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Plasmid Files

pCR-XL-2-TOPO™ (linearized)

Linearized vector with bound topoisomerase, for TOPO™ cloning of long blunt-ended PCR products. Positive clones disrupt a lethal lacZα-ccdB fusion gene.

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pCR-XL-2-TOPO (linearized).dna
Map and Sequence File:    Download    Open   
Sequence Author:  Thermo Fisher (Invitrogen)
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AflIII - PciI (3537) SspI (2786) XmnI (2581) ScaI (2462) BglI (2102) BpmI (2052) AhdI (1982) RsrII (1509) SpeI (3924) PstI - SbfI (3935) PmeI (3940) EcoRI (3945) End (3956) Start (0) EcoRI (7) NotI (14) BsiWI - SnaBI (207) BsrGI (267) BmgBI (328) BstXI (411) XcmI (809) BclI * (834) PflFI - Tth111I (1111) BanII (1358) SphI (1398) BtgI - NcoI (1425) NgoMIV (1493) NaeI (1495) pCR-XL-2-TOPO™ (linearized) 3956 bp
AflIII  (3537)
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  (3537)
1 site
A C A T G T T G T A C A

PciI is inhibited by nonionic detergents.
SspI  (2786)
1 site
A A T A T T T T A T A A
XmnI  (2581)
1 site
G A A N N N N T T C C T T N N N N A A G
ScaI  (2462)
1 site
A G T A C T T C A T G A
BglI  (2102)
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  (2052)
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  (1982)
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  (1509)
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  (3924)
1 site
A C T A G T T G A T C A
PstI  (3935)
1 site
C T G C A G G A C G T C
SbfI  (3935)
1 site
C C T G C A G G G G A C G T C C
PmeI  (3940)
1 site
G T T T A A A C C A A A T T T G
EcoRI  (3945)
2 sites
G A A T T C C T T A A G
End  (3956)
0 sites
Start  (0)
0 sites
EcoRI  (7)
2 sites
G A A T T C C T T A A G
NotI  (14)
1 site
G C G G C C G C C G C C G G C G
BsiWI  (207)
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  (207)
1 site
T A C G T A A T G C A T
BsrGI  (267)
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  (328)
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  (411)
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  (809)
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  (834)
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  (1111)
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  (1111)
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  (1358)
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  (1398)
1 site
G C A T G C C G T A C G
BtgI  (1425)
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  (1425)
1 site
C C A T G G G G T A C C
NgoMIV  (1493)
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  (1495)
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
1909 .. 2769  =  861 bp
286 amino acids  =  31.6 kDa
   Segment 2:  
   1909 .. 2700  =  792 bp
   263 amino acids  =  28.9 kDa
Product: β-lactamase
confers resistance to ampicillin, carbenicillin, and related antibiotics
AmpR
1909 .. 2769  =  861 bp
286 amino acids  =  31.6 kDa
   Segment 1:  signal sequence  
   2701 .. 2769  =  69 bp
   23 amino acids  =  2.6 kDa
Product: β-lactamase
confers resistance to ampicillin, carbenicillin, and related antibiotics
AmpR
1909 .. 2769  =  861 bp
286 amino acids  =  31.6 kDa
2 segments
Product: β-lactamase
confers resistance to ampicillin, carbenicillin, and related antibiotics
NeoR/KanR
865 .. 1659  =  795 bp
264 amino acids  =  29.0 kDa
Product: aminoglycoside phosphotransferase from Tn5
confers resistance to neomycin, kanamycin, and G418 (Geneticin®)
NeoR/KanR
865 .. 1659  =  795 bp
264 amino acids  =  29.0 kDa
Product: aminoglycoside phosphotransferase from Tn5
confers resistance to neomycin, kanamycin, and G418 (Geneticin®)
ori
2893 .. 3481  =  589 bp
high-copy-number ColE1/pMB1/pBR322/pUC origin of replication
ori
2893 .. 3481  =  589 bp
high-copy-number ColE1/pMB1/pBR322/pUC origin of replication
ccdB
214 .. 516  =  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
214 .. 516  =  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
1 .. 204  =  204 bp
68 amino acids  =  7.8 kDa
Product: LacZα fragment of β-galactosidase
lacZα
1 .. 204  =  204 bp
68 amino acids  =  7.8 kDa
Product: LacZα fragment of β-galactosidase
lacZα
3879 .. 3956  =  78 bp
26 amino acids  =  2.7 kDa
Product: LacZα fragment of β-galactosidase
lacZα
3879 .. 3956  =  78 bp
26 amino acids  =  2.7 kDa
Product: LacZα fragment of β-galactosidase
lac promoter
3805 .. 3835  =  31 bp
   Segment 1:  -35  
   3805 .. 3810  =  6 bp
promoter for the E. coli lac operon
lac promoter
3805 .. 3835  =  31 bp
   Segment 2:  
   3811 .. 3828  =  18 bp
promoter for the E. coli lac operon
lac promoter
3805 .. 3835  =  31 bp
   Segment 3:  -10  
   3829 .. 3835  =  7 bp
promoter for the E. coli lac operon
lac promoter
3805 .. 3835  =  31 bp
3 segments
promoter for the E. coli lac operon
lac operator
3843 .. 3859  =  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
3843 .. 3859  =  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).
T7 promoter
35 .. 53  =  19 bp
promoter for bacteriophage T7 RNA polymerase
T7 promoter
35 .. 53  =  19 bp
promoter for bacteriophage T7 RNA polymerase
T3 promoter
3904 .. 3922  =  19 bp
promoter for bacteriophage T3 RNA polymerase
T3 promoter
3904 .. 3922  =  19 bp
promoter for bacteriophage T3 RNA polymerase
M13 fwd
60 .. 76  =  17 bp
common sequencing primer, one of multiple similar variants
M13 fwd
60 .. 76  =  17 bp
common sequencing primer, one of multiple similar variants
M13 rev
3867 .. 3883  =  17 bp
common sequencing primer, one of multiple similar variants
M13 rev
3867 .. 3883  =  17 bp
common sequencing primer, one of multiple similar variants
ORF:  1 .. 516  =  516 bp
ORF:  171 amino acids  =  19.8 kDa  (no start codon)
ORF:  865 .. 1659  =  795 bp
ORF:  264 amino acids  =  29.0 kDa
ORF:  2 .. 310  =  309 bp
ORF:  102 amino acids  =  11.4 kDa  (no start codon)
ORF:  1037 .. 1423  =  387 bp
ORF:  128 amino acids  =  14.6 kDa
ORF:  2039 .. 2305  =  267 bp
ORF:  88 amino acids  =  9.2 kDa
ORF:  1174 .. 1707  =  534 bp
ORF:  177 amino acids  =  19.9 kDa
ORF:  1909 .. 2769  =  861 bp
ORF:  286 amino acids  =  31.6 kDa
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