pcDNA5 FRT V5-His-TOPO (linearized)

Linearized mammalian vector with 3'-T overhangs and bound topoisomerase, for TOPO® TA cloning of PCR products, FLP recombination, and expression of tagged proteins.

Sequence Author: Thermo Fisher (Invitrogen)

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HomePlasmidsTOPO Cloning VectorspcDNA5 FRT V5-His-TOPO (linearized)
SgrDI (4170) SspI (4053) XmnI (3848) FspI (3471) AhdI (3249) PciI (2356) BspQI - SapI (2240) BstZ17I (1977) BsmI (1925) PsiI (1864) PfoI (1780) BglII (4184) MfeI (4333) Bpu10I (4352) NruI (4380) SpeI (4421) SnaBI (4762) NheI (5067) BmtI (5071) AflII (5080) End (5094) Start (1) Acc65I (13) KpnI (17) BamHI (25) BseRI (46) AgeI (79) BclI * (118) BbsI (320) SphI (331) EcoO109I - KflI - PpuMI (557) XbaI (665) MscI (690) PshAI (717) BsmBI - Esp3I (758) EcoRI (933) BfuAI - BspMI (1003) PstI (1027) AsiSI (1054) RsrII (1098) SacII (1470) pcDNA™5/FRT/V5-His-TOPO® 5093 bp
SgrDI  (4170)
1 site		 C G T C G A C G G C A G C T G C
SspI  (4053)
1 site		 A A T A T T T T A T A A
XmnI  (3848)
1 site		 G A A N N N N T T C C T T N N N N A A G
FspI  (3471)
1 site		 T G C G C A A C G C G T
AhdI  (3249)
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.
PciI  (2356)
1 site		 A C A T G T T G T A C A
PciI is inhibited by nonionic detergents.
BspQI  (2240)
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  (2240)
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  (1977)
1 site		 G T A T A C C A T A T G
BsmI  (1925)
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  (1864)
1 site		 T T A T A A A A T A T T
PfoI  (1780)
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.
BglII  (4184)
1 site		 A G A T C T T C T A G A
MfeI  (4333)
1 site		 C A A T T G G T T A A C
Bpu10I  (4352)
1 site		 C C T N A G C G G A N T C G
Cleavage may be enhanced when more than one copy of the Bpu10I recognition sequence is present.
This recognition sequence is asymmetric, so ligating sticky ends generated by Bpu10I will not always regenerate a Bpu10I site.
Sticky ends from different Bpu10I sites may not be compatible.
NruI  (4380)
1 site		 T C G C G A A G C G C T
SpeI  (4421)
1 site		 A C T A G T T G A T C A
SnaBI  (4762)
1 site		 T A C G T A A T G C A T
NheI  (5067)
1 site		 G C T A G C C G A T C G
BmtI  (5071)
1 site		 G C T A G C C G A T C G
AflII  (5080)
1 site		 C T T A A G G A A T T C
End  (5094)
0 sites
Start  (1)
0 sites
Acc65I  (13)
1 site		 G G T A C C C C A T G G
KpnI  (17)
1 site		 G G T A C C C C A T G G
BamHI  (25)
1 site		 G G A T C C C C T A G G
After cleavage, BamHI-HF® (but not the original BamHI) can remain bound to DNA and alter its electrophoretic mobility.
BseRI  (46)
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.
AgeI  (79)
1 site		 A C C G G T T G G C C A
BclI  (118)
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  (320)
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  (331)
1 site		 G C A T G C C G T A C G
EcoO109I  (557)
1 site		 R G G N C C Y Y C C N G G R
Sticky ends from different EcoO109I sites may not be compatible.
KflI  (557)
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  (557)
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.
XbaI  (665)
1 site		 T C T A G A A G A T C T
MscI  (690)
1 site		 T G G C C A A C C G G T
PshAI  (717)
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  (758)
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  (758)
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.
EcoRI  (933)
1 site		 G A A T T C C T T A A G
BfuAI  (1003)
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  (1003)
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.
PstI  (1027)
1 site		 C T G C A G G A C G T C
AsiSI  (1054)
1 site		 G C G A T C G C C G C T A G C G
RsrII  (1098)
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.
SacII  (1470)
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.
HygR
693 .. 1715  =  1023 bp
340 amino acids  =  37.9 kDa
Product: aminoglycoside phosphotransferase from E. coli
confers resistance to hygromycin
HygR
693 .. 1715  =  1023 bp
340 amino acids  =  37.9 kDa
Product: aminoglycoside phosphotransferase from E. coli
confers resistance to hygromycin
AmpR
3176 .. 4036  =  861 bp
286 amino acids  =  31.6 kDa
2 segments
   Segment 2:  
   3176 .. 3967  =  792 bp
   263 amino acids  =  28.9 kDa
Product: β-lactamase
confers resistance to ampicillin, carbenicillin, and related antibiotics
AmpR
3176 .. 4036  =  861 bp
286 amino acids  =  31.6 kDa
2 segments
   Segment 1:  signal sequence  
   3968 .. 4036  =  69 bp
   23 amino acids  =  2.6 kDa
Product: β-lactamase
confers resistance to ampicillin, carbenicillin, and related antibiotics
AmpR
3176 .. 4036  =  861 bp
286 amino acids  =  31.6 kDa
2 segments
Product: β-lactamase
confers resistance to ampicillin, carbenicillin, and related antibiotics
ori
2417 .. 3005  =  589 bp
high-copy-number ColE1/pMB1/pBR322/pUC origin of replication
ori
2417 .. 3005  =  589 bp
high-copy-number ColE1/pMB1/pBR322/pUC origin of replication
CMV enhancer
4407 .. 4786  =  380 bp
human cytomegalovirus immediate early enhancer
CMV enhancer
4407 .. 4786  =  380 bp
human cytomegalovirus immediate early enhancer
bGH poly(A) signal
131 .. 355  =  225 bp
bovine growth hormone polyadenylation signal
bGH poly(A) signal
131 .. 355  =  225 bp
bovine growth hormone polyadenylation signal
CMV promoter
4787 .. 4990  =  204 bp
human cytomegalovirus (CMV) immediate early promoter
CMV promoter
4787 .. 4990  =  204 bp
human cytomegalovirus (CMV) immediate early promoter
SV40 poly(A) signal
1845 .. 1966  =  122 bp
SV40 polyadenylation signal
SV40 poly(A) signal
1845 .. 1966  =  122 bp
SV40 polyadenylation signal
AmpR promoter
4037 .. 4141  =  105 bp
AmpR promoter
4037 .. 4141  =  105 bp
V5 tag
34 .. 75  =  42 bp
14 amino acids  =  1.4 kDa
Product: epitope tag from simian virus 5
V5 tag
34 .. 75  =  42 bp
14 amino acids  =  1.4 kDa
Product: epitope tag from simian virus 5
6xHis
85 .. 102  =  18 bp
6 amino acids  =  840.9 Da
Product: 6xHis affinity tag
6xHis
85 .. 102  =  18 bp
6 amino acids  =  840.9 Da
Product: 6xHis affinity tag
FRT
638 .. 685  =  48 bp
FLP-mediated recombination occurs in the 8-bp core sequence TCTAGAAA (Turan and Bode, 2011).
FRT
638 .. 685  =  48 bp
FLP-mediated recombination occurs in the 8-bp core sequence TCTAGAAA (Turan and Bode, 2011).
T7 promoter
5035 .. 5053  =  19 bp
promoter for bacteriophage T7 RNA polymerase
T7 promoter
5035 .. 5053  =  19 bp
promoter for bacteriophage T7 RNA polymerase
ORF:  765 .. 1715  =  951 bp
ORF:  316 amino acids  =  35.2 kDa
ORF:  3306 .. 3572  =  267 bp
ORF:  88 amino acids  =  9.2 kDa
ORF:  666 .. 1448  =  783 bp
ORF:  260 amino acids  =  29.2 kDa
ORF:  3176 .. 4036  =  861 bp
ORF:  286 amino acids  =  31.6 kDa
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Individual Sequences & Maps

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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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