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pIEx/Bac™-4 Ek/LIC (linearized)

Linearized insect cell and baculovirus vector for ligation-independent cloning (LIC), encoding a cleavable N-terminal GST tag and a C-terminal 10xHis tag.

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pIEx Bac-4 Ek_LIC (linearized) Sequence and MappIEx Bac-4 Ek_LIC (linearized).dna
Map and Sequence File   
Sequence Author:  Novagen (EMD Millipore)
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 BstXI (4633) BanII (3952) NmeAIII (3022) BpmI (2944) AlwNI (2397) PspFI (2289) BseYI (2285) PmeI (1861) SgrAI (1573) AleI (1564) AgeI (1333) BmgBI (989) AatII (766) ZraI (764) SnaBI (485) NsiI (4650) BbvCI - Bpu10I (4882) BglII (5479) EcoRV (5752) NheI (6119) BmtI (6123) PvuII (6289) BsiWI (6297) EagI (6355) PfoI (6596) PpuMI (6600) BspEI (6713) BamHI (6747) BtgI - NcoI (6751) EcoNI (6763) BspQI - SapI (6832) MscI (6960) BstBI (7150) End (7439) Start (0) AvrII (74) StuI (79) Bsu36I (81) PflFI - Tth111I (334) BlpI (398) pIEx/Bac™-4 Ek/LIC 7439 bp
BstXI  (4633)
1 site
C C A N N N N N N T G G G G T N N N N N N A C C

Sticky ends from different BstXI sites may not be compatible.
BanII  (3952)
1 site
G R G C Y C C Y C G R G

Sticky ends from different BanII sites may not be compatible.
NmeAIII  (3022)
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).
BpmI  (2944)
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.
AlwNI  (2397)
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.
PspFI  (2289)
1 site
C C C A G C G G G T C G
BseYI  (2285)
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.
PmeI  (1861)
1 site
G T T T A A A C C A A A T T T G
SgrAI  (1573)
1 site
C R C C G G Y G G Y G G C C R C

Efficient cleavage requires at least two copies of the SgrAI
recognition sequence.
AleI  (1564)
1 site
C A C N N N N G T G G T G N N N N C A C
AgeI  (1333)
1 site
A C C G G T T G G C C A

AgeI quickly loses activity at 37°C, but can be used at 25°C for
long incubations.
BmgBI  (989)
1 site
C A C G T C G T G C A G

This recognition sequence is asymmetric, so ligating blunt ends
generated by BmgBI will not always regenerate a BmgBI site.
AatII  (766)
1 site
G A C G T C C T G C A G
ZraI  (764)
1 site
G A C G T C C T G C A G
SnaBI  (485)
1 site
T A C G T A A T G C A T
NsiI  (4650)
1 site
A T G C A T T A C G T A
BbvCI  (4882)
1 site
C C T C A G C G G A G T C G
Bpu10I  (4882)
1 site
C C T N A G C G G A N T C G

Efficient cleavage requires at least two copies of the Bpu10I
recognition sequence.
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.
BglII  (5479)
1 site
A G A T C T T C T A G A
EcoRV  (5752)
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.
NheI  (6119)
1 site
G C T A G C C G A T C G
BmtI  (6123)
1 site
G C T A G C C G A T C G
PvuII  (6289)
1 site
C A G C T G G T C G A C
BsiWI  (6297)
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.
EagI  (6355)
1 site
C G G C C G G C C G G C
PfoI  (6596)
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.
PpuMI  (6600)
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.
BspEI  (6713)
1 site
T C C G G A A G G C C T
BamHI  (6747)
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.
BtgI  (6751)
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  (6751)
1 site
C C A T G G G G T A C C
EcoNI  (6763)
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.
BspQI  (6832)
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  (6832)
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.
MscI  (6960)
1 site
T G G C C A A C C G G T
BstBI  (7150)
1 site
T T C G A A A A G C T T
End  (7439)
0 sites
Start  (0)
0 sites
AvrII  (74)
1 site
C C T A G G G G A T C C
StuI  (79)
1 site
A G G C C T T C C G G A
Bsu36I  (81)
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.
PflFI  (334)
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  (334)
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.
BlpI  (398)
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.
baculovirus recombination region (ORF1629)
422 .. 1779  =  1358 bp
contains part of ORF1629
baculovirus recombination region (ORF1629)
422 .. 1779  =  1358 bp
contains part of ORF1629
AmpR
2801 .. 3661  =  861 bp
286 amino acids  =  31.6 kDa
   Segment 2:  
   2801 .. 3592  =  792 bp
   263 amino acids  =  28.9 kDa
Product: β-lactamase
confers resistance to ampicillin, carbenicillin, and
related antibiotics
AmpR
2801 .. 3661  =  861 bp
286 amino acids  =  31.6 kDa
   Segment 1:  signal sequence  
   3593 .. 3661  =  69 bp
   23 amino acids  =  2.6 kDa
Product: β-lactamase
confers resistance to ampicillin, carbenicillin, and
related antibiotics
AmpR
2801 .. 3661  =  861 bp
286 amino acids  =  31.6 kDa
2 segments
Product: β-lactamase
confers resistance to ampicillin, carbenicillin, and
related antibiotics
baculovirus recombination region (lef2/ORF603)
4647 .. 5474  =  828 bp
contains ORF603 and part of lef2
baculovirus recombination region (lef2/ORF603)
4647 .. 5474  =  828 bp
contains ORF603 and part of lef2
GST
6753 .. 7406  =  654 bp
218 amino acids  =  25.5 kDa
Product: glutathione S-transferase from
Schistosoma japonicum
GST
6753 .. 7406  =  654 bp
218 amino acids  =  25.5 kDa
Product: glutathione S-transferase from
Schistosoma japonicum
IE1 promoter
6000 .. 6591  =  592 bp
promoter of the ie1 gene from the baculovirus
Autographa californica
IE1 promoter
6000 .. 6591  =  592 bp
promoter of the ie1 gene from the baculovirus
Autographa californica
ori
2042 .. 2630  =  589 bp
high-copy-number ColE1/pMB1/pBR322/pUC origin
of replication
ori
2042 .. 2630  =  589 bp
high-copy-number ColE1/pMB1/pBR322/pUC origin
of replication
hr5 enhancer
5514 .. 5996  =  483 bp
baculovirus early transcription enhancer
hr5 enhancer
5514 .. 5996  =  483 bp
baculovirus early transcription enhancer
f1 ori
3793 .. 4248  =  456 bp
f1 bacteriophage origin of replication; arrow
indicates direction of (+) strand synthesis
f1 ori
3793 .. 4248  =  456 bp
f1 bacteriophage origin of replication; arrow
indicates direction of (+) strand synthesis
IE1 terminator
87 .. 393  =  307 bp
terminator of the ie1 gene from the baculovirus
Autographa californica
IE1 terminator
87 .. 393  =  307 bp
terminator of the ie1 gene from the baculovirus
Autographa californica
p10 promoter
6601 .. 6710  =  110 bp
baculovirus promoter for expression in insect cells
p10 promoter
6601 .. 6710  =  110 bp
baculovirus promoter for expression in insect cells
AmpR promoter
3662 .. 3766  =  105 bp
AmpR promoter
3662 .. 3766  =  105 bp
10xHis
25 .. 54  =  30 bp
10 amino acids  =  1.4 kDa
Product: 10xHis affinity tag
10xHis
25 .. 54  =  30 bp
10 amino acids  =  1.4 kDa
Product: 10xHis affinity tag
enterokinase site
7437 .. 7451  =  15 bp
5 amino acids  =  606.5 Da
Product: enterokinase recognition and cleavage site
enterokinase site
7437 .. 7451  =  15 bp
5 amino acids  =  606.5 Da
Product: enterokinase recognition and cleavage site
ORF1629
438 .. 1779  =  1342 bp
446 amino acids  =  49.2 kDa
Product: baculovirus capsid-associated protein
required for viral replication
ORF1629
438 .. 1779  =  1342 bp
446 amino acids  =  49.2 kDa
Product: baculovirus capsid-associated protein
required for viral replication
ORF603
4809 .. 5414  =  606 bp
201 amino acids  =  23.6 kDa
Product: baculovirus ORF603 protein
ORF603
4809 .. 5414  =  606 bp
201 amino acids  =  23.6 kDa
Product: baculovirus ORF603 protein
lef2
4647 .. 4771  =  125 bp
40 amino acids  =  4.5 kDa
Product: baculovirus late expression factor 2
lef2
4647 .. 4771  =  125 bp
40 amino acids  =  4.5 kDa
Product: baculovirus late expression factor 2
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