pMCSG68 (linearized)

Linearized compact bacterial vector for ligation-independent cloning (LIC), with a 6xHis-Strep-Tag® II-TEV leader plus tRNA genes for rare Arg and Ile codons.

Sequence Author: Midwest Center for Structural Genomics

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BlpI (4693) T7 terminator BspEI (4615) ScaI (3918) PvuI (3808) FspI (3660) BglI (3558) AhdI (3438) AlwNI (2961) DrdI (2653) PciI (2545) BspQI - SapI (2429) BstZ17I (2316) BsaAI (2297) PshAI (2290) BsmBI - Esp3I (2060) HpaI (1951) AvaI - BsoBI - PaeR7I - PspXI - XhoI (4771) EagI - NotI (4779) HindIII (4786) SalI (4792) Eco53kI (4801) SacI (4803) EcoRI (4805) BamHI (4811) End (4817) Start (0) TEV site Acc65I (25) KpnI (29) Strep-Tag II BglII (56) ATG NdeI (92) RBS XbaI (130) T7 promoter BspDI * - ClaI * (199) AgeI (241) AscI (458) SbfI (469) BfuAI - BspMI (487) MreI (606) AfeI (692) NcoI (876) PsiI (891) EcoNI (980) PfoI (1012) PflMI (1027) BstAPI (1128) MluI (1445) BclI * (1459) BstEII (1626) PspOMI (1652) ApaI (1656) EcoRV (1895) pMCSG68 4817 bp
BlpI  (4693)
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.
BspEI  (4615)
1 site
T C C G G A A G G C C T
ScaI  (3918)
1 site
A G T A C T T C A T G A
PvuI  (3808)
1 site
C G A T C G G C T A G C
FspI  (3660)
1 site
T G C G C A A C G C G T
BglI  (3558)
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.
AhdI  (3438)
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  (2961)
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.
DrdI  (2653)
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  (2545)
1 site
A C A T G T T G T A C A

PciI is inhibited by nonionic detergents.
BspQI  (2429)
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  (2429)
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  (2316)
1 site
G T A T A C C A T A T G
BsaAI  (2297)
1 site
Y A C G T R R T G C A Y
PshAI  (2290)
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  (2060)
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  (2060)
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  (1951)
1 site
G T T A A C C A A T T G
AvaI  (4771)
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  (4771)
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  (4771)
1 site
C T C G A G G A G C T C

PaeR7I does not recognize the sequence CTCTCGAG.
PspXI  (4771)
1 site
V C T C G A G B B G A G C T C V
XhoI  (4771)
1 site
C T C G A G G A G C T C
EagI  (4779)
1 site
C G G C C G G C C G G C
NotI  (4779)
1 site
G C G G C C G C C G C C G G C G
HindIII  (4786)
1 site
A A G C T T T T C G A A
SalI  (4792)
1 site
G T C G A C C A G C T G
Eco53kI  (4801)
1 site
G A G C T C C T C G A G
SacI  (4803)
1 site
G A G C T C C T C G A G
EcoRI  (4805)
1 site
G A A T T C C T T A A G
BamHI  (4811)
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  (4817)
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
BglII  (56)
1 site
A G A T C T T C T A G A
NdeI  (92)
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  (130)
1 site
T C T A G A A G A T C T
BspDI  (199)
1 site
A T C G A T T A G C T A
* Blocked by Dam methylation.
ClaI  (199)
1 site
A T C G A T T A G C T A
* Blocked by Dam methylation.
AgeI  (241)
1 site
A C C G G T T G G C C A
AscI  (458)
1 site
G G C G C G C C C C G C G C G G
SbfI  (469)
1 site
C C T G C A G G G G A C G T C C
BfuAI  (487)
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  (487)
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  (606)
1 site
C G C C G G C G G C G G C C G C
AfeI  (692)
1 site
A G C G C T T C G C G A
NcoI  (876)
1 site
C C A T G G G G T A C C
PsiI  (891)
1 site
T T A T A A A A T A T T
EcoNI  (980)
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  (1012)
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  (1027)
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  (1128)
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  (1445)
1 site
A C G C G T T G C G C A
BclI  (1459)
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  (1626)
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  (1652)
1 site
G G G C C C C C C G G G
ApaI  (1656)
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  (1895)
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.
lacI
1095 .. 2177  =  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
1095 .. 2177  =  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
3365 .. 4225  =  861 bp
286 amino acids  =  31.5 kDa
2 segments
   Segment 2:  
   3365 .. 4156  =  792 bp
   263 amino acids  =  28.9 kDa
Product: β-lactamase
confers resistance to ampicillin, carbenicillin, and related antibiotics
AmpR
3365 .. 4225  =  861 bp
286 amino acids  =  31.5 kDa
2 segments
   Segment 1:  signal sequence  
   4157 .. 4225  =  69 bp
   23 amino acids  =  2.6 kDa
Product: β-lactamase
confers resistance to ampicillin, carbenicillin, and related antibiotics
AmpR
3365 .. 4225  =  861 bp
286 amino acids  =  31.5 kDa
2 segments
Product: β-lactamase
confers resistance to ampicillin, carbenicillin, and related antibiotics
ori
2606 .. 3194  =  589 bp
high-copy-number ColE1/pMB1/pBR322/pUC origin of replication
ori
2606 .. 3194  =  589 bp
high-copy-number ColE1/pMB1/pBR322/pUC origin of replication
AmpR promoter
4226 .. 4329  =  104 bp
AmpR promoter
4226 .. 4329  =  104 bp
TEV site
4 .. 24  =  21 bp
7 amino acids  =  900.0 Da