pEF5 FRT V5-D-TOPO

Directional TOPO® cloning 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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HomePlasmidsTOPO Cloning VectorspEF5 FRT V5-D-TOPO
SspI (4092) FspI (3510) AhdI (3288) BspQI - SapI (2279) BstZ17I (2016) BsmI (1964) PsiI (1903) PfoI (1819) NdeI (1179) RsrII (1137) SgrDI (4209) NruI (4419) AflII (4943) FseI (5268) PasI (5312) BbvCI (5466) KasI (5502) NarI (5503) SfoI (5504) PluTI (5506) Acc65I (5804) KpnI (5808) SpeI (7) EcoRV (36) NotI (51) PspXI (57) BstBI (80) BseRI (97) PmeI (148) BclI * (157) BbsI (359) SphI (370) KflI - PpuMI (596) MscI (729) PshAI (756) BsmBI - Esp3I (797) NcoI (1081) AsiSI (1093) pEF5/FRT/V5-D-TOPO® 5827 bp
SspI  (4092)
1 site		 A A T A T T T T A T A A
FspI  (3510)
1 site		 T G C G C A A C G C G T
AhdI  (3288)
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  (2279)
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  (2279)
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.
BstZ17I  (2016)
1 site		 G T A T A C C A T A T G
BsmI  (1964)
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.
PsiI  (1903)
1 site		 T T A T A A A A T A T T
PfoI  (1819)
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  (1179)
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  (1137)
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.
SgrDI  (4209)
1 site		 C G T C G A C G G C A G C T G C
NruI  (4419)
1 site		 T C G C G A A G C G C T
AflII  (4943)
1 site		 C T T A A G G A A T T C
FseI  (5268)
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.
PasI  (5312)
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.
BbvCI  (5466)
1 site		 C C T C A G C G G A G T C G
KasI  (5502)
1 site		 G G C G C C C C G C G G
NarI  (5503)
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  (5504)
1 site		 G G C G C C C C G C G G
PluTI  (5506)
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  (5804)
1 site		 G G T A C C C C A T G G
KpnI  (5808)
1 site		 G G T A C C C C A T G G
SpeI  (7)
1 site		 A C T A G T T G A T C A
EcoRV  (36)
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.
NotI  (51)
1 site		 G C G G C C G C C G C C G G C G
PspXI  (57)
1 site		 V C T C G A G B B G A G C T C V
BstBI  (80)
1 site		 T T C G A A A A G C T T
BseRI  (97)
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  (148)
1 site		 G T T T A A A C C A A A T T T G
BclI  (157)
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  (359)
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  (370)
1 site		 G C A T G C C G T A C G
KflI  (596)
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  (596)
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.
MscI  (729)
1 site		 T G G C C A A C C G G T
PshAI  (756)
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.
BsmBI  (797)
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.
BsmBI-v2 is an improved version of BsmBI.
Esp3I  (797)
1 site		 C G T C T C N G C A G A G N ( N ) 4
Sticky ends from different Esp3I sites may not be compatible.
NcoI  (1081)
1 site		 C C A T G G G G T A C C
AsiSI  (1093)
1 site		 G C G A T C G C C G C T A G C G
EF-1α promoter
4564 .. 5742  =  1179 bp
strong constitutive promoter for human elongation factor EF-1α
EF-1α promoter
4564 .. 5742  =  1179 bp
strong constitutive promoter for human elongation factor EF-1α
HygR
732 .. 1754  =  1023 bp
340 amino acids  =  37.9 kDa
Product: aminoglycoside phosphotransferase from E. coli
confers resistance to hygromycin
HygR
732 .. 1754  =  1023 bp
340 amino acids  =  37.9 kDa
Product: aminoglycoside phosphotransferase from E. coli
confers resistance to hygromycin
AmpR
3215 .. 4075  =  861 bp
286 amino acids  =  31.6 kDa
2 segments
   Segment 2:  
   3215 .. 4006  =  792 bp
   263 amino acids  =  28.9 kDa
Product: β-lactamase
confers resistance to ampicillin, carbenicillin, and related antibiotics
AmpR
3215 .. 4075  =  861 bp
286 amino acids  =  31.6 kDa
2 segments
   Segment 1:  signal sequence  
   4007 .. 4075  =  69 bp
   23 amino acids  =  2.6 kDa
Product: β-lactamase
confers resistance to ampicillin, carbenicillin, and related antibiotics
AmpR
3215 .. 4075  =  861 bp
286 amino acids  =  31.6 kDa
2 segments
Product: β-lactamase
confers resistance to ampicillin, carbenicillin, and related antibiotics
ori
2456 .. 3044  =  589 bp
high-copy-number ColE1/pMB1/pBR322/pUC origin of replication
ori
2456 .. 3044  =  589 bp
high-copy-number ColE1/pMB1/pBR322/pUC origin of replication
bGH poly(A) signal
170 .. 394  =  225 bp
bovine growth hormone polyadenylation signal
bGH poly(A) signal
170 .. 394  =  225 bp
bovine growth hormone polyadenylation signal
SV40 poly(A) signal
1884 .. 2005  =  122 bp
SV40 polyadenylation signal
SV40 poly(A) signal
1884 .. 2005  =  122 bp
SV40 polyadenylation signal
AmpR promoter
4076 .. 4180  =  105 bp
AmpR promoter
4076 .. 4180  =  105 bp
FRT
677 .. 724  =  48 bp
FLP-mediated recombination occurs in the 8-bp core sequence TCTAGAAA (Turan and Bode, 2011).
FRT
677 .. 724  =  48 bp
FLP-mediated recombination occurs in the 8-bp core sequence TCTAGAAA (Turan and Bode, 2011).
V5 tag
85 .. 126  =  42 bp
14 amino acids  =  1.4 kDa
Product: epitope tag from simian virus 5
V5 tag
85 .. 126  =  42 bp
14 amino acids  =  1.4 kDa
Product: epitope tag from simian virus 5
T7 promoter
5759 .. 5777  =  19 bp
promoter for bacteriophage T7 RNA polymerase
T7 promoter
5759 .. 5777  =  19 bp
promoter for bacteriophage T7 RNA polymerase
EF-1α intron A
4795 .. 5733  =  939 bp
intron upstream of the start codon of human EF-1α
EF-1α intron A
4795 .. 5733  =  939 bp
intron upstream of the start codon of human EF-1α
ORF:  5120 .. 5557  =  438 bp
ORF:  145 amino acids  =  15.9 kDa
ORF:  804 .. 1754  =  951 bp
ORF:  316 amino acids  =  35.2 kDa
ORF:  3345 .. 3611  =  267 bp
ORF:  88 amino acids  =  9.2 kDa
ORF:  5199 .. 5432  =  234 bp
ORF:  77 amino acids  =  7.7 kDa
ORF:  705 .. 1487  =  783 bp
ORF:  260 amino acids  =  29.2 kDa
ORF:  3215 .. 4075  =  861 bp
ORF:  286 amino acids  =  31.6 kDa
ORF:  4862 .. 5482  =  621 bp
ORF:  206 amino acids  =  23.0 kDa
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Individual Sequences & Maps

pBAD-TOPO (linearized)
pcDNA5 FRT TO-TOPO (linearized)
pEF6 V5-His-TOPO (linearized)
pGlow-TOPO (linearized)
pBAD Thio-TOPO (linearized)
pcDNA5 FRT V5-His-TOPO (linearized)
pENTR D-TOPO
pIB V5-His-TOPO (linearized)
pBAD202 D-TOPO
pcDNA6.2 C-EmGFP-GW TOPO (linearized)
pENTR SD D-TOPO
pLenti6.3 V5-TOPO (linearized)
pBlue-TOPO (linearized)
pcDNA6.2 C-YFP-GW TOPO (linearized)
pENTR TEV D-TOPO
pLenti6 V5-D-TOPO
pcDNA3.1D V5-His-TOPO
pcDNA6.2 N-EmGFP-GW TOPO (linearized)
pENTR5'-TOPO (linearized)
pLenti7.3 V5-TOPO (linearized)
pcDNA3.2 V5 GW D-TOPO
pCR-XL-2-TOPO (linearized)
pET100 D-TOPO
pLPS-B (linearized)
pcDNA6.2 cGeneBLAzer-GW D-TOPO
pCR-XL-TOPO (linearized)
pET101 D-TOPO
pMT V5-His-TOPO (linearized)
pcDNA6.2 V5 GW D-TOPO
pCR2.1-TOPO (linearized)
pET102 D-TOPO
pOptiVEC-TOPO (linearized)
pcDNA3.1 CT-GFP-TOPO (linearized)
pCR4-TOPO (linearized)
pET151 D-TOPO
pSecTag FRT V5-His-TOPO (linearized)
pcDNA3.1 NT-GFP-TOPO (linearized)
pCR4Blunt-TOPO (linearized)
pET160 GW D-TOPO
pTOP Blunt V2 (linearized)
pcDNA3.1 V5-His-TOPO (linearized)
pCR8 GW TOPO (linearized)
pET161 GW D-TOPO
pTrcHis-TOPO (linearized)
pcDNA3.3-TOPO (linearized)
pCR-Blunt II-TOPO (linearized)
pET200 D-TOPO
pTrcHis2-TOPO (linearized)
pcDNA3.4-TOPO (linearized)
pCRII-TOPO (linearized)
pEXP5-CT TOPO (linearized)
pYES2.1 V5-His-TOPO (linearized)
pcDNA4 HisMax-TOPO (linearized)
pEF5 FRT V5-D-TOPO
pEXP5-NT TOPO (linearized)

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