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Plasmid Files

pBlue-TOPO® (linearized)

Linearized vector with 3'-T overhangs and bound topoisomerase, for TOPO® TA cloning of PCR products to measure promoter activity using β-galactosidase.

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pBlue-TOPO (linearized) Sequence and MappBlue-TOPO (linearized).dna
Map and Sequence File   
Sequence Author:  Invitrogen (Life Technologies)
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 BglII (7689) SgrDI (7675) ScaI (7234) AhdI (6754) BsmI (5430) AanI (5369) BstBI (5192) RsrII (5026) NcoI (4942) PflFI - Tth111I (4628) MscI (4592) PstI (4563) PluTI (4513) SfoI (4511) NarI (4510) KasI (4509) SmaI (4323) TspMI - XmaI (4321) AvrII (4300) StuI (4299) BseRI (4296) NcoI (4207) SexAI * (4067) AanI (3901) T7 promoter PmeI (7735) AflII (7739) HindIII (7742) BamHI (7760) SpeI (7766) End (7793) Start (1) PstI (15) HindIII (18) NcoI (25) ATG 6xHis Xpress™ tag Bpu10I (642) BspDI - ClaI (961) EcoRV (1250) MauBI (1635) AfeI (1972) BsiWI (2908) NdeI (3094) BlpI (3150) NotI (3224) PaeR7I - PspXI - XhoI (3230) XbaI (3236) EcoO109I - PspOMI (3242) ApaI (3246) PmeI (3251) pBlue-TOPO® 7792 bp
BglII  (7689)
1 site
A G A T C T T C T A G A
SgrDI  (7675)
1 site
C G T C G A C G G C A G C T G C
ScaI  (7234)
1 site
A G T A C T T C A T G A
AhdI  (6754)
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.
BsmI  (5430)
1 site
G A A T G C N C T T A C G N

Sticky ends from different BsmI sites may not be compatible.
AanI  (5369)
2 sites
T T A T A A A A T A T T
BstBI  (5192)
1 site
T T C G A A A A G C T T
RsrII  (5026)
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.
NcoI  (4942)
3 sites
C C A T G G G G T A C C
PflFI  (4628)
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  (4628)
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.
MscI  (4592)
1 site
T G G C C A A C C G G T
PstI  (4563)
2 sites
C T G C A G G A C G T C
PluTI  (4513)
1 site
G G C G C C C C G C G G

Efficient cleavage requires at least two copies of the PluTI
recognition sequence.
SfoI  (4511)
1 site
G G C G C C C C G C G G
NarI  (4510)
1 site
G G C G C C C C G C G G

Efficient cleavage requires at least two copies of the NarI
recognition sequence.
KasI  (4509)
1 site
G G C G C C C C G C G G
SmaI  (4323)
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  (4321)
1 site
C C C G G G G G G C C C
XmaI  (4321)
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.
AvrII  (4300)
1 site
C C T A G G G G A T C C
StuI  (4299)
1 site
A G G C C T T C C G G A
BseRI  (4296)
1 site
G A G G A G ( N ) 8 N N C T C C T C ( N ) 8

Sticky ends from different BseRI sites may not be compatible.
BseRI quickly loses activity at 37°C.
Prolonged incubation with BseRI may lead to degradation of the
DNA.
NcoI  (4207)
3 sites
C C A T G G G G T A C C
SexAI  (4067)
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.
AanI  (3901)
2 sites
T T A T A A A A T A T T
PmeI  (7735)
2 sites
G T T T A A A C C A A A T T T G
AflII  (7739)
1 site
C T T A A G G A A T T C

The sticky ends produced by AflII are hard to ligate.
HindIII  (7742)
2 sites
A A G C T T T T C G A A
BamHI  (7760)
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  (7766)
1 site
A C T A G T T G A T C A
End  (7793)
0 sites
Start  (1)
0 sites
PstI  (15)
2 sites
C T G C A G G A C G T C
HindIII  (18)
2 sites
A A G C T T T T C G A A
NcoI  (25)
3 sites
C C A T G G G G T A C C
Bpu10I  (642)
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.
BspDI  (961)
1 site
A T C G A T T A G C T A
ClaI  (961)
1 site
A T C G A T T A G C T A
EcoRV  (1250)
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.
MauBI  (1635)
1 site
C G C G C G C G G C G C G C G C
AfeI  (1972)
1 site
A G C G C T T C G C G A
BsiWI  (2908)
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.
NdeI  (3094)
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.
BlpI  (3150)
1 site
G C T N A G C C G A N T C G

Sticky ends from different BlpI sites may not be compatible.
NotI  (3224)
1 site
G C G G C C G C C G C C G G C G
PaeR7I  (3230)
1 site
C T C G A G G A G C T C

PaeR7I does not recognize the sequence CTCTCGAG.
PspXI  (3230)
1 site
V C T C G A G B B G A G C T C V
XhoI  (3230)
1 site
C T C G A G G A G C T C
XbaI  (3236)
1 site
T C T A G A A G A T C T
EcoO109I  (3242)
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  (3242)
1 site
G G G C C C C C C G G G
ApaI  (3246)
1 site
G G G C C C C C C G G G

ApaI can be used between 25°C and 37°C.
PmeI  (3251)
2 sites
G T T T A A A C C A A A T T T G
lacZ
150 .. 3197  =  3048 bp
1015 amino acids  =  115.5 kDa
Product: β-galactosidase
lacZ
150 .. 3197  =  3048 bp
1015 amino acids  =  115.5 kDa
Product: β-galactosidase
AmpR
6681 .. 7541  =  861 bp
286 amino acids  =  31.6 kDa
   Segment 2:  
   6681 .. 7472  =  792 bp
   263 amino acids  =  28.9 kDa
Product: β-lactamase
confers resistance to ampicillin, carbenicillin, and
related antibiotics
AmpR
6681 .. 7541  =  861 bp
286 amino acids  =  31.6 kDa
   Segment 1:  signal sequence  
   7473 .. 7541  =  69 bp
   23 amino acids  =  2.6 kDa
Product: β-lactamase
confers resistance to ampicillin, carbenicillin, and
related antibiotics
AmpR
6681 .. 7541  =  861 bp
286 amino acids  =  31.6 kDa
2 segments
Product: β-lactamase
confers resistance to ampicillin, carbenicillin, and
related antibiotics
NeoR/KanR
4382 .. 5176  =  795 bp
264 amino acids  =  29.0 kDa
Product: aminoglycoside phosphotransferase from
Tn5
confers resistance to neomycin, kanamycin, and
G418 (Geneticin)
NeoR/KanR
4382 .. 5176  =  795 bp
264 amino acids  =  29.0 kDa
Product: aminoglycoside phosphotransferase from
Tn5
confers resistance to neomycin, kanamycin, and
G418 (Geneticin)
ori
5922 .. 6510  =  589 bp
high-copy-number colE1/pMB1/pBR322/pUC origin
of replication
ori
5922 .. 6510  =  589 bp
high-copy-number colE1/pMB1/pBR322/pUC origin
of replication
f1 ori
3543 .. 3971  =  429 bp
f1 bacteriophage origin of replication; arrow
indicates direction of (+) strand synthesis
f1 ori
3543 .. 3971  =  429 bp
f1 bacteriophage origin of replication; arrow
indicates direction of (+) strand synthesis
SV40 promoter
3985 .. 4315  =  331 bp
SV40 enhancer and early promoter
SV40 promoter
3985 .. 4315  =  331 bp
SV40 enhancer and early promoter
bGH poly(A) signal
3273 .. 3497  =  225 bp
bovine growth hormone polyadenylation signal
bGH poly(A) signal
3273 .. 3497  =  225 bp
bovine growth hormone polyadenylation signal
SV40 poly(A) signal
5350 .. 5471  =  122 bp
SV40 polyadenylation signal
SV40 poly(A) signal
5350 .. 5471  =  122 bp
SV40 polyadenylation signal
AmpR promoter
7542 .. 7646  =  105 bp
AmpR promoter
7542 .. 7646  =  105 bp
T7 tag
60 .. 92  =  33 bp
11 amino acids  =  1.1 kDa
Product: epitope tag from a T7 major capsid protein
T7 tag
60 .. 92  =  33 bp
11 amino acids  =  1.1 kDa
Product: epitope tag from a T7 major capsid protein
Xpress™ tag
96 .. 119  =  24 bp
8 amino acids  =  998.0 Da
   Segment 1:  
   96 .. 104  =  9 bp
   3 amino acids  =  409.5 Da
Product: Xpress™ epitope tag, including an
enterokinase recognition and cleavage site
Xpress™ tag
96 .. 119  =  24 bp
8 amino acids  =  998.0 Da
   Segment 2:  enterokinase site  
   105 .. 119  =  15 bp
   5 amino acids  =  606.5 Da
Product: Xpress™ epitope tag, including an
enterokinase recognition and cleavage site
Xpress™ tag
96 .. 119  =  24 bp
8 amino acids  =  998.0 Da
2 segments
Product: Xpress™ epitope tag, including an
enterokinase recognition and cleavage site
T7 promoter
7694 .. 7712  =  19 bp
promoter for bacteriophage T7 RNA polymerase
T7 promoter
7694 .. 7712  =  19 bp
promoter for bacteriophage T7 RNA polymerase
6xHis
39 .. 56  =  18 bp
6 amino acids  =  840.9 Da
Product: 6xHis affinity tag
6xHis
39 .. 56  =  18 bp
6 amino acids  =  840.9 Da
Product: 6xHis affinity tag
ATG
27 .. 29  =  3 bp
1 amino acid  =  149.2 Da
Product: start codon
ATG
27 .. 29  =  3 bp
1 amino acid  =  149.2 Da
Product: start codon
SV40 ori
4166 .. 4301  =  136 bp
SV40 origin of replication
SV40 ori
4166 .. 4301  =  136 bp
SV40 origin of replication
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