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Plasmid Files

pENTR™5'-TOPO® (linearized)

Linearized vector with 3'-T overhangs and bound topoisomerase, for TOPO® TA cloning of PCR products to create a multisite Gateway® entry clone.

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pENTR5pENTR5'-TOPO (linearized).dna
Map and Sequence File   
Sequence Author:  Invitrogen (Life Technologies)
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 PciI (1969) DrdI (1867) BssS α I (1796) BciVI (1771) PspFI (1669) BseYI (1665) ApaLI (1655) AlwNI (1560) AcuI - Eco57MI (1427) PflMI (1057) Bpu10I (811) BsmBI (810) AsiSI - PvuI (794) BsrFI (748) SspI (719) EcoNI (706) NspI (1973) BspQI - SapI (2086) PvuII (2149) BsaHI (2302) AclI (2339) BbsI (2412) M13 fwd AhdI (2524) AflII (2529) PspOMI (2538) ApaI (2542) BtgI - NcoI - StyI (2556) PsiI (2636) End (2680) Start (1) BsrGI - TatI (23) NdeI (125) XmnI (178) XbaI (188) PstI (199) EagI - NotI (203) EcoRV (212) NruI (451) pENTR™5'-TOPO® (linearized) 2679 bp
PciI  (1969)
1 site
A C A T G T T G T A C A

PciI is inhibited by nonionic detergents.
DrdI  (1867)
1 site
G A C N N N N N N G T C C T G N N N N N N C A G

Sticky ends from different DrdI sites may not be compatible.
BssSαI  (1796)
1 site
C A C G A G G T G C T C
BciVI  (1771)
1 site
G T A T C C ( N ) 5 N C A T A G G ( N ) 5

The 1-base overhangs produced by BciVI may be hard to ligate.
Sticky ends from different BciVI sites may not be compatible.
PspFI  (1669)
1 site
C C C A G C G G G T C G
BseYI  (1665)
1 site
C C C A G C G G G T C G

After cleavage, BseYI can remain bound to DNA and alter its
electrophoretic mobility.
ApaLI  (1655)
1 site
G T G C A C C A C G T G
AlwNI  (1560)
1 site
C A G N N N C T G G T C N N N G A C

Sticky ends from different AlwNI sites may not be compatible.
AcuI  (1427)
1 site
C T G A A G ( N ) 14 N N G A C T T C ( N ) 14

Efficient cleavage requires at least two copies of the AcuI
recognition sequence.
Sticky ends from different AcuI sites may not be compatible.
After cleavage, AcuI can remain bound to DNA and alter its
electrophoretic mobility.
For full activity, add fresh S-adenosylmethionine (SAM).
Eco57MI  (1427)
1 site
C T G R A G ( N ) 14 N N G A C Y T C ( N ) 14

Sticky ends from different Eco57MI sites may not be compatible.
After cleavage, Eco57MI can remain bound to DNA and alter its
electrophoretic mobility.
For full activity, add fresh S-adenosylmethionine (SAM).
PflMI  (1057)
1 site
C C A N N N N N T G G G G T N N N N N A C C

Sticky ends from different PflMI sites may not be compatible.
Bpu10I  (811)
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.
BsmBI  (810)
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.
AsiSI  (794)
1 site
G C G A T C G C C G C T A G C G
PvuI  (794)
1 site
C G A T C G G C T A G C
BsrFI  (748)
1 site
R C C G G Y Y G G C C R

Efficient cleavage requires at least two copies of the BsrFI
recognition sequence.
After cleavage, BsrFI can remain bound to DNA and alter its
electrophoretic mobility.
SspI  (719)
1 site
A A T A T T T T A T A A
EcoNI  (706)
1 site
C C T N N N N N A G G G G A N N N N N T C C

The 1-base overhangs produced by EcoNI may be hard to ligate.
Sticky ends from different EcoNI sites may not be compatible.
NspI  (1973)
1 site
R C A T G Y Y G T A C R
BspQI  (2086)
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  (2086)
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.
PvuII  (2149)
1 site
C A G C T G G T C G A C
BsaHI  (2302)
1 site
G R C G Y C C Y G C R G

BsaHI is typically used at 37°C, but is even more active at 60°C.
AclI  (2339)
1 site
A A C G T T T T G C A A
BbsI  (2412)
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.
AhdI  (2524)
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.
AflII  (2529)
1 site
C T T A A G G A A T T C

The sticky ends produced by AflII are hard to ligate.
PspOMI  (2538)
1 site
G G G C C C C C C G G G
ApaI  (2542)
1 site
G G G C C C C C C G G G

ApaI can be used between 25°C and 37°C.
BtgI  (2556)
1 site
C C R Y G G G G Y R C C

Sticky ends from different BtgI sites may not be compatible.
NcoI  (2556)
1 site
C C A T G G G G T A C C
StyI  (2556)
1 site
C C W W G G G G W W C C

Sticky ends from different StyI sites may not be compatible.
PsiI  (2636)
1 site
T T A T A A A A T A T T
End  (2680)
0 sites
Start  (1)
0 sites
BsrGI  (23)
1 site
T G T A C A A C A T G T

BsrGI is typically used at 37°C, but is even more active at 60°C.
TatI  (23)
1 site
W G T A C W W C A T G W
NdeI  (125)
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.
XmnI  (178)
1 site
G A A N N N N T T C C T T N N N N A A G
XbaI  (188)
1 site
T C T A G A A G A T C T
PstI  (199)
1 site
C T G C A G G A C G T C
EagI  (203)
1 site
C G G C C G G C C G G C
NotI  (203)
1 site
G C G G C C G C C G C C G G C G
EcoRV  (212)
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.
NruI  (451)
1 site
T C G C G A A G C G C T
KanR
369 .. 1178  =  810 bp
269 amino acids  =  30.8 kDa
Product: aminoglycoside phosphotransferase
confers resistance to kanamycin in bacteria or G418
(Geneticin®) in eukaryotes
KanR
369 .. 1178  =  810 bp
269 amino acids  =  30.8 kDa
Product: aminoglycoside phosphotransferase
confers resistance to kanamycin in bacteria or G418
(Geneticin®) in eukaryotes
ori
1325 .. 1913  =  589 bp
high-copy-number ColE1/pMB1/pBR322/pUC origin
of replication
ori
1325 .. 1913  =  589 bp
high-copy-number ColE1/pMB1/pBR322/pUC origin
of replication
attR1
17 .. 140  =  124 bp
recombination site for the Gateway® LR reaction
attR1
17 .. 140  =  124 bp
recombination site for the Gateway® LR reaction
attL4
2568 .. 2663  =  96 bp
recombination site for the Gateway® LR reaction
attL4
2568 .. 2663  =  96 bp
recombination site for the Gateway® LR reaction
rrnB T1 terminator
2362 .. 2448  =  87 bp
transcription terminator T1 from the E. coli rrnB
gene
rrnB T1 terminator
2362 .. 2448  =  87 bp
transcription terminator T1 from the E. coli rrnB
gene
rrnB T2 terminator
2243 .. 2270  =  28 bp
transcription terminator T2 from the E. coli rrnB
gene
rrnB T2 terminator
2243 .. 2270  =  28 bp
transcription terminator T2 from the E. coli rrnB
gene
T7 promoter
217 .. 235  =  19 bp
promoter for bacteriophage T7 RNA polymerase
T7 promoter
217 .. 235  =  19 bp
promoter for bacteriophage T7 RNA polymerase
M13 rev
240 .. 256  =  17 bp
common sequencing primer, one of multiple similar
variants
M13 rev
240 .. 256  =  17 bp
common sequencing primer, one of multiple similar
variants
M13 fwd
2512 .. 2528  =  17 bp
common sequencing primer, one of multiple similar
variants
M13 fwd
2512 .. 2528  =  17 bp
common sequencing primer, one of multiple similar
variants
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