A A G C T T T T C G A A
SalI  (4699)
1 site
G T C G A C C A G C T G
Eco53kI  (4708)
1 site
G A G C T C C T C G A G
SacI  (4710)
1 site
G A G C T C C T C G A G
EcoRI  (4712)
1 site
G A A T T C C T T A A G
BsmI  (5171)
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.
DraIII  (5599)
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.
End  (5866)
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.
AgeI  (148)
1 site
A C C G G T T G G C C A
AscI  (365)
1 site
G G C G C G C C C C G C G C G G
SbfI  (376)
1 site
C C T G C A G G G G A C G T C C
BfuAI  (394)
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  (394)
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.
MreI  (513)
1 site
C G C C G G C G G C G G C C G C
AfeI  (599)
1 site
A G C G C T T C G C G A
NcoI  (783)
1 site
C C A T G G G G T A C C
PsiI  (798)
1 site
T T A T A A A A T A T T
EcoNI  (887)
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.
PfoI  (919)
1 site
T C C N G G A A G G N C C T

Sticky ends from different PfoI sites may not be compatible.
PflMI  (934)
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  (1035)
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  (1352)
1 site
A C G C G T T G C G C A
BclI  (1366)
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.
lacI
1002 .. 2084  =  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
1002 .. 2084  =  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).
BirA
4761 .. 5726  =  966 bp
321 amino acids  =  35.3 kDa
Product: E. coli biotin ligase
BirA
4761 .. 5726  =  966 bp
321 amino acids  =  35.3 kDa
Product: E. coli biotin ligase
AmpR
3272 .. 4132  =  861 bp
286 amino acids  =  31.5 kDa
2 segments
   Segment 2:  
   3272 .. 4063  =  792 bp
   263 amino acids  =  28.9 kDa
Product: β-lactamase
confers resistance to ampicillin, carbenicillin, and related antibiotics
AmpR
3272 .. 4132  =  861 bp
286 amino acids  =  31.5 kDa
2 segments
   Segment 1:  signal sequence  
   4064 .. 4132  =