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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.dna
Map and Sequence File:    Download    Open   
Sequence Author:  Thermo Fisher (Invitrogen)
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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) KflI - PpuMI (3913) SphI (3687) NruI (208) AflII (732) FseI (1057) KasI (1291) NarI (1292) SfoI (1293) PluTI (1295) Acc65I (1593) KpnI (1597) SpeI (1611) PflMI * (2323) BssHII (2585) TspMI - XmaI (2960) SmaI - SrfI (2962) BmgBI (2996) PspXI (3374) BstBI (3397) BseRI (3414) PmeI (3465) BclI * (3474) BbsI (3676) 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.
SphI  (3687)
1 site
G C A T G C C G T A C G
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
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