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Plasmid Files

pCR®-Blunt II-TOPO® (linearized)

Linearized vector for TOPO® cloning of blunt PCR products.

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pCR-Blunt II-TOPO (linearized) Sequence and MappCR-Blunt II-TOPO (linearized).dna
Map and Sequence File   
Sequence Author:  Invitrogen (Life Technologies)
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 PciI (3058) DraI (2303) DraIII (2255) FseI (2178) SexAI * (2065) SgrAI (2016) SmaI (1992) TspMI - XmaI (1990) AatII (1948) ZraI (1946) BssHII - MauBI (1938) HincII (1912) SfiI (1902) TsoI (1558) RsrII (1545) CAP binding site NsiI (3456) HindIII (3459) Acc65I (3465) KpnI (3469) Eco53kI (3473) SacI (3475) BamHI (3477) SpeI (3483) EcoRI (3508) End (3519) Start (0) EcoRI (7) PstI (16) EcoRV (19) NotI (34) PaeR7I - PspXI - XhoI (40) NsiI (51) XbaI (52) EcoO109I - PspOMI (58) ApaI (62) PvuI (213) BsiWI - SnaBI (243) BsrGI - TatI (303) BsaI (468) XcmI (845) BclI * (870) PflFI - Tth111I (1147) BsrDI (1262) pCR®-Blunt II-TOPO® 3519 bp
PciI  (3058)
1 site
A C A T G T T G T A C A

PciI is inhibited by nonionic detergents.
DraI  (2303)
1 site
T T T A A A A A A T T T
DraIII  (2255)
1 site
C A C N N N G T G G T G N N N C A C

Sticky ends from different DraIII sites may not be compatible.
FseI  (2178)
1 site
G G C C G G C C C C G G C C G G

FseI gradually loses activity when stored at -20°C.
SexAI  (2065)
1 site
A C C W G G T T G G W C C A
* Blocked by Dcm methylation.
Sticky ends from different SexAI sites may not be compatible.
SgrAI  (2016)
1 site
C R C C G G Y G G Y G G C C R C

Efficient cleavage requires at least two copies of the SgrAI
recognition sequence.
SmaI  (1992)
1 site
C C C G G G G G G C C C

SmaI can be used at 37°C for brief incubations.
TspMI  (1990)
1 site
C C C G G G G G G C C C
XmaI  (1990)
1 site
C C C G G G G G G C C C

Efficient cleavage requires at least two copies of the XmaI
recognition sequence.
Full cleavage with XmaI may require a long incubation.
AatII  (1948)
1 site
G A C G T C C T G C A G
ZraI  (1946)
1 site
G A C G T C C T G C A G
BssHII  (1938)
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.
MauBI  (1938)
1 site
C G C G C G C G G C G C G C G C
HincII  (1912)
1 site
G T Y R A C C A R Y T G
SfiI  (1902)
1 site
G G C C N N N N N G G C C C C G G N N N N N C C G G

Efficient cleavage requires at least two copies of the SfiI
recognition sequence.
Sticky ends from different SfiI sites may not be compatible.
TsoI  (1558)
1 site
T A R C C A ( N ) 9 N N A T Y G G T ( N ) 9

Sticky ends from different TsoI sites may not be compatible.
After cleavage, TsoI can remain bound to DNA and alter its
electrophoretic mobility.
For full activity, add fresh S-adenosylmethionine (SAM).
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.
NsiI  (3456)
2 sites
A T G C A T T A C G T A
HindIII  (3459)
1 site
A A G C T T T T C G A A
Acc65I  (3465)
1 site
G G T A C C C C A T G G
KpnI  (3469)
1 site
G G T A C C C C A T G G
Eco53kI  (3473)
1 site
G A G C T C C T C G A G
SacI  (3475)
1 site
G A G C T C C T C G A G
BamHI  (3477)
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  (3483)
1 site
A C T A G T T G A T C A
EcoRI  (3508)
2 sites
G A A T T C C T T A A G
End  (3519)
0 sites
Start  (0)
0 sites
EcoRI  (7)
2 sites
G A A T T C C T T A A G
PstI  (16)
1 site
C T G C A G G A C G T C
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.
NotI  (34)
1 site
G C G G C C G C C G C C G G C G
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
NsiI  (51)
2 sites
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.
PvuI  (213)
1 site
C G A T C G G C T A G 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.
TatI  (303)
1 site
W G T A C W W C A T G W
BsaI  (468)
1 site
G G T C T C N C C A G A G N ( N ) 4

Sticky ends from different BsaI sites may not be compatible.
BsaI can be used between 37°C and 50°C.
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.
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.
BsrDI  (1262)
1 site
G C A A T G N N C G T T A C

Sticky ends from different BsrDI sites may not be compatible.
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
2414 .. 3002  =  589 bp
high-copy-number ColE1/pMB1/pBR322/pUC origin
of replication
ori
2414 .. 3002  =  589 bp
high-copy-number ColE1/pMB1/pBR322/pUC origin
of replication
BleoR
1902 .. 2276  =  375 bp
124 amino acids  =  13.8 kDa
Product: antibiotic-binding protein
confers resistance to bleomycin, phleomycin, and
Zeocin™
BleoR
1902 .. 2276  =  375 bp
124 amino acids  =  13.8 kDa
Product: antibiotic-binding protein
confers resistance to bleomycin, phleomycin, and
Zeocin™
ccdB
250 .. 552  =  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
250 .. 552  =  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 .. 240  =  240 bp
80 amino acids  =  9.4 kDa
Product: LacZα fragment of β-galactosidase
lacZα
1 .. 240  =  240 bp
80 amino acids  =  9.4 kDa
Product: LacZα fragment of β-galactosidase
lacZα
3400 .. 3519  =  120 bp
40 amino acids  =  4.2 kDa
Product: LacZα fragment of β-galactosidase
lacZα
3400 .. 3519  =  120 bp
40 amino acids  =  4.2 kDa
Product: LacZα fragment of β-galactosidase
lac promoter
3326 .. 3356  =  31 bp
   Segment 1:  -35  
   3326 .. 3331  =  6 bp
promoter for the E. coli lac operon
lac promoter
3326 .. 3356  =  31 bp
   Segment 2:  
   3332 .. 3349  =  18 bp
promoter for the E. coli lac operon
lac promoter
3326 .. 3356  =  31 bp
   Segment 3:  -10  
   3350 .. 3356  =  7 bp
promoter for the E. coli lac operon
lac promoter
3326 .. 3356  =  31 bp
3 segments
promoter for the E. coli lac operon
CAP binding site
3290 .. 3311  =  22 bp
CAP binding activates transcription in the presence
of cAMP.
CAP binding site
3290 .. 3311  =  22 bp
CAP binding activates transcription in the presence
of cAMP.
lac operator
3364 .. 3380  =  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
3364 .. 3380  =  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
71 .. 89  =  19 bp
promoter for bacteriophage T7 RNA polymerase
T7 promoter
71 .. 89  =  19 bp
promoter for bacteriophage T7 RNA polymerase
SP6 promoter
3422 .. 3440  =  19 bp
promoter for bacteriophage SP6 RNA polymerase
SP6 promoter
3422 .. 3440  =  19 bp
promoter for bacteriophage SP6 RNA polymerase
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
3388 .. 3404  =  17 bp
common sequencing primer, one of multiple similar
variants
M13 rev
3388 .. 3404  =  17 bp
common sequencing primer, one of multiple similar
variants
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