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pIEx™-7 Ek/LIC (linearized)

Linearized insect cell vector for ligation-independent cloning (LIC) to express proteins with an N-terminal 6xHis-enterokinase cassette and a C-terminal S-Tag.

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pIEx-7 Ek_LIC (linearized) Sequence and MappIEx-7 Ek_LIC (linearized).dna
Map and Sequence File   
Sequence Author:  Novagen (EMD Millipore)
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 TspMI - XmaI (2590) BspQI - SapI (2346) PciI (2229) DrdI (2127) PspFI (1929) BseYI (1925) AlwNI (1820) AhdI (1341) BsaI (1275) BpmI (1272) BsrFI (1256) NmeAIII (1194) ScaI (860) XmnI (741) SmaI (2592) BspDI * - ClaI * (2802) EcoRV (2834) PsiI (3004) NheI (3201) BmtI (3205) SphI (3209) BsmI (3370) BclI * (3373) BsiWI (3379) EagI (3437) MluI (3467) HincII (3639) BtgI - NcoI (3677) ATG PmlI (3702) End (3708) enterokinase site Start (0) PaeR7I - XhoI (19) AccI (76) DraIII (96) Bsu36I (106) PacI (135) PflFI - Tth111I (359) BtgZI (382) XbaI (414) ZraI (420) AatII (422) pIEx™-7 Ek/LIC 3708 bp
TspMI  (2590)
1 site
C C C G G G G G G C C C
XmaI  (2590)
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  (2346)
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  (2346)
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  (2229)
1 site
A C A T G T T G T A C A

PciI is inhibited by nonionic detergents.
DrdI  (2127)
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  (1929)
1 site
C C C A G C G G G T C G
BseYI  (1925)
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  (1820)
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  (1341)
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  (1275)
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  (1272)
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.
BsrFI  (1256)
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  (1194)
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  (860)
1 site
A G T A C T T C A T G A
XmnI  (741)
1 site
G A A N N N N T T C C T T N N N N A A G
SmaI  (2592)
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  (2802)
1 site
A T C G A T T A G C T A
* Blocked by Dam methylation.
ClaI  (2802)
1 site
A T C G A T T A G C T A
* Blocked by Dam methylation.
EcoRV  (2834)
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  (3004)
1 site
T T A T A A A A T A T T
NheI  (3201)
1 site
G C T A G C C G A T C G
BmtI  (3205)
1 site
G C T A G C C G A T C G
SphI  (3209)
1 site
G C A T G C C G T A C G
BsmI  (3370)
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  (3373)
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  (3379)
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  (3437)
1 site
C G G C C G G C C G G C
MluI  (3467)
1 site
A C G C G T T G C G C A
HincII  (3639)
1 site
G T Y R A C C A R Y T G
BtgI  (3677)
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  (3677)
1 site
C C A T G G G G T A C C
PmlI  (3702)
1 site
C A C G T G G T G C A C

PmlI gradually loses activity when stored at -20°C.
End  (3708)
0 sites
Start  (0)
0 sites
PaeR7I  (19)
1 site
C T C G A G G A G C T C

PaeR7I does not recognize the sequence CTCTCGAG.
XhoI  (19)
1 site
C T C G A G G A G C T C
AccI  (76)
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.
DraIII  (96)
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  (106)
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  (135)
1 site
T T A A T T A A A A T T A A T T
PflFI  (359)
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  (359)
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  (382)
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  (414)
1 site
T C T A G A A G A T C T
ZraI  (420)
1 site
G A C G T C C T G C A G
AatII  (422)
1 site
G A C G T C C T G C A G
AmpR
554 .. 1414  =  861 bp
286 amino acids  =  31.6 kDa
   Segment 1:  signal sequence  
   554 .. 622  =  69 bp
   23 amino acids  =  2.6 kDa
Product: β-lactamase
confers resistance to ampicillin, carbenicillin, and
related antibiotics
AmpR
554 .. 1414  =  861 bp
286 amino acids  =  31.6 kDa
   Segment 2:  
   623 .. 1414  =  792 bp
   263 amino acids  =  28.9 kDa
Product: β-lactamase
confers resistance to ampicillin, carbenicillin, and
related antibiotics
AmpR
554 .. 1414  =  861 bp
286 amino acids  =  31.6 kDa
2 segments
Product: β-lactamase
confers resistance to ampicillin, carbenicillin, and
related antibiotics
IE1 promoter
3082 .. 3673  =  592 bp
promoter of the ie1 gene from the baculovirus
Autographa californica
IE1 promoter
3082 .. 3673  =  592 bp
promoter of the ie1 gene from the baculovirus
Autographa californica
ori
1585 .. 2173  =  589 bp
high-copy-number ColE1/pMB1/pBR322/pUC origin
of replication
ori
1585 .. 2173  =  589 bp
high-copy-number ColE1/pMB1/pBR322/pUC origin
of replication
hr5 enhancer
2596 .. 3078  =  483 bp
baculovirus early transcription enhancer
hr5 enhancer
2596 .. 3078  =  483 bp
baculovirus early transcription enhancer
IE1 terminator
112 .. 419  =  308 bp
terminator of the ie1 gene from the baculovirus
Autographa californica
IE1 terminator
112 .. 419  =  308 bp
terminator of the ie1 gene from the baculovirus
Autographa californica
AmpR promoter
449 .. 553  =  105 bp
AmpR promoter
449 .. 553  =  105 bp
S-Tag
31 .. 75  =  45 bp
15 amino acids  =  1.7 kDa
Product: affinity and epitope tag derived from
pancreatic ribonuclease A
S-Tag
31 .. 75  =  45 bp
15 amino acids  =  1.7 kDa
Product: affinity and epitope tag derived from
pancreatic ribonuclease A
6xHis
3685 .. 3702  =  18 bp
6 amino acids  =  840.9 Da
Product: 6xHis affinity tag
6xHis
3685 .. 3702  =  18 bp
6 amino acids  =  840.9 Da
Product: 6xHis affinity tag
enterokinase site
3706 .. 3720  =  15 bp
5 amino acids  =  606.5 Da
Product: enterokinase recognition and cleavage site
enterokinase site
3706 .. 3720  =  15 bp
5 amino acids  =  606.5 Da
Product: enterokinase recognition and cleavage site
ATG
3679 .. 3681  =  3 bp
1 amino acid  =  149.2 Da
Product: start codon
ATG
3679 .. 3681  =  3 bp
1 amino acid  =  149.2 Da
Product: start codon
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