pET-43.1 Ek_LIC (linearized)

Linearized bacterial vector for ligation-independent cloning (LIC) to express NusA-tagged proteins with an enterokinase site.

Sequence Author: MilliporeSigma (Novagen)

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DraIII (6354) PsiI (6226) AhdI (5942) BglI (5824) FspI (5719) ScaI (5461) AlwNI (4748) PciI (4332) BspQI - SapI (4216) PflFI - Tth111I (4077) Bpu10I (3438) PpuMI (3338) HpaI (2974) ApaI (2679) T7 terminator AvrII (7079) PacI (7099) PaeR7I - XhoI (7126) PmlI (7172) EagI - NotI (7188) HindIII (7195) Acc65I (7216) KpnI (7220) SalI - SgrDI (7222) PstI - SbfI (7235) AscI (7237) BsrGI (7244) EcoRI (7250) BamHI (7256) Eco53kI (7266) SacI (7268) End (7279) Start (0) TspMI - XmaI (40) SmaI (42) thrombin site SacII (118) 6xHis SpeI (149) BglII (264) BsmI (308) MscI (396) StuI (449) BfuAI - BspMI (598) NcoI (724) NdeI (1638) RBS XbaI (1676) T7 promoter SphI (1943) EcoNI (2003) BstEII (2649) PspOMI (2675) pET-43.1 Ek/LIC 7279 bp
DraIII  (6354)
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.
PsiI  (6226)
1 site
T T A T A A A A T A T T
AhdI  (5942)
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.
BglI  (5824)
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.
FspI  (5719)
1 site
T G C G C A A C G C G T
ScaI  (5461)
1 site
A G T A C T T C A T G A
AlwNI  (4748)
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.
PciI  (4332)
1 site
A C A T G T T G T A C A

PciI is inhibited by nonionic detergents.
BspQI  (4216)
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  (4216)
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.
PflFI  (4077)
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  (4077)
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  (3438)
1 site
C C T N A G C G G A N T C G

Cleavage may be enhanced when more than one copy of the Bpu10I recognition sequence is present.
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  (3338)
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.
HpaI  (2974)
1 site
G T T A A C C A A T T G
ApaI  (2679)
1 site
G G G C C C C C C G G G

ApaI can be used between 25°C and 37°C.
AvrII  (7079)
1 site
C C T A G G G G A T C C
PacI  (7099)
1 site
T T A A T T A A A A T T A A T T
PaeR7I  (7126)
1 site
C T C G A G G A G C T C

PaeR7I does not recognize the sequence CTCTCGAG.
XhoI  (7126)
1 site
C T C G A G G A G C T C
PmlI  (7172)
1 site
C A C G T G G T G C A C
EagI  (7188)
1 site
C G G C C G G C C G G C
NotI  (7188)
1 site
G C G G C C G C C G C C G G C G
HindIII  (7195)
1 site
A A G C T T T T C G A A
Acc65I  (7216)
1 site
G G T A C C C C A T G G
KpnI  (7220)
1 site
G G T A C C C C A T G G
SalI  (7222)
1 site
G T C G A C C A G C T G
SgrDI  (7222)
1 site
C G T C G A C G G C A G C T G C
PstI  (7235)
1 site
C T G C A G G A C G T C
SbfI  (7235)
1 site
C C T G C A G G G G A C G T C C
AscI  (7237)
1 site
G G C G C G C C C C G C G C G G
BsrGI  (7244)
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.
EcoRI  (7250)
1 site
G A A T T C C T T A A G
BamHI  (7256)
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.
Eco53kI  (7266)
1 site
G A G C T C C T C G A G
SacI  (7268)
1 site
G A G C T C C T C G A G
End  (7279)
0 sites
Start  (0)
0 sites
TspMI  (40)
1 site
C C C G G G G G G C C C
XmaI  (40)
1 site
C C C G G G G G G C C C

Cleavage may be enhanced when more than one copy of the XmaI recognition sequence is present.
SmaI  (42)
1 site
C C C G G G G G G C C C

SmaI can be used at 37°C for brief incubations.
SacII  (118)
1 site
C C G C G G G G C G C C

Efficient cleavage requires at least two copies of the SacII recognition sequence.
SpeI  (149)
1 site
A C T A G T T G A T C A
BglII  (264)
1 site
A G A T C T T C T A G