pIEx-3

Vector for high-level expression in insect cells of secreted proteins with a cleavable N-terminal GST-6xHis-S-Tag cassette plus a C-terminal HSV tag.

Sequence Author: MilliporeSigma (Novagen)

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DrdI (4161) AhdI (3375) BsaI (3309) BpmI (3306) NmeAIII (3228) AatII (2456) ZraI (2454) XbaI (2448) PflFI - Tth111I (2393) BspDI * - ClaI * (207) EcoRV (239) PsiI (409) NheI (606) BmtI (610) SphI (614) BsmI (775) BsiWI (784) MluI (872) BsaBI (1111) BbvCI - Bpu10I (1133) BtgI - NcoI (1148) EcoNI (1163) EcoO109I (1185) MscI (1360) BsgI (1439) SwaI (1580) SpeI (1819) 6xHis XcmI (1852) PflMI (1896) thrombin site PshAI (1970) BseRI (1974) BamHI (1992) MfeI (1998) Eco53kI (2007) BanII - SacI (2009) AscI (2020) PstI - SbfI (2030) SalI (2032) Acc65I (2038) AgeI (2041) KpnI (2042) HindIII (2050) NotI (2057) BstZ17I (2072) PmlI (2077) PaeR7I - XhoI (2119) DraIII (2130) Bsu36I (2140) PacI (2169) IE1 terminator pIEx™-3 4629 bp
DrdI  (4161)
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.
AhdI  (3375)
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.
BsaI  (3309)
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.
BpmI  (3306)
1 site
C T G G A G ( N ) 14 N N G A C C T C ( N ) 14

Efficient cleavage requires at least two copies of the BpmI recognition sequence.
Sticky ends from different BpmI sites may not be compatible.
After cleavage, BpmI can remain bound to DNA and alter its electrophoretic mobility.
BpmI quickly loses activity at 37°C.
NmeAIII  (3228)
1 site
G C C G A G ( N ) 18-19 N N C G G C T C ( N ) 18-19

Efficient cleavage requires at least two copies of the NmeAIII recognition sequence.
Sticky ends from different NmeAIII sites may not be compatible.
For full activity, add fresh S-adenosylmethionine (SAM).
AatII  (2456)
1 site
G A C G T C C T G C A G
ZraI  (2454)
1 site
G A C G T C C T G C A G
XbaI  (2448)
1 site
T C T A G A A G A T C T
PflFI  (2393)
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  (2393)
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.
BspDI  (207)
1 site
A T C G A T T A G C T A
* Blocked by Dam methylation.
ClaI  (207)
1 site
A T C G A T T A G C T A
* Blocked by Dam methylation.
EcoRV  (239)
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.
PsiI  (409)
1 site
T T A T A A A A T A T T
NheI  (606)
1 site
G C T A G C C G A T C G
BmtI  (610)
1 site
G C T A G C C G A T C G
SphI  (614)
1 site
G C A T G C C G T A C G
BsmI  (775)
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.
BsiWI  (784)
1 site
C G T A C G G C A T G C

BsiWI is typically used at 55°C, but is 50% active at 37°C.
MluI  (872)
1 site
A C G C G T T G C G C A
BsaBI  (1111)
1 site
G A T N N N N A T C C T A N N N N T A G
BbvCI  (1133)
1 site
C C T C A G C G G A G T C G
Bpu10I  (1133)
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.
BtgI  (1148)
1 site
C C R Y G G G G Y R C C

Sticky ends from different BtgI sites may not be compatible.
NcoI  (1148)
1 site
C C A T G G G G T A C C
EcoNI  (1163)
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.
EcoO109I  (1185)
1 site
R G G N C C Y Y C C N G G R

Sticky ends from different EcoO109I sites may not be compatible.
MscI  (1360)
1 site
T G G C C A A C C G G T
BsgI  (1439)
1 site
G T G C A G ( N ) 14 N N C A C G T C ( N ) 14

Efficient cleavage requires at least two copies of the BsgI recognition sequence.
Sticky ends from different BsgI sites may not be compatible.
For full activity, add fresh S-adenosylmethionine (SAM).
SwaI  (1580)
1 site
A T T T A A A T T A A A T T T A

SwaI is typically used at 25°C, but is 50% active at 37°C.
SpeI  (1819)
1 site
A C T A G T T G A T C A
XcmI  (1852)
1 site
C C A N N N N N N N N N T G G G G T N N N N N N N N N A C C

The 1-base overhangs produced by XcmI may be hard to ligate.
Sticky ends from different XcmI sites may not be compatible.
PflMI  (1896)
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.
PshAI  (1970)
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.
BseRI  (1974)
1 site
G A G G A G ( N ) 8 N N C T C C T C ( N ) 8

Sticky ends from different BseRI sites may not be compatible.
BseRI quickly loses activity at 37°C.
Prolonged incubation with BseRI may lead to degradation of the DNA.
BamHI  (1992)
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.
MfeI  (1998)
1 site
C A A T T G G T T A A C
Eco53kI  (2007)
1 site
G A G C T C C T C G A G
BanII  (2009)
1 site
G R G C Y C C Y C G R G

Sticky ends from different BanII sites may not be compatible.
SacI  (2009)
1 site
G A G C T C C T C G A G
AscI  (2020)
1 site
G G C G C G C C C C G C G C G G
PstI  (2030)
1 site
C T G C A G G A C G T C
SbfI  (2030)
1 site
C C T G C A G G G G A C G T C C
SalI  (2032)
1 site
G T C G A C C A G C T G
Acc65I  (2038)
1 site
G G T A C C C C A T G G
AgeI  (2041)
1 site
A C C G G T T G G C C A
KpnI  (2042)
1 site
G G T A C C C C A T G G
HindIII  (2050)
1 site
A A G C T T T T C