pIEx-9 3C_LIC (linearized)

Linearized insect cell vector for ligation-independent cloning (LIC) to express proteins with an N-terminal Strep-Tag® II-HRV 3C cassette and a C-terminal 10xHis tag.

Sequence Author: MilliporeSigma (Novagen)

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AvaI - BsoBI - TspMI - XmaI (2555) BspQI - SapI (2311) PciI (2194) DrdI (2092) PspFI (1894) BseYI (1890) AlwNI (1785) AhdI (1306) BsaI (1240) BsrFI (1221) NmeAIII (1159) ScaI (825) XmnI (706) SmaI (2557) BspDI * - ClaI * (2767) EcoRV (2799) PsiI (2969) NheI (3166) BmtI (3170) SphI (3174) BsmI (3335) BclI * (3338) BsiWI (3344) EagI (3402) MluI (3432) HincII (3604) BtgI - NcoI (3642) ATG BstBI (3669) End (3697) Start (0) DraIII (61) Bsu36I (71) PacI (100) PflFI - Tth111I (324) BtgZI (347) XbaI (379) ZraI (385) AatII (387) pIEx™-9 3C/LIC 3697 bp
AvaI  (2555)
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  (2555)
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  (2555)
1 site
C C C G G G G G G C C C
XmaI  (2555)
1 site
C C C G G G G G G C C C

Cleavage may be enhanced when more than one copy of the XmaI recognition sequence is present.
BspQI  (2311)
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  (2311)
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  (2194)
1 site
A C A T G T T G T A C A

PciI is inhibited by nonionic detergents.
DrdI  (2092)
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  (1894)
1 site
C C C A G C G G G T C G
BseYI  (1890)
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  (1785)
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  (1306)
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  (1240)
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  (1221)
1 site
R C C G G Y Y G G C C R

Cleavage may be enhanced when more than one copy of the BsrFI recognition sequence is present.
After cleavage, BsrFI can remain bound to DNA and alter its electrophoretic mobility.
NmeAIII  (1159)
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  (825)
1 site
A G T A C T T C A T G A
XmnI  (706)
1 site
G A A N N N N T T C C T T N N N N A A G
SmaI  (2557)
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  (2767)
1 site
A T C G A T T A G C T A
* Blocked by Dam methylation.
ClaI  (2767)
1 site
A T C G A T T A G C T A
* Blocked by Dam methylation.
EcoRV  (2799)
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  (2969)
1 site
T T A T A A A A T A T T
NheI  (3166)
1 site
G C T A G C C G A T C G
BmtI  (3170)
1 site
G C T A G C C G A T C G
SphI  (3174)
1 site
G C A T G C C G T A C G
BsmI  (3335)
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  (3338)
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  (3344)
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  (3402)
1 site
C G G C C G G C C G G C
MluI  (3432)
1 site
A C G C G T T G C G C A
HincII  (3604)
1 site
G T Y R A C C A R Y T G
BtgI  (3642)
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  (3642)
1 site
C C A T G G G G T A C C
BstBI  (3669)
1 site
T T C G A A A A G C T T
End  (3697)
0 sites
Start  (0)
0 sites
DraIII  (61)
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  (71)
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  (100)
1 site
T T A A T T A A A A T T A A T T
PflFI  (324)
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  (324)
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  (347)
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  (379)
1 site
T C T A G A A G A T C T
ZraI  (385)
1 site
G A C G T C C T G C A G
AatII  (387)
1 site
G A C G T C C T G C A G
AmpR
519 .. 1379  =  861 bp
286 amino acids  =  31.6 kDa
2 segments
   Segment 1:  signal sequence  
   519 .. 587  =  69 bp
   23 amino acids  =  2.6 kDa
Product: β-lactamase
confers resistance to ampicillin, carbenicillin, and related antibiotics
AmpR
519 .. 1379  =  861 bp
286 amino acids  =  31.6 kDa
2 segments
   Segment 2:  
   588 .. 1379  =  792 bp
   263 amino acids  =  28.9 kDa
Product: β-lactamase
confers resistance to ampicillin, carbenicillin, and related antibiotics
AmpR
519 .. 1379  =  861 bp
286 amino acids  =  31.6 kDa
2 segments
Product: β-lactamase
confers resistance to ampicillin, carbenicillin, and related antibiotics
IE1 promoter
3047 .. 3638  =  592 bp
promoter of the ie1 gene from the baculovirus Autographa californica
IE1 promoter
3047 .. 3638  =  592 bp
promoter of the ie1 gene from the baculovirus Autographa californica
ori
1550 .. 2138  =  589 bp
high-copy-number ColE1/pMB1/pBR322/pUC origin of replication
ori
1550 .. 2138  =  589 bp
high-copy-number ColE1/pMB1/pBR322/pUC origin of replication
hr5 enhancer
2561 .. 3043  =  483 bp
baculovirus early transcription enhancer
hr5 enhancer
2561 .. 3043  =  483 bp
baculovirus early transcription enhancer
IE1 terminator
77 .. 384  =  308 bp
terminator of the ie1 gene from the baculovirus Autographa californica
IE1 terminator
77 .. 384  =  308 bp
terminator of the ie1 gene from the baculovirus Autographa californica
AmpR promoter
414 .. 518  =  105 bp
AmpR promoter
414 .. 518  =  105 bp
ATG
3644 .. 3646  =  3 bp
1 amino acid  =  149.2 Da
Product: start codon
ATG
3644 .. 3646  =  3 bp
1 amino acid  =  149.2 Da
Product: start codon
Strep-Tag II
3653 .. 3676  =  24 bp
8 amino acids  =  1.1 kDa
Product: peptide that binds Strep-Tactin®, an engineered form of streptavidin
Strep-Tag II
3653 .. 3676  =  24 bp
8 amino acids  =  1.1 kDa
Product: peptide that binds Strep-Tactin®, an engineered form of streptavidin
HRV 3C site
3683 .. 3706  =  24 bp
8 amino acids  =  902.1 Da
Product: recognition and cleavage site for human rhinovirus 3C and PreScission proteases
HRV 3C site
3683 .. 3706  =  24 bp
8 amino acids  =  902.1 Da
Product: recognition and cleavage site for human rhinovirus 3C and PreScission proteases
thrombin site
5 .. 22  =  18 bp
6 amino acids  =  627.7 Da
Product: thrombin recognition and cleavage site
thrombin site
5 .. 22  =  18 bp
6 amino acids  =  627.7 Da
Product: thrombin recognition and cleavage site
10xHis
29 .. 58  =  30 bp
10 amino acids  =  1.4 kDa
Product: 10xHis affinity tag
10xHis
29 .. 58  =  30 bp
10 amino acids  =  1.4 kDa
Product: 10xHis affinity tag
ORF:  519 .. 1379  =  861 bp
ORF:  286 amino acids  =  31.6 kDa
ORF:  3012 .. 3707  =  696 bp
ORF:  231 amino acids  =  26.2 kDa  (no start codon)
ORF:  983 .. 1249  =  267 bp
ORF:  88 amino acids  =  9.2 kDa
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