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pIEx™-8 Ek/LIC

Insect cell vector for ligation-independent cloning (LIC) to express proteins with an N-terminal Strep-Tag® II-enterokinase cassette and a C-terminal 10xHis tag.

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pIEx-8 Ek_LIC Sequence and MappIEx-8 Ek_LIC.dna
Map and Sequence File   
Sequence Author:  Novagen (EMD Millipore)
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 BmeT110I (3734) AvaI - BsoBI - TspMI - XmaI (3733) BspQI - SapI (3489) PciI (3372) DrdI (3270) PspFI (3072) BseYI (3068) AlwNI (2963) AhdI (2484) BsaI (2418) BsrFI (2399) NmeAIII (2337) ScaI (2003) SmaI (3735) BspDI * - ClaI * (207) EcoRV (239) PsiI (409) NheI (606) BmtI (610) SphI (614) BsmI (775) BclI * (778) BsiWI (784) MluI (872) BtgI - NcoI (1082) ATG BstBI (1109) Acc65I (1137) BsgI (1139) KpnI (1141) BamHI (1143) PshAI (1153) SalI (1156) AccI (1157) HindIII (1162) NotI (1169) Eco53kI (1180) BanII - BseRI - SacI (1182) DraIII (1239) Bsu36I (1249) PacI (1278) PflFI - Tth111I (1502) BtgZI (1525) XbaI (1557) XmnI (1884) pIEx™-8 Ek/LIC 3738 bp
BmeT110I  (3734)
1 site
C Y C G R G G R G C Y C
AvaI  (3733)
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  (3733)
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.
TspMI  (3733)
1 site
C C C G G G G G G C C C
XmaI  (3733)
1 site
C C C G G G G G G C C C

Efficient cleavage requires at least two copies of the XmaI
recognition sequence.
Full cleavage with XmaI may require a long incubation.
BspQI  (3489)
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  (3489)
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  (3372)
1 site
A C A T G T T G T A C A

PciI is inhibited by nonionic detergents.
DrdI  (3270)
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  (3072)
1 site
C C C A G C G G G T C G
BseYI  (3068)
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.
AlwNI  (2963)
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.
AhdI  (2484)
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  (2418)
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.
BsrFI  (2399)
1 site
R C C G G Y Y G G C C R

Efficient cleavage requires at least two copies of the BsrFI
recognition sequence.
After cleavage, BsrFI can remain bound to DNA and alter its
electrophoretic mobility.
NmeAIII  (2337)
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  (2003)
1 site
A G T A C T T C A T G A
SmaI  (3735)
1 site
C C C G G G G G G C C C

SmaI can be used at 37°C for brief incubations.
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
BstBI  (1109)
1 site
T T C G A A A A G C T T
Acc65I  (1137)
1 site
G G T A C C C C A T G G
BsgI  (1139)
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).
KpnI  (1141)
1 site
G G T A C C C C A T G G
BamHI  (1143)
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.
PshAI  (1153)
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.
SalI  (1156)
1 site
G T C G A C C A G C T G
AccI  (1157)
1 site
G T M K A C C A K M T G

Efficient cleavage with AccI requires ≥13 bp on each side of the
recognition sequence.
Sticky ends from different AccI sites may not be compatible.
HindIII  (1162)
1 site
A A G C T T T T C G A A
NotI  (1169)
1 site
G C G G C C G C C G C C G G C G
Eco53kI  (1180)
1 site
G A G C T C C T C G A G
BanII  (1182)
1 site
G R G C Y C C Y C G R G

Sticky ends from different BanII sites may not be compatible.
BseRI  (1182)
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.
SacI  (1182)
1 site
G A G C T C C T C G A G
DraIII  (1239)
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  (1249)
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  (1278)
1 site
T T A A T T A A A A T T A A T T
PflFI  (1502)
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  (1502)
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  (1525)
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  (1557)
1 site
T C T A G A A G A T C T
XmnI  (1884)
1 site
G A A N N N N T T C C T T N N N N A A G
AmpR
1697 .. 2557  =  861 bp
286 amino acids  =  31.6 kDa
   Segment 1:  signal sequence  
   1697 .. 1765  =  69 bp
   23 amino acids  =  2.6 kDa
Product: β-lactamase
confers resistance to ampicillin, carbenicillin, and
related antibiotics
AmpR
1697 .. 2557  =  861 bp
286 amino acids  =  31.6 kDa
   Segment 2:  
   1766 .. 2557  =  792 bp
   263 amino acids  =  28.9 kDa
Product: β-lactamase
confers resistance to ampicillin, carbenicillin, and
related antibiotics
AmpR
1697 .. 2557  =  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
2728 .. 3316  =  589 bp
high-copy-number ColE1/pMB1/pBR322/pUC origin
of replication
ori
2728 .. 3316  =  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
1255 .. 1562  =  308 bp
terminator of the ie1 gene from the baculovirus
Autographa californica
IE1 terminator
1255 .. 1562  =  308 bp
terminator of the ie1 gene from the baculovirus
Autographa californica
AmpR promoter
1592 .. 1696  =  105 bp
AmpR promoter
1592 .. 1696  =  105 bp
10xHis
1207 .. 1236  =  30 bp
10 amino acids  =  1.4 kDa
Product: 10xHis affinity tag
10xHis
1207 .. 1236  =  30 bp
10 amino acids  =  1.4 kDa
Product: 10xHis affinity tag
Strep-Tag II
1093 .. 1116  =  24 bp
8 amino acids  =  1.1 kDa
Product: peptide that binds Strep-Tactin®, an
engineered form of streptavidin
Strep-Tag II
1093 .. 1116  =  24 bp
8 amino acids  =  1.1 kDa
Product: peptide that binds Strep-Tactin®, an
engineered form of streptavidin
enterokinase site
1123 .. 1137  =  15 bp
5 amino acids  =  606.5 Da
Product: enterokinase recognition and cleavage site
enterokinase site
1123 .. 1137  =  15 bp
5 amino acids  =  606.5 Da
Product: enterokinase recognition and cleavage site
ATG
1084 .. 1086  =  3 bp
1 amino acid  =  149.2 Da
Product: start codon
ATG
1084 .. 1086  =  3 bp
1 amino acid  =  149.2 Da
Product: start codon
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