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pIB/V5-His-DEST

Gateway® destination vector for stable constitutive expression of C-terminally V5-6xHis-tagged proteins in insect cells.

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pIB V5-His-DEST Sequence and MappIB V5-His-DEST.dna
Map and Sequence File   
Sequence Author:  Invitrogen (Life Technologies)
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 AhdI (4870) BglI (4752) XmnI (4270) EarI (4071) BglII (4003) DraIII (3970) BlpI (3960) NruI * (3796) PpuMI (3724) MreI - SgrAI (3658) BbsI (3650) SpeI (3415) BciVI (2826) PciI (2623) BspDI * - ClaI * (2560) BspHI (0) FseI (293) BsiWI (317) RsrII (335) KasI (474) NarI (475) SfoI (476) PluTI (478) HindIII (557) EcoRV (565) AfeI (586) NotI (728) BspEI (1044) BsmBI (1272) PasI - PflMI * (1280) SspI (1360) BssHII (1542) BbvCI (1773) SrfI (1919) BstXI (2036) BfuAI - BspMI (2163) SalI (2176) PshAI (2315) XbaI (2319) SacII (2332) AgeI (2386) 6xHis PsiI (2493) pIB/V5-His-DEST 5038 bp
AhdI  (4870)
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.
BglI  (4752)
1 site
G C C N N N N N G G C C G G N N N N N C C G

Sticky ends from different BglI sites may not be compatible.
XmnI  (4270)
1 site
G A A N N N N T T C C T T N N N N A A G
EarI  (4071)
1 site
C T C T T C N G A G A A G N N N N

Efficient cleavage requires at least two copies of the EarI
recognition sequence.
Sticky ends from different EarI sites may not be compatible.
BglII  (4003)
1 site
A G A T C T T C T A G A
DraIII  (3970)
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.
BlpI  (3960)
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.
NruI  (3796)
1 site
T C G C G A A G C G C T
* Blocked by Dam methylation.
PpuMI  (3724)
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.
MreI  (3658)
1 site
C G C C G G C G G C G G C C G C
SgrAI  (3658)
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.
BbsI  (3650)
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.
SpeI  (3415)
1 site
A C T A G T T G A T C A
BciVI  (2826)
1 site
G T A T C C ( N ) 5 N C A T A G G ( N ) 5

The 1-base overhangs produced by BciVI may be hard to ligate.
Sticky ends from different BciVI sites may not be compatible.
PciI  (2623)
1 site
A C A T G T T G T A C A

PciI is inhibited by nonionic detergents.
BspDI  (2560)
1 site
A T C G A T T A G C T A
* Blocked by Dam methylation.
ClaI  (2560)
1 site
A T C G A T T A G C T A
* Blocked by Dam methylation.
BspHI  (0)
1 site
T C A T G A A G T A C T
FseI  (293)
1 site
G G C C G G C C C C G G C C G G

FseI gradually loses activity when stored at -20°C.
BsiWI  (317)
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.
RsrII  (335)
1 site
C G G W C C G G C C W G G C

Efficient cleavage requires at least two copies of the RsrII
recognition sequence.
Sticky ends from different RsrII sites may not be compatible.
For full activity, add fresh DTT.
KasI  (474)
1 site
G G C G C C C C G C G G
NarI  (475)
1 site
G G C G C C C C G C G G

Efficient cleavage requires at least two copies of the NarI
recognition sequence.
SfoI  (476)
1 site
G G C G C C C C G C G G
PluTI  (478)
1 site
G G C G C C C C G C G G

Efficient cleavage requires at least two copies of the PluTI
recognition sequence.
HindIII  (557)
1 site
A A G C T T T T C G A A
EcoRV  (565)
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.
AfeI  (586)
1 site
A G C G C T T C G C G A
NotI  (728)
1 site
G C G G C C G C C G C C G G C G
BspEI  (1044)
1 site
T C C G G A A G G C C T
BsmBI  (1272)
1 site
C G T C T C N G C A G A G N ( N ) 4

Sticky ends from different BsmBI sites may not be compatible.
PasI  (1280)
1 site
C C C W G G G G G G W C C C

Sticky ends from different PasI sites may not be compatible.
PflMI  (1280)
1 site
C C A N N N N N T G G G G T N N N N N A C C
* Blocked by Dcm methylation.
Sticky ends from different PflMI sites may not be compatible.
SspI  (1360)
1 site
A A T A T T T T A T A A
BssHII  (1542)
1 site
G C G C G C C G C G C G

BssHII is typically used at 50°C, but is 75% active at 37°C.
BbvCI  (1773)
1 site
C C T C A G C G G A G T C G
SrfI  (1919)
1 site
G C C C G G G C C G G G C C C G
BstXI  (2036)
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.
BfuAI  (2163)
1 site
A C C T G C ( N ) 4 T G G A C G ( N ) 4 ( N ) 4

Efficient cleavage requires at least two copies of the BfuAI
recognition sequence.
Sticky ends from different BfuAI sites may not be compatible.
BfuAI is typically used at 50°C, but is 50% active at 37°C.
BspMI  (2163)
1 site
A C C T G C ( N ) 4 T G G A C G ( N ) 4 ( N ) 4

Efficient cleavage requires at least two copies of the BspMI
recognition sequence.
Sticky ends from different BspMI sites may not be compatible.
SalI  (2176)
1 site
G T C G A C C A G C T G
PshAI  (2315)
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.
XbaI  (2319)
1 site
T C T A G A A G A T C T
SacII  (2332)
1 site
C C G C G G G G C G C C

Efficient cleavage requires at least two copies of the SacII
recognition sequence.
AgeI  (2386)
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.
PsiI  (2493)
1 site
T T A T A A A A T A T T
AmpR
4083 .. 4943  =  861 bp
286 amino acids  =  31.6 kDa
   Segment 1:  signal sequence  
   4083 .. 4151  =  69 bp
   23 amino acids  =  2.6 kDa
Product: β-lactamase
confers resistance to ampicillin, carbenicillin, and
related antibiotics
AmpR
4083 .. 4943  =  861 bp
286 amino acids  =  31.6 kDa
   Segment 2:  
   4152 .. 4943  =  792 bp
   263 amino acids  =  28.9 kDa
Product: β-lactamase
confers resistance to ampicillin, carbenicillin, and
related antibiotics
AmpR
4083 .. 4943  =  861 bp
286 amino acids  =  31.6 kDa
2 segments
Product: β-lactamase
confers resistance to ampicillin, carbenicillin, and
related antibiotics
CmR
835 .. 1494  =  660 bp
219 amino acids  =  25.7 kDa
Product: chloramphenicol acetyltransferase
confers resistance to chloramphenicol
CmR
835 .. 1494  =  660 bp
219 amino acids  =  25.7 kDa
Product: chloramphenicol acetyltransferase
confers resistance to chloramphenicol
ori
2684 .. 3272  =  589 bp
high-copy-number ColE1/pMB1/pBR322/pUC origin
of replication
ori
2684 .. 3272  =  589 bp
high-copy-number ColE1/pMB1/pBR322/pUC origin
of replication
OpIE-2 promoter
1 .. 548  =  548 bp
strong constitutive baculovirus promoter for insect
cell expression
OpIE-2 promoter
1 .. 548  =  548 bp
strong constitutive baculovirus promoter for insect
cell expression
BSD
3565 .. 3963  =  399 bp
132 amino acids  =  13.6 kDa
Product: blasticidin S deaminase
confers resistance to blasticidin
BSD
3565 .. 3963  =  399 bp
132 amino acids  =  13.6 kDa
Product: blasticidin S deaminase
confers resistance to blasticidin
ccdB
1836 .. 2141  =  306 bp
101 amino acids  =  11.7 kDa
Product: CcdB, a bacterial toxin that poisons DNA
gyrase
Plasmids containing the ccdB gene cannot be
propagated in standard E. coli strains.
ccdB
1836 .. 2141  =  306 bp
101 amino acids  =  11.7 kDa
Product: CcdB, a bacterial toxin that poisons DNA
gyrase
Plasmids containing the ccdB gene cannot be
propagated in standard E. coli strains.
OpIE-2 poly(A) signal
2427 .. 2556  =  130 bp
baculovirus polyadenylation signal
OpIE-2 poly(A) signal
2427 .. 2556  =  130 bp
baculovirus polyadenylation signal
attR1
602 .. 726  =  125 bp
recombination site for the Gateway® LR reaction
attR1
602 .. 726  =  125 bp
recombination site for the Gateway® LR reaction
attR2
2182 .. 2306  =  125 bp
recombination site for the Gateway® LR reaction
attR2
2182 .. 2306  =  125 bp
recombination site for the Gateway® LR reaction
gp64 promoter
3364 .. 3463  =  100 bp
baculovirus promoter for constitutive expression in
insect cells
gp64 promoter
3364 .. 3463  =  100 bp
baculovirus promoter for constitutive expression in
insect cells
EM7 promoter
3499 .. 3546  =  48 bp
synthetic bacterial promoter
EM7 promoter
3499 .. 3546  =  48 bp
synthetic bacterial promoter
V5 tag
2341 .. 2382  =  42 bp
14 amino acids  =  1.4 kDa
Product: epitope tag from simian virus 5
V5 tag
2341 .. 2382  =  42 bp
14 amino acids  =  1.4 kDa
Product: epitope tag from simian virus 5
lac UV5 promoter
751 .. 781  =  31 bp
   Segment 1:  -35  
   751 .. 756  =  6 bp
E. coli lac promoter with an "up" mutation
lac UV5 promoter
751 .. 781  =  31 bp
   Segment 2:  
   757 .. 774  =  18 bp
E. coli lac promoter with an "up" mutation
lac UV5 promoter
751 .. 781  =  31 bp
   Segment 3:  -10  
   775 .. 781  =  7 bp
E. coli lac promoter with an "up" mutation
lac UV5 promoter
751 .. 781  =  31 bp
3 segments
E. coli lac promoter with an "up" mutation
6xHis
2392 .. 2409  =  18 bp
6 amino acids  =  840.9 Da
Product: 6xHis affinity tag
6xHis
2392 .. 2409  =  18 bp
6 amino acids  =  840.9 Da
Product: 6xHis affinity tag
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