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Plasmid Files

pCR®4-TOPO®

Parental sequencing vector for TOPO® TA cloning of PCR products.

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pCR4-TOPO Sequence and MappCR4-TOPO.dna
Map and Sequence File   
Sequence Author:  Invitrogen (Life Technologies)
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 AflIII - PciI (3831) SspI (3080) XmnI (2875) ScaI (2756) BglI (2396) BpmI (2346) AhdI (2276) RsrII (1803) lac operator SpeI (262) PstI - SbfI (273) PmeI (278) EcoRI (283) AflII (292) EcoRI (301) NotI (308) BsiWI - SnaBI (501) BsrGI (561) BmgBI (622) BstXI (705) XcmI (1103) BclI * (1128) PflFI - Tth111I (1405) BanII (1652) SphI (1692) BtgI - NcoI (1719) NgoMIV (1787) NaeI (1789) pCR®4-TOPO® 3956 bp
AflIII  (3831)
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  (3831)
1 site
A C A T G T T G T A C A

PciI is inhibited by nonionic detergents.
SspI  (3080)
1 site
A A T A T T T T A T A A
XmnI  (2875)
1 site
G A A N N N N T T C C T T N N N N A A G
ScaI  (2756)
1 site
A G T A C T T C A T G A
BglI  (2396)
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.
BpmI  (2346)
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.
AhdI  (2276)
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.
RsrII  (1803)
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.
SpeI  (262)
1 site
A C T A G T T G A T C A
PstI  (273)
1 site
C T G C A G G A C G T C
SbfI  (273)
1 site
C C T G C A G G G G A C G T C C
PmeI  (278)
1 site
G T T T A A A C C A A A T T T G
EcoRI  (283)
2 sites
G A A T T C C T T A A G
AflII  (292)
1 site
C T T A A G G A A T T C

The sticky ends produced by AflII are hard to ligate.
EcoRI  (301)
2 sites
G A A T T C C T T A A G
NotI  (308)
1 site
G C G G C C G C C G C C G G C G
BsiWI  (501)
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  (501)
1 site
T A C G T A A T G C A T
BsrGI  (561)
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  (622)
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  (705)
1 site
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  (1103)
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.
BclI  (1128)
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  (1405)
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  (1405)
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.
BanII  (1652)
1 site
G R G C Y C C Y C G R G

Sticky ends from different BanII sites may not be compatible.
SphI  (1692)
1 site
G C A T G C C G T A C G
BtgI  (1719)
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  (1719)
1 site
C C A T G G G G T A C C
NgoMIV  (1787)
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.
NaeI  (1789)
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.
AmpR
2203 .. 3063  =  861 bp
286 amino acids  =  31.6 kDa
   Segment 2:  
   2203 .. 2994  =  792 bp
   263 amino acids  =  28.9 kDa
Product: β-lactamase
confers resistance to ampicillin, carbenicillin, and
related antibiotics
AmpR
2203 .. 3063  =  861 bp
286 amino acids  =  31.6 kDa
   Segment 1:  signal sequence  
   2995 .. 3063  =  69 bp
   23 amino acids  =  2.6 kDa
Product: β-lactamase
confers resistance to ampicillin, carbenicillin, and
related antibiotics
AmpR
2203 .. 3063  =  861 bp
286 amino acids  =  31.6 kDa
2 segments
Product: β-lactamase
confers resistance to ampicillin, carbenicillin, and
related antibiotics
NeoR/KanR
1159 .. 1953  =  795 bp
264 amino acids  =  29.0 kDa
Product: aminoglycoside phosphotransferase from
Tn5
confers resistance to neomycin, kanamycin, and
G418 (Geneticin®)
NeoR/KanR
1159 .. 1953  =  795 bp
264 amino acids  =  29.0 kDa
Product: aminoglycoside phosphotransferase from
Tn5
confers resistance to neomycin, kanamycin, and
G418 (Geneticin®)
ori
3187 .. 3775  =  589 bp
high-copy-number ColE1/pMB1/pBR322/pUC origin
of replication
ori
3187 .. 3775  =  589 bp
high-copy-number ColE1/pMB1/pBR322/pUC origin
of replication
ccdB
508 .. 810  =  303 bp
100 amino acids  =  11.6 kDa
Product: CcdB, a bacterial toxin that poisons DNA
gyrase
Plasmids containing the ccdB gene cannot be
propagated in standard E. coli strains.
ccdB
508 .. 810  =  303 bp
100 amino acids  =  11.6 kDa
Product: CcdB, a bacterial toxin that poisons DNA
gyrase
Plasmids containing the ccdB gene cannot be
propagated in standard E. coli strains.
lacZα
217 .. 498  =  282 bp
94 amino acids  =  10.5 kDa
Product: LacZα fragment of β-galactosidase
lacZα
217 .. 498  =  282 bp
94 amino acids  =  10.5 kDa
Product: LacZα fragment of β-galactosidase
lac promoter
143 .. 173  =  31 bp
   Segment 1:  -35  
   143 .. 148  =  6 bp
promoter for the E. coli lac operon
lac promoter
143 .. 173  =  31 bp
   Segment 2:  
   149 .. 166  =  18 bp
promoter for the E. coli lac operon
lac promoter
143 .. 173  =  31 bp
   Segment 3:  -10  
   167 .. 173  =  7 bp
promoter for the E. coli lac operon
lac promoter
143 .. 173  =  31 bp
3 segments
promoter for the E. coli lac operon
CAP binding site
107 .. 128  =  22 bp
CAP binding activates transcription in the presence
of cAMP.
CAP binding site
107 .. 128  =  22 bp
CAP binding activates transcription in the presence
of cAMP.
lac operator
181 .. 197  =  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
181 .. 197  =  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).
T3 promoter
242 .. 260  =  19 bp
promoter for bacteriophage T3 RNA polymerase
T3 promoter
242 .. 260  =  19 bp
promoter for bacteriophage T3 RNA polymerase
T7 promoter
329 .. 347  =  19 bp
promoter for bacteriophage T7 RNA polymerase
T7 promoter
329 .. 347  =  19 bp
promoter for bacteriophage T7 RNA polymerase
M13 rev
205 .. 221  =  17 bp
common sequencing primer, one of multiple similar
variants
M13 rev
205 .. 221  =  17 bp
common sequencing primer, one of multiple similar
variants
M13 fwd
354 .. 370  =  17 bp
common sequencing primer, one of multiple similar
variants
M13 fwd
354 .. 370  =  17 bp
common sequencing primer, one of multiple similar
variants
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