pEF5 FRT V5-DEST

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

Sequence Author: Thermo Fisher (Invitrogen)

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HomePlasmidsGateway® Cloning VectorspEF5 FRT V5-DEST
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
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
Cleavage may be enhanced when more than one copy of the XmaI recognition sequence is present.
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
2 segments
   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
2 segments
   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
3 segments
   Segment 1:  -35  
   1794 .. 1799  =  6 bp
E. coli lac promoter with an "up" mutation
lac UV5 promoter
1794 .. 1824  =  31 bp
3 segments
   Segment 2:  
   1800 .. 1817  =  18 bp
E. coli lac promoter with an "up" mutation
lac UV5 promoter
1794 .. 1824  =  31 bp
3 segments
   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
ORF:  988 .. 1221  =  234 bp
ORF:  77 amino acids  =  7.7 kDa
ORF:  2879 .. 3184  =  306 bp
ORF:  101 amino acids  =  11.7 kDa
ORF:  4121 .. 5071  =  951 bp
ORF:  316 amino acids  =  35.2 kDa
ORF:  6662 .. 6928  =  267 bp
ORF:  88 amino acids  =  9.2 kDa
ORF:  909 .. 1346  =  438 bp
ORF:  145 amino acids  =  15.9 kDa
ORF:  1878 .. 2537  =  660 bp
ORF:  219 amino acids  =  25.7 kDa
ORF:  4022 .. 4804  =  783 bp
ORF:  260 amino acids  =  29.2 kDa
ORF:  6532 .. 7392  =  861 bp
ORF:  286 amino acids  =  31.6 kDa
ORF:  651 .. 1271  =  621 bp
ORF:  206 amino acids  =  23.0 kDa
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Download pEF5 FRT V5-DEST.dna file

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Individual Sequences & Maps

pAd BLOCK-iT-DEST
pDEST R4-R3 Vector II
pENTR11
pHELLSGATE 4
pAd CMV V5-DEST
pDEST10
pENTR1A
pHELLSGATE 8
pAd PL-DEST
pDEST14
pENTR2B
pIB V5-His-DEST
pBAD-DEST49
pDEST15
pENTR3C
pLenti4 BLOCK-iT-DEST
pcDNA-DEST40
pDEST17
pENTR4
pLenti4 V5-DEST
pcDNA-DEST47
pDEST20
pET-53-DEST
pLenti6.2 C-Lumio V5-DEST
pcDNA-DEST53
pDEST24
pET-54-DEST
pLenti6.2 N-Lumio V5-DEST
pcDNA3.1 nV5-DEST
pDEST26
pET-55-DEST
pLenti6.2 V5-DEST
pcDNA3.2 capTEV-CT V5-DEST
pDEST27
pET-56-DEST
pLenti6.3 TO V5-DEST
pcDNA3.2 capTEV-NT V5-DEST
pDEST8
pET-57-DEST
pLenti6.3 V5-DEST
pcDNA3.2 V5-DEST
pDONR221 P5-P2
pET-58-DEST
pLenti6.4 R4R2 V5-DEST
pcDNA6.2 C-EmGFP-DEST
pDONR P2r-P3
pET-59-DEST
pLenti6 BLOCK-iT-DEST
pcDNA6.2 C-YFP-DEST
pDONR P4-P1r
pET-60-DEST
pLenti6 capTEV-CT-DEST
pcDNA6.2 cGeneBLAzer-DEST
pDONR Zeo
pET-61-DEST
pLenti6 capTEV-NT-DEST1
pcDNA6.2 cLumio-DEST
pDONR201
pET-62-DEST
pLenti6 UbC V5-DEST
pcDNA6.2 EmGFP-Bsd V5-DEST
pDONR207
pET-DEST42
pLenti6 V5-DEST
pcDNA6.2 N-EmGFP-DEST
pDONR221
pET104-DEST
pLenti7.3 V5-DEST
pcDNA6.2 N-YFP-DEST
pDONR221 P1-P4
pET104.1-DEST
pMT-DEST48
pcDNA6.2 nGeneBLAzer-DEST
pDONR221 P1-P5r
pET160-DEST
pMT BioEase-DEST
pcDNA6.2 nLumio-DEST
pDONR221 P3-P2
pET161-DEST
pSCREEN-iT lacZ-DEST
pcDNA6.2 V5-DEST
pDONR221 P4r-P3r
pEXP1-DEST
pT-REx-DEST30
pcDNA6.2 V5-pL-DEST
pDONR221 P5-P4
pEXP2-DEST
pT-REx-DEST31
pCMV SPORT6
pDONR222
pEXP3-DEST
pYES-DEST52
pCMV SPORT6.1
pDONR223
pEXP4-DEST
Reading Frame Cassette A
pCOLA-2-DEST
pEF-DEST51
pHELLSGATE
Reading Frame Cassette B
pCOLA-3-DEST
pEF5 FRT V5-DEST
pHELLSGATE 12
Reading Frame Cassette C.1
pDEST R4-R3
pENTR GeneBLAzer

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