pIEx-1

Vector for high-level expression in insect cells of proteins with a cleavable N-terminal 10xHis-S-Tag cassette plus a C-terminal HSV tag.

Sequence Author: MilliporeSigma (Novagen)

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BspQI - SapI (3648) PciI (3531) DrdI (3429) PspFI (3231) BseYI (3227) AhdI (2643) BsaI (2577) BpmI (2574) NmeAIII (2496) ScaI (2162) XmnI (2043) BspDI * - ClaI * (207) EcoRV (239) PsiI (409) NheI (606) BmtI (610) SphI (614) BsmI (775) BclI * (778) BsiWI (784) MluI (872) BtgI - NcoI (1082) XcmI (1120) PflMI (1164) thrombin site PshAI (1238) BseRI (1242) BamHI (1260) MfeI (1266) Eco53kI (1275) BanII - SacI (1277) BsrGI (1280) AscI (1288) PstI - SbfI (1298) SalI (1300) Acc65I (1306) AgeI (1309) KpnI (1310) BstBI (1315) HindIII (1318) NotI (1325) BstZ17I (1340) PmlI (1345) BbsI (1379) PaeR7I - XhoI (1387) DraIII (1398) Bsu36I (1408) PacI (1437) PflFI - Tth111I (1661) BtgZI (1684) XbaI (1716) ZraI (1722) AatII (1724) pIEx™-1 3897 bp
BspQI  (3648)
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  (3648)
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.
PciI  (3531)
1 site
A C A T G T T G T A C A

PciI is inhibited by nonionic detergents.
DrdI  (3429)
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.
PspFI  (3231)
1 site
C C C A G C G G G T C G
BseYI  (3227)
1 site
C C C A G C G G G T C G

After cleavage, BseYI can remain bound to DNA and alter its electrophoretic mobility.
AhdI  (2643)
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  (2577)
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  (2574)
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  (2496)
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).
ScaI  (2162)
1 site
A G T A C T T C A T G A
XmnI  (2043)
1 site
G A A N N N N T T C C T T N N N N A A G
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.
BclI  (778)
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.
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
BtgI  (1082)
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  (1082)
1 site
C C A T G G G G T A C C
XcmI  (1120)
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  (1164)
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  (1238)
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  (1242)
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  (1260)
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  (1266)
1 site
C A A T T G G T T A A C
Eco53kI  (1275)
1 site
G A G C T C C T C G A G
BanII  (1277)
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  (1277)
1 site
G A G C T C C T C G A G
BsrGI  (1280)
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.
AscI  (1288)
1 site
G G C G C G C C C C G C G C G G
PstI  (1298)
1 site
C T G C A G G A C G T C
SbfI  (1298)
1 site
C C T G C A G G G G A C G T C C
SalI  (1300)
1 site
G T C G A C C A G C T G
Acc65I  (1306)
1 site
G G T A C C C C A T G G
AgeI  (1309)
1 site
A C C G G T T G G C C A
KpnI  (1310)
1 site
G G T A C C C C A T G G
BstBI  (1315)
1 site
T T C G A A A A G C T T
HindIII  (1318)
1 site
A A G C T T T T C G A A
NotI  (1325)
1 site
G C G G C C G C C G C C G G C G
BstZ17I  (1340)
1 site
G T A T A C C A T A T G
PmlI  (1345)
1 site
C A C G T G G T G C A C
BbsI  (1379)
1 site
G A A G A C N N C T T C T G N N ( N ) 4

Sticky ends from different BbsI sites may not be compatible.
BbsI gradually loses activity when stored at -20°C.
PaeR7I  (1387)
1 site
C T C G A G G A G C T C

PaeR7I does not recognize the sequence CTCTCGAG.
XhoI  (1387)
1 site
C T C G A G G A G C T C
DraIII  (1398)
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.
Bsu36I  (1408)
1 site
C C T N A G G G G A N T C C

Sticky ends from different Bsu36I sites may not be compatible.
PacI  (1437)
1 site
T T A A T T A A A A T T A A T T
PflFI  (1661)
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  (1661)
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.
BtgZI  (1684)
1 site
G C G A T G ( N ) 10 C G C T A C ( N ) 10 ( N ) 4

Sticky ends from different BtgZI sites may not be compatible.
After cleavage, BtgZI can remain bound to DNA and alter its electrophoretic mobility.
BtgZI is typically used at 60°C, but is 75% active at 37°C.
XbaI  (1716)
1 site
T C T A G A A G A T C T
ZraI  (1722)
1 site
G A C G T C C T G C A G
AatII  (1724)
1 site
G A C G T C C T G C A G
AmpR
1856 .. 2716  =  861 bp
286 amino acids  =  31.6 kDa
2 segments
   Segment 1:  signal sequence  
   1856 .. 1924  =  69 bp
   23 amino acids  =  2.6 kDa
Product: β-lactamase
confers resistance to ampicillin, carbenicillin, and related antibiotics
AmpR
1856 .. 2716  =  861 bp
286 amino acids  =  31.6 kDa
2 segments
   Segment 2:  
   1925 .. 2716  =  792 bp
   263 amino acids  =  28.9 kDa
Product: β-lactamase
confers resistance to ampicillin, carbenicillin, and related antibiotics
AmpR
1856 .. 2716  =  861 bp
286 amino acids  =  31.6 kDa
2 segments
Product: β-lactamase
confers resistance to ampicillin, carbenicillin, and related antibiotics
IE1 promoter
487 .. 1078  =  592 bp
promoter of the ie1 gene from the baculovirus Autographa californica
IE1 promoter
487 .. 1078  =  592 bp
promoter of the ie1 gene from the baculovirus Autographa californica
ori
2887 .. 3475  =  589 bp
high-copy-number ColE1/pMB1/pBR322/pUC origin of replication
ori
2887 .. 3475  =  589 bp
high-copy-number ColE1/pMB1/pBR322/pUC origin of replication
hr5 enhancer
1 .. 483  =  483 bp
baculovirus early transcription enhancer
hr5 enhancer
1 .. 483  =  483 bp
baculovirus early transcription enhancer
IE1 terminator
1414 .. 1721  =  308 bp
terminator of the ie1 gene from the baculovirus Autographa californica
IE1 terminator
1414 .. 1721  =  308 bp
terminator of the ie1 gene from the baculovirus Autographa californica
ATG
1084 .. 1086  =  3 bp
1 amino acid  =  149.2 Da
Product: start codon