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pcDNA™3.1/V5-His-TOPO® (linearized)

Linearized mammalian vector with 3'-T overhangs and bound topoisomerase, for TOPO® TA cloning of PCR products and expression of C-terminally V5- and 6xHis-tagged proteins.

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pcDNA3.1 V5-His-TOPO (linearized).dna
Map and Sequence File:    Download    Open   
Sequence Author:  Thermo Fisher (Invitrogen)
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SgrDI (4568) SspI (4451) ScaI (4127) PvuI (4017) AhdI (3647) PciI (2754) BstZ17I (2375) BsmI (2323) PfoI (2178) RsrII (1917) BssHII (1798) BglII (4582) MfeI (4731) Bpu10I (4750) NdeI (5054) SnaBI (5160) HindIII (5472) Acc65I (5478) KpnI (5482) BamHI (5490) End (5523) Start (1) EcoRV (18) NotI (33) PaeR7I - PspXI - XhoI (39) XbaI (45) EcoO109I - PspOMI (51) ApaI (55) SacII (58) BstBI (62) AgeI (112) PmeI (142) BbsI (353) DraIII (667) SexAI * (958) StuI (1190) AvrII (1191) TspMI - XmaI (1212) SmaI (1214) BsaBI * (1260) KasI (1400) NarI (1401) SfoI (1402) PluTI (1404) MscI (1483) PflFI - Tth111I (1519) pcDNA™3.1/V5-His-TOPO® 5522 bp
SgrDI  (4568)
1 site
C G T C G A C G G C A G C T G C
SspI  (4451)
1 site
A A T A T T T T A T A A
ScaI  (4127)
1 site
A G T A C T T C A T G A
PvuI  (4017)
1 site
C G A T C G G C T A G C
AhdI  (3647)
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  (2754)
1 site
A C A T G T T G T A C A

PciI is inhibited by nonionic detergents.
BstZ17I  (2375)
1 site
G T A T A C C A T A T G
BsmI  (2323)
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.
PfoI  (2178)
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.
RsrII  (1917)
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.
BssHII  (1798)
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.
BglII  (4582)
1 site
A G A T C T T C T A G A
MfeI  (4731)
1 site
C A A T T G G T T A A C
Bpu10I  (4750)
1 site
C C T N A G C G G A N T C G

Efficient cleavage requires at least two copies of the Bpu10I recognition sequence.
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.
NdeI  (5054)
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.
SnaBI  (5160)
1 site
T A C G T A A T G C A T
HindIII  (5472)
1 site
A A G C T T T T C G A A
Acc65I  (5478)
1 site
G G T A C C C C A T G G
KpnI  (5482)
1 site
G G T A C C C C A T G G
BamHI  (5490)
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.
End  (5523)
0 sites
Start  (1)
0 sites
EcoRV  (18)
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  (33)
1 site
G C G G C C G C C G C C G G C G
PaeR7I  (39)
1 site
C T C G A G G A G C T C

PaeR7I does not recognize the sequence CTCTCGAG.