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pMCSG68

Compact bacterial vector encoding tRNA genes for rare Arg and Ile codons, with a 6xHis-Strep-Tag® II-TEV leader for high-throughput purification of recombinant proteins.

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pMCSG68.dna
Map and Sequence File:    Download    Open   
Sequence Author:  Midwest Center for Structural Genomics
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ScaI (4137) PvuI (4027) FspI (3879) BglI (3777) AhdI (3657) AlwNI (3180) DrdI (2872) PciI (2764) BspQI - SapI (2648) BstZ17I (2535) BsaAI (2516) PshAI (2509) BspEI (2) BlpI (80) AvaI - BsoBI - PaeR7I - PspXI - XhoI (158) BmeT110I (159) EagI - NotI (166) HindIII (173) SalI (179) Eco53kI (188) SacI (190) EcoRI (192) BamHI (198) SspI (219) Acc65I (244) KpnI (248) BglII (275) NdeI (311) XbaI (349) BspDI * - ClaI * (418) AgeI (460) AscI (677) SbfI (688) BfuAI - BspMI (706) MreI (825) AfeI (911) NcoI (1095) PsiI (1110) EcoNI (1199) PfoI (1231) PflMI (1246) BstAPI (1347) MluI (1664) BclI * (1678) BstEII (1845) PspOMI (1871) ApaI (1875) EcoRV (2114) HpaI (2170) BsmBI (2279) pMCSG68 4832 bp
ScaI  (4137)
1 site
A G T A C T T C A T G A
PvuI  (4027)
1 site
C G A T C G G C T A G C
FspI  (3879)
1 site
T G C G C A A C G C G T
BglI  (3777)
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  (3657)
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  (3180)
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  (2872)
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  (2764)
1 site
A C A T G T T G T A C A

PciI is inhibited by nonionic detergents.
BspQI  (2648)
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  (2648)
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  (2535)
1 site
G T A T A C C A T A T G
BsaAI  (2516)
1 site
Y A C G T R R T G C A Y
PshAI  (2509)
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.
BspEI  (2)
1 site
T C C G G A A G G C C T
BlpI  (80)
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  (158)
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  (158)
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  (158)
1 site
C T C G A G G A G C T C

PaeR7I does not recognize the sequence CTCTCGAG.
PspXI  (158)
1 site
V C T C G A G B B G A G C T C V
XhoI  (158)
1 site
C T C G A G G A G C T C
BmeT110I  (159)
1 site
C Y C G R G G R G C Y C
EagI  (166)
1 site
C G G C C G G C C G G C
NotI  (166)
1 site
G C G G C C G C C G C C G G C G
HindIII  (173)
1 site
A A G C T T T T C G A A
SalI  (179)
1 site
G T C G A C C A G C T G
Eco53kI  (188)
1 site
G A G C T C C T C G A G
SacI  (190)
1 site
G A G C T C C T C G A G
EcoRI  (192)
1 site
G A A T T C C T T A A G
BamHI  (198)
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.
SspI  (219)
1 site
A A T A T T T T A T A A
Acc65I  (244)
1 site
G G T A C C C C A T G G
KpnI  (248)
1 site
G G T A C C C C A T G G
BglII  (275)
1 site
A G A T C T T C T A G A
NdeI  (311)
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  (349)
1 site
T C T A G A A G A T C T
BspDI  (418)
1 site
A T C G A T T A G C T A
* Blocked by Dam methylation.
ClaI  (418)
1 site
A T C G A T T A G C T A
* Blocked by Dam methylation.
AgeI  (460)
1 site
A C C G G T T G G C C A
AscI  (677)
1 site
G G C G C G C C C C G C G C G G
SbfI  (688)
1 site
C C T G C A G G G G A C G T C C
BfuAI  (706)
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  (706)
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  (825)
1 site
C G C C G G C G G C G G C C G C
AfeI  (911)
1 site
A G C G C T T C G C G A
NcoI  (1095)
1 site
C C A T G G G G T A C C
PsiI  (1110)
1 site
T T A T A A A A T A T T
EcoNI  (1199)
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  (1231)
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  (1246)
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  (1347)
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  (1664)
1 site
A C G C G T T G C G C A
BclI  (1678)
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  (1845)
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  (1871)
1 site
G G G C C C C C C G G G
ApaI  (1875)
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  (2114)
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  (2170)
1 site
G T T A A C C A A T T G
BsmBI  (2279)
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.
lacI
1314 .. 2396  =  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
1314 .. 2396  =  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
3584 .. 4444  =  861 bp
286 amino acids  =  31.5 kDa
   Segment 2:  
   3584 ..