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Plasmid Files

pCR®2.1-TOPO® (linearized)

Linearized vector with 3'-T overhangs and bound topoisomerase, for TOPO® TA cloning of PCR products. Similar to pCR®II-TOPO® but lacking an SP6 promoter.

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pCR2.1-TOPO (linearized) Sequence and MappCR2.1-TOPO (linearized).dna
Map and Sequence File   
Sequence Author:  Invitrogen (Life Technologies)
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 PciI (3512) BspHI (2792) AhdI (2624) BsaI (2558) BpmI (2555) ScaI (2143) TatI (2141) XmnI (2024) RsrII (1669) BtgI - NcoI (1585) HindIII (3871) Acc65I (3877) KpnI (3881) Eco53kI (3885) SacI (3887) BamHI (3889) SpeI (3895) End (3931) Start (1) EcoRV (19) NotI (34) AvaI - BsoBI - PaeR7I - PspXI - XhoI (40) BmeT110I (41) NsiI (51) XbaI (52) EcoO109I - PspOMI (58) ApaI (62) DraIII (486) PsiI (611) BstAPI (705) XcmI (969) BglII (989) BclI * (994) MscI (1235) PflFI - Tth111I (1271) BssHII (1550) pCR®2.1-TOPO® 3930 bp
PciI  (3512)
1 site
A C A T G T T G T A C A

PciI is inhibited by nonionic detergents.
BspHI  (2792)
1 site
T C A T G A A G T A C T
AhdI  (2624)
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.
BsaI  (2558)
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.
BpmI  (2555)
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.
ScaI  (2143)
1 site
A G T A C T T C A T G A
TatI  (2141)
1 site
W G T A C W W C A T G W
XmnI  (2024)
1 site
G A A N N N N T T C C T T N N N N A A G
RsrII  (1669)
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.
BtgI  (1585)
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  (1585)
1 site
C C A T G G G G T A C C
HindIII  (3871)
1 site
A A G C T T T T C G A A
Acc65I  (3877)
1 site
G G T A C C C C A T G G
KpnI  (3881)
1 site
G G T A C C C C A T G G
Eco53kI  (3885)
1 site
G A G C T C C T C G A G
SacI  (3887)
1 site
G A G C T C C T C G A G
BamHI  (3889)
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  (3895)
1 site
A C T A G T T G A T C A
End  (3931)
0 sites
Start  (1)
0 sites
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
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.
DraIII  (486)
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.
PsiI  (611)
1 site
T T A T A A A A T A T T
BstAPI  (705)
1 site
G C A N N N N N T G C C G T N N N N N A C G

Sticky ends from different BstAPI sites may not be compatible.
XcmI  (969)
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  (989)
1 site
A G A T C T T C T A G A
BclI  (994)
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.
MscI  (1235)
1 site
T G G C C A A C C G G T
PflFI  (1271)
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  (1271)
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  (1550)
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.
AmpR
1837 .. 2697  =  861 bp
286 amino acids  =  31.6 kDa
   Segment 1:  signal sequence  
   1837 .. 1905  =  69 bp
   23 amino acids  =  2.6 kDa
Product: β-lactamase
confers resistance to ampicillin, carbenicillin, and
related antibiotics
AmpR
1837 .. 2697  =  861 bp
286 amino acids  =  31.6 kDa
   Segment 2:  
   1906 .. 2697  =  792 bp
   263 amino acids  =  28.9 kDa
Product: β-lactamase
confers resistance to ampicillin, carbenicillin, and
related antibiotics
AmpR
1837 .. 2697  =  861 bp
286 amino acids  =  31.6 kDa
2 segments
Product: β-lactamase
confers resistance to ampicillin, carbenicillin, and
related antibiotics
NeoR/KanR
1025 .. 1819  =  795 bp
264 amino acids  =  29.0 kDa
Product: aminoglycoside phosphotransferase from
Tn5
confers resistance to neomycin, kanamycin, and
G418 (Geneticin)
NeoR/KanR
1025 .. 1819  =  795 bp
264 amino acids  =  29.0 kDa
Product: aminoglycoside phosphotransferase from
Tn5
confers resistance to neomycin, kanamycin, and
G418 (Geneticin)
ori
2868 .. 3456  =  589 bp
high-copy-number colE1/pMB1/pBR322/pUC origin
of replication
ori
2868 .. 3456  =  589 bp
high-copy-number colE1/pMB1/pBR322/pUC origin
of replication
f1 ori
253 .. 681  =  429 bp
f1 bacteriophage origin of replication; arrow
indicates direction of (+) strand synthesis
f1 ori
253 .. 681  =  429 bp
f1 bacteriophage origin of replication; arrow
indicates direction of (+) strand synthesis
lacZα
3854 .. 3931  =  78 bp
26 amino acids  =  2.7 kDa
Product: LacZα fragment of β-galactosidase
lacZα
3854 .. 3931  =  78 bp
26 amino acids  =  2.7 kDa
Product: LacZα fragment of β-galactosidase
lac promoter
3780 .. 3810  =  31 bp
   Segment 1:  -35  
   3780 .. 3785  =  6 bp
promoter for the E. coli lac operon
lac promoter
3780 .. 3810  =  31 bp
   Segment 2:  
   3786 .. 3803  =  18 bp
promoter for the E. coli lac operon
lac promoter
3780 .. 3810  =  31 bp
   Segment 3:  -10  
   3804 .. 3810  =  7 bp
promoter for the E. coli lac operon
lac promoter
3780 .. 3810  =  31 bp
3 segments
promoter for the E. coli lac operon
T7 promoter
71 .. 89  =  19 bp
promoter for bacteriophage T7 RNA polymerase
T7 promoter
71 .. 89  =  19 bp
promoter for bacteriophage T7 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
lac operator
3818 .. 3834  =  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
3818 .. 3834  =  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).
lacZα
1 .. 276  =  276 bp
91 amino acids  =  10.6 kDa
Product: LacZα fragment of β-galactosidase
lacZα
1 .. 276  =  276 bp
91 amino acids  =  10.6 kDa
Product: LacZα fragment of β-galactosidase
M13 rev
3842 .. 3858  =  17 bp
common sequencing primer, one of multiple similar
variants
M13 rev
3842 .. 3858  =  17 bp
common sequencing primer, one of multiple similar
variants
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