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Plasmid Files

pEF5/FRT/V5-DEST

Gateway® destination vector for constitutive high-level expression of proteins in mammalian cells using the Flp-In™ system.

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pEF5 FRT V5-DEST Sequence and MappEF5 FRT V5-DEST.dna
Map and Sequence File   
Sequence Author:  Invitrogen (Life Technologies)
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 SgrDI (7526) FspI (6827) AhdI (6605) BspQI - SapI (5596) PsiI (5220) PfoI (5136) NdeI (4496) RsrII (4454) AsiSI (4410) PshAI (4073) FRT KflI - PpuMI (3913) NruI (208) AflII (732) FseI (1057) KasI (1291) NarI (1292) SfoI (1293) PluTI (1295) Acc65I (1593) KpnI (1597) SpeI (1611) lac UV5 promoter PflMI * (2323) BssHII (2585) TspMI - XmaI (2960) SmaI - SrfI (2962) BmgBI (2996) PspXI (3374) BstBI (3397) BseRI (3414) PmeI (3465) BclI * (3474) BbsI (3676) SphI (3687) pEF5/FRT/V5-DEST 7528 bp
SgrDI  (7526)
1 site
C G T C G A C G G C A G C T G C
FspI  (6827)
1 site
T G C G C A A C G C G T
AhdI  (6605)
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.
BspQI  (5596)
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  (5596)
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.
PsiI  (5220)
1 site
T T A T A A A A T A T T
PfoI  (5136)
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.
NdeI  (4496)
1 site
C A T A T G G T A T A C

Prolonged incubation with NdeI may lead to removal of additional
nucleotides.
RsrII  (4454)
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.
AsiSI  (4410)
1 site
G C G A T C G C C G C T A G C G
PshAI  (4073)
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.
KflI  (3913)
1 site
G G G W C C C C C C W G G G

Sticky ends from different KflI sites may not be compatible.
PpuMI  (3913)
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.
NruI  (208)
1 site
T C G C G A A G C G C T
AflII  (732)
1 site
C T T A A G G A A T T C

The sticky ends produced by AflII are hard to ligate.
FseI  (1057)
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.
KasI  (1291)
1 site
G G C G C C C C G C G G
NarI  (1292)
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  (1293)
1 site
G G C G C C C C G C G G
PluTI  (1295)
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.
Acc65I  (1593)
1 site
G G T A C C C C A T G G
KpnI  (1597)
1 site
G G T A C C C C A T G G
SpeI  (1611)
1 site
A C T A G T T G A T C A
PflMI  (2323)
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.
BssHII  (2585)
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.
TspMI  (2960)
1 site
C C C G G G G G G C C C
XmaI  (2960)
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.
SmaI  (2962)
1 site
C C C G G G G G G C C C

SmaI can be used at 37°C for brief incubations.
SrfI  (2962)
1 site
G C C C G G G C C G G G C C C G
BmgBI  (2996)
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.
PspXI  (3374)
1 site
V C T C G A G B B G A G C T C V
BstBI  (3397)
1 site
T T C G A A A A G C T T
BseRI  (3414)
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.
PmeI  (3465)
1 site
G T T T A A A C C A A A T T T G
BclI  (3474)
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.
BbsI  (3676)
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.
SphI  (3687)
1 site
G C A T G C C G T A C G
EF-1α promoter
353 .. 1531  =  1179 bp
strong constitutive promoter for human elongation
factor EF-1α
EF-1α promoter
353 .. 1531  =  1179 bp
strong constitutive promoter for human elongation
factor EF-1α
HygR
4049 .. 5071  =  1023 bp
340 amino acids  =  37.9 kDa
Product: aminoglycoside phosphotransferase from E.
coli

confers resistance to hygromycin
HygR
4049 .. 5071  =  1023 bp
340 amino acids  =  37.9 kDa
Product: aminoglycoside phosphotransferase from E.
coli

confers resistance to hygromycin
AmpR
6532 .. 7392  =  861 bp
286 amino acids  =  31.6 kDa
   Segment 2:  
   6532 .. 7323  =  792 bp
   263 amino acids  =  28.9 kDa
Product: β-lactamase
confers resistance to ampicillin, carbenicillin, and
related antibiotics
AmpR
6532 .. 7392  =  861 bp
286 amino acids  =  31.6 kDa
   Segment 1:  signal sequence  
   7324 .. 7392  =  69 bp
   23 amino acids  =  2.6 kDa
Product: β-lactamase
confers resistance to ampicillin, carbenicillin, and
related antibiotics
AmpR
6532 .. 7392  =  861 bp
286 amino acids  =  31.6 kDa
2 segments
Product: β-lactamase
confers resistance to ampicillin, carbenicillin, and
related antibiotics
CmR
1878 .. 2537  =  660 bp
219 amino acids  =  25.7 kDa
Product: chloramphenicol acetyltransferase
confers resistance to chloramphenicol
CmR
1878 .. 2537  =  660 bp
219 amino acids  =  25.7 kDa
Product: chloramphenicol acetyltransferase
confers resistance to chloramphenicol
ori
5773 .. 6361  =  589 bp
high-copy-number ColE1/pMB1/pBR322/pUC origin
of replication
ori
5773 .. 6361  =  589 bp
high-copy-number ColE1/pMB1/pBR322/pUC origin
of replication
ccdB
2879 .. 3184  =  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
2879 .. 3184  =  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.
bGH poly(A) signal
3487 .. 3711  =  225 bp
bovine growth hormone polyadenylation signal
bGH poly(A) signal
3487 .. 3711  =  225 bp
bovine growth hormone polyadenylation signal
attR1
1645 .. 1769  =  125 bp
recombination site for the Gateway® LR reaction
attR1
1645 .. 1769  =  125 bp
recombination site for the Gateway® LR reaction
attR2
3225 .. 3349  =  125 bp
recombination site for the Gateway® LR reaction
attR2
3225 .. 3349  =  125 bp
recombination site for the Gateway® LR reaction
SV40 poly(A) signal
5201 .. 5322  =  122 bp
SV40 polyadenylation signal
SV40 poly(A) signal
5201 .. 5322  =  122 bp
SV40 polyadenylation signal
AmpR promoter
7393 .. 7497  =  105 bp
AmpR promoter
7393 .. 7497  =  105 bp
FRT
3994 .. 4041  =  48 bp
FLP-mediated recombination occurs in the 8-bp core
sequence TCTAGAAA (Turan and Bode, 2011).
FRT
3994 .. 4041  =  48 bp
FLP-mediated recombination occurs in the 8-bp core
sequence TCTAGAAA (Turan and Bode, 2011).
V5 tag
3402 .. 3443  =  42 bp
14 amino acids  =  1.4 kDa
Product: epitope tag from simian virus 5
V5 tag
3402 .. 3443  =  42 bp
14 amino acids  =  1.4 kDa
Product: epitope tag from simian virus 5
lac UV5 promoter
1794 .. 1824  =  31 bp
   Segment 1:  -35  
   1794 .. 1799  =  6 bp
E. coli lac promoter with an "up" mutation
lac UV5 promoter
1794 .. 1824  =  31 bp
   Segment 2:  
   1800 .. 1817  =  18 bp
E. coli lac promoter with an "up" mutation
lac UV5 promoter
1794 .. 1824  =  31 bp
   Segment 3:  -10  
   1818 .. 1824  =  7 bp
E. coli lac promoter with an "up" mutation
lac UV5 promoter
1794 .. 1824  =  31 bp
3 segments
E. coli lac promoter with an "up" mutation
T7 promoter
1548 .. 1566  =  19 bp
promoter for bacteriophage T7 RNA polymerase
T7 promoter
1548 .. 1566  =  19 bp
promoter for bacteriophage T7 RNA polymerase
EF-1α intron A
584 .. 1522  =  939 bp
intron upstream of the start codon of human EF-1α
EF-1α intron A
584 .. 1522  =  939 bp
intron upstream of the start codon of human EF-1α
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