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Plasmid Files

pTOP TA V2 (linearized)

Linearized vector with 3'-T overhangs and bound topoisomerase, for efficient TOPO® TA cloning of PCR products.

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pTOP TA V2 (linearized).dna
Map and Sequence File:    Download    Open   
Sequence Author:  Enzynomics
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AflIII - PciI (3388) BspHI (2668) BpmI (2431) BglI (2382) ScaI (2019) XmnI (1900) RsrII (1545) NaeI (1531) HindIII (3747) Acc65I (3753) KpnI (3757) Eco53kI (3761) SacI (3763) BamHI (3765) SpeI (3771) BstXI (3792) EcoRI (3796) End (3807) Start (1) EcoRI (7) EcoRV (19) BstXI (29) NotI (34) AvaI - BsoBI - PaeR7I - PspXI - XhoI (40) BmeT110I (41) NsiI (51) XbaI (52) EcoO109I - PspOMI (58) ApaI (62) BsiWI - SnaBI (243) BsrGI (303) BmgBI (364) BstXI (447) XcmI (845) BglII (865) BclI * (870) PflFI - Tth111I (1147) BssHII (1426) BtgI - NcoI (1461) NgoMIV (1529) pTOP TA V2 3806 bp
AflIII  (3388)
1 site
A C R Y G T T G Y R C A

Sticky ends from different AflIII sites may not be compatible.
PciI  (3388)
1 site
A C A T G T T G T A C A

PciI is inhibited by nonionic detergents.
BspHI  (2668)
1 site
T C A T G A A G T A C T
BpmI  (2431)
1 site
C T G G A G ( N ) 14 N N G A C C T C ( N ) 14

Efficient cleavage requires at least two copies of the BpmI recognition sequence.
Sticky ends from different BpmI sites may not be compatible.
After cleavage, BpmI can remain bound to DNA and alter its electrophoretic mobility.
BpmI quickly loses activity at 37°C.
BglI  (2382)
1 site
G C C N N N N N G G C C G G N N N N N C C G

Sticky ends from different BglI sites may not be compatible.
ScaI  (2019)
1 site
A G T A C T T C A T G A
XmnI  (1900)
1 site
G A A N N N N T T C C T T N N N N A A G
RsrII  (1545)
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.
NaeI  (1531)
1 site
G C C G G C C G G C C G

Efficient cleavage requires at least two copies of the NaeI recognition sequence.
HindIII  (3747)
1 site
A A G C T T T T C G A A
Acc65I  (3753)
1 site
G G T A C C C C A T G G
KpnI  (3757)
1 site
G G T A C C C C A T G G
Eco53kI  (3761)
1 site
G A G C T C C T C G A G
SacI  (3763)
1 site
G A G C T C C T C G A G
BamHI  (3765)
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.
SpeI  (3771)
1 site
A C T A G T T G A T C A
BstXI  (3792)
3 sites
C C A N N N N N N T G G G G T N N N N N N A C C

Sticky ends from different BstXI sites may not be compatible.
EcoRI  (3796)
2 sites
G A A T T C C T T A A G
End  (3807)
0 sites
Start  (1)
0 sites
EcoRI  (7)
2 sites
G A A T T C C T T A A G
EcoRV  (19)
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.
BstXI  (29)
3 sites
C C A N N N N N N T G G G G T N N N N N N A C C

Sticky ends from different BstXI sites may not be compatible.
NotI  (34)
1 site
G C G G C C G C C G C C G G C G
AvaI  (40)
1 site
C Y C G R G G R G C Y C

Sticky ends from different AvaI sites may not be compatible.
BsoBI  (40)
1 site
C Y C G R G G R G C Y C

Sticky ends from different BsoBI sites may not be compatible.
BsoBI is typically used at 37°C, but can be used at temperatures up to 65°C.
PaeR7I  (40)
1 site
C T C G A G G A G C T C

PaeR7I does not recognize the sequence CTCTCGAG.
PspXI  (40)
1 site
V C T C G A G B B G A G C T C V
XhoI  (40)
1 site
C T C G A G G A G C T C
BmeT110I  (41)
1 site
C Y C G R G G R G C Y C
NsiI  (51)
1 site
A T G C A T T A C G T A
XbaI  (52)
1 site
T C T A G A A G A T C T
EcoO109I  (58)
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.
PspOMI  (58)
1 site
G G G C C C C C C G G G
ApaI  (62)
1 site
G G G C C C C C C G G G

ApaI can be used between 25°C and 37°C.
BsiWI  (243)
1 site
C G T A C G G C A T G C

BsiWI is typically used at 55°C, but is 50% active at 37°C.
SnaBI  (243)
1 site
T A C G T A A T G C A T
BsrGI  (303)
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.
BmgBI  (364)
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.
BstXI  (447)
3 sites
C C A N N N N N N T G G G G T N N N N N N A C C

Sticky ends from different BstXI sites may not be compatible.
XcmI  (845)
1 site
C C A N N N N N N N N N T G G G G T N N N N N N N N N A C C

The 1-base overhangs produced by XcmI may be hard to ligate.
Sticky ends from different XcmI sites may not be compatible.
BglII  (865)
1 site
A G A T C T T C T A G A
BclI  (870)
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.
PflFI  (1147)
1 site
G A C N N N G T C C T G N N N C A G

The 1-base overhangs produced by PflFI may be hard to ligate.
Sticky ends from different PflFI sites may not be compatible.
Tth111I  (1147)
1 site
G A C N N N G T C C T G N N N C A G

The 1-base overhangs produced by Tth111I may be hard to ligate.
Sticky ends from different Tth111I sites may not be compatible.
BssHII  (1426)
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.
BtgI  (1461)
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  (1461)
1 site
C C A T G G G G T A C C
NgoMIV  (1529)
1 site
G C C G G C C G G C C G

Efficient cleavage requires at least two copies of the NgoMIV recognition sequence.
AmpR
1713 .. 2573  =  861 bp
286 amino acids  =  31.6 kDa
   Segment 1:  signal sequence  
   1713 .. 1781  =  69 bp
   23 amino acids  =  2.6 kDa
Product: β-lactamase
confers resistance to ampicillin, carbenicillin, and related antibiotics
AmpR
1713 .. 2573  =  861 bp
286 amino acids  =  31.6 kDa
   Segment 2:  
   1782 .. 2573  =  792 bp
   263 amino acids  =  28.9 kDa
Product: β-lactamase
confers resistance to ampicillin, carbenicillin, and related antibiotics
AmpR
1713 .. 2573  =  861 bp
286 amino acids  =  31.6 kDa
2 segments
Product: β-lactamase
confers resistance to ampicillin, carbenicillin, and related antibiotics
NeoR/KanR
901 .. 1695  =  795 bp
264 amino acids  =  29.0 kDa
Product: aminoglycoside phosphotransferase from Tn5
confers resistance to neomycin, kanamycin, and G418 (Geneticin®)
NeoR/KanR
901 .. 1695  =  795 bp
264 amino acids  =  29.0 kDa
Product: aminoglycoside phosphotransferase from Tn5
confers resistance to neomycin, kanamycin, and G418 (Geneticin®)
ori
2744 .. 3332  =  589 bp
high-copy-number ColE1/pMB1/pBR322/pUC origin of replication
ori
2744 .. 3332  =  589 bp
high-copy-number ColE1/pMB1/pBR322/pUC origin of replication
lacZα
1 .. 552  =  552 bp
183 amino acids  =  21.3 kDa
Product: LacZα fragment of β-galactosidase
lacZα
1 .. 552  =  552 bp
183 amino acids  =  21.3 kDa
Product: LacZα fragment of β-galactosidase
lacZα
3730 .. 3807  =  78 bp
26 amino acids  =  2.7 kDa
Product: LacZα fragment of β-galactosidase
lacZα
3730 .. 3807  =  78 bp
26 amino acids  =  2.7 kDa
Product: LacZα fragment of β-galactosidase
lac promoter
3656 .. 3686  =  31 bp
   Segment 1:  -35  
   3656 .. 3661  =  6 bp
promoter for the E. coli lac operon
lac promoter
3656 .. 3686  =  31 bp
   Segment 2:  
   3662 .. 3679  =  18 bp
promoter for the E. coli lac operon
lac promoter
3656 .. 3686  =  31 bp
   Segment 3:  -10  
   3680 .. 3686  =  7 bp
promoter for the E. coli lac operon
lac promoter
3656 .. 3686  =  31 bp
3 segments
promoter for the E. coli lac operon
lac operator
3694 .. 3710  =  17 bp
The lac repressor binds to the lac operator to inhibit transcription in E. coli. This inhibition can be relieved by adding lactose or isopropyl-β-D-thiogalactopyranoside (IPTG).
lac operator
3694 .. 3710  =  17 bp
The lac repressor binds to the lac operator to inhibit transcription in E. coli. This inhibition can be relieved by adding lactose or isopropyl-β-D-thiogalactopyranoside (IPTG).
MCS
1 .. 63  =  63 bp
multiple cloning site
MCS
1 .. 63  =  63 bp
multiple cloning site
MCS
3747 .. 3807  =  61 bp
multiple cloning site
MCS
3747 .. 3807  =  61 bp
multiple cloning site
M13 fwd
96 .. 112  =  17 bp
common sequencing primer, one of multiple similar variants
M13 fwd
96 .. 112  =  17 bp
common sequencing primer, one of multiple similar variants
M13 rev
3718 .. 3734  =  17 bp
common sequencing primer, one of multiple similar variants
M13 rev
3718 .. 3734  =  17 bp
common sequencing primer, one of multiple similar variants
ORF:  1 .. 552  =  552 bp
ORF:  183 amino acids  =  21.3 kDa  (no start codon)
ORF:  901 .. 1695  =  795 bp
ORF:  264 amino acids  =  29.0 kDa
ORF:  2 .. 346  =  345 bp
ORF:  114 amino acids  =  12.6 kDa  (no start codon)
ORF:  1073 .. 1459  =  387 bp
ORF:  128 amino acids  =  14.6 kDa
ORF:  1713 .. 2573  =  861 bp
ORF:  286 amino acids  =  31.6 kDa
ORF:  1210 .. 1464  =  255 bp
ORF:  84 amino acids  =  9.5 kDa
ORF:  963 .. 1322  =  360 bp
ORF:  119 amino acids  =  12.5 kDa
ORF:  2177 .. 2443  =  267 bp
ORF:  88 amino acids  =  9.2 kDa
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