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pcDNA™5/FRT/TO-TOPO® (linearized)

Linearized vector with 3'-T overhangs and bound topoisomerase, for TOPO® TA cloning of PCR products, FLP recombination, and tetracycline-inducible expression.

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pcDNA5 FRT TO-TOPO (linearized) Sequence and MappcDNA5 FRT TO-TOPO (linearized).dna
Map and Sequence File   
Sequence Author:  Invitrogen (Life Technologies)
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 SspI (4047) XmnI (3842) FspI (3465) BsaI (3304) AhdI (3243) PciI (2350) BspQI - SapI (2234) BstZ17I (1971) BsmI (1919) AanI - PsiI (1858) PfoI (1774) SacII (1464) MfeI (4327) Bpu10I (4346) NruI (4374) MluI (4394) CMV enhancer SnaBI (4756) tet operator tet operator PmeI (5137) AflII (5141) End (5155) Start (1) Acc65I (13) KpnI (17) BamHI (25) BstXI (44) EcoRV (60) BstXI (70) NotI (75) AvaI - BsoBI - PaeR7I - PspXI - XhoI (81) BmeT110I (82) EcoO109I - PspOMI (93) ApaI (97) PmeI (102) BclI * (111) SphI (324) MscI (684) PshAI (711) BfuAI - BspMI (997) AsiSI (1048) RsrII (1092) pcDNA™5/FRT/TO-TOPO® 5154 bp
SspI  (4047)
1 site
A A T A T T T T A T A A
XmnI  (3842)
1 site
G A A N N N N T T C C T T N N N N A A G
FspI  (3465)
1 site
T G C G C A A C G C G T
BsaI  (3304)
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.
AhdI  (3243)
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.
PciI  (2350)
1 site
A C A T G T T G T A C A

PciI is inhibited by nonionic detergents.
BspQI  (2234)
1 site
G C T C T T C N C G A G A A G N N N N

Sticky ends from different BspQI sites may not be compatible.
SapI  (2234)
1 site
G C T C T T C N C G A G A A G N N N N

Sticky ends from different SapI sites may not be compatible.
SapI gradually settles in solution, so a tube of SapI should be
mixed before removing an aliquot.
BstZ17I  (1971)
1 site
G T A T A C C A T A T G
BsmI  (1919)
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  (1858)
1 site
T T A T A A A A T A T T
PsiI  (1858)
1 site
T T A T A A A A T A T T
PfoI  (1774)
1 site
T C C N G G A A G G N C C T

Sticky ends from different PfoI sites may not be compatible.
SacII  (1464)
1 site
C C G C G G G G C G C C

Efficient cleavage requires at least two copies of the SacII
recognition sequence.
MfeI  (4327)
1 site
C A A T T G G T T A A C
Bpu10I  (4346)
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.
NruI  (4374)
1 site
T C G C G A A G C G C T
MluI  (4394)
1 site
A C G C G T T G C G C A
SnaBI  (4756)
1 site
T A C G T A A T G C A T
PmeI  (5137)
2 sites
G T T T A A A C C A A A T T T G
AflII  (5141)
1 site
C T T A A G G A A T T C

The sticky ends produced by AflII are hard to ligate.
End  (5155)
0 sites
Start  (1)
0 sites
Acc65I  (13)
1 site
G G T A C C C C A T G G
KpnI  (17)
1 site
G G T A C C C C A T G G
BamHI  (25)
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.
BstXI  (44)
2 sites
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.
EcoRV  (60)
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.
BstXI  (70)
2 sites
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.
NotI  (75)
1 site
G C G G C C G C C G C C G G C G
AvaI  (81)
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  (81)
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  (81)
1 site
C T C G A G G A G C T C

PaeR7I does not recognize the sequence CTCTCGAG.
PspXI  (81)
1 site
V C T C G A G B B G A G C T C V
XhoI  (81)
1 site
C T C G A G G A G C T C
BmeT110I  (82)
1 site
C Y C G R G G R G C Y C
EcoO109I  (93)
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  (93)
1 site
G G G C C C C C C G G G
ApaI  (97)
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  (102)
2 sites
G T T T A A A C C A A A T T T G
BclI  (111)
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.
SphI  (324)
1 site
G C A T G C C G T A C G
MscI  (684)
1 site
T G G C C A A C C G G T
PshAI  (711)
1 site
G A C N N N N G T C C T G N N N N C A G

PshAI quickly loses activity at 37°C, but can be used at 25°C for
long incubations.
BfuAI  (997)
1 site
A C C T G C ( N ) 4 T G G A C G ( N ) 4 ( N ) 4

Efficient cleavage requires at least two copies of the BfuAI
recognition sequence.
Sticky ends from different BfuAI sites may not be compatible.
BfuAI is typically used at 50°C, but is 50% active at 37°C.
BspMI  (997)
1 site
A C C T G C ( N ) 4 T G G A C G ( N ) 4 ( N ) 4

Efficient cleavage requires at least two copies of the BspMI
recognition sequence.
Sticky ends from different BspMI sites may not be compatible.
AsiSI  (1048)
1 site
G C G A T C G C C G C T A G C G
RsrII  (1092)
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.
HygR
687 .. 1709  =  1023 bp
340 amino acids  =  37.9 kDa
Product: aminoglycoside phosphotransferase from E.
coli

confers resistance to hygromycin
HygR
687 .. 1709  =  1023 bp
340 amino acids  =  37.9 kDa
Product: aminoglycoside phosphotransferase from E.
coli

confers resistance to hygromycin
AmpR
3170 .. 4030  =  861 bp
286 amino acids  =  31.6 kDa
   Segment 2:  
   3170 .. 3961  =  792 bp
   263 amino acids  =  28.9 kDa
Product: β-lactamase
confers resistance to ampicillin, carbenicillin, and
related antibiotics
AmpR
3170 .. 4030  =  861 bp
286 amino acids  =  31.6 kDa
   Segment 1:  signal sequence  
   3962 .. 4030  =  69 bp
   23 amino acids  =  2.6 kDa
Product: β-lactamase
confers resistance to ampicillin, carbenicillin, and
related antibiotics
AmpR
3170 .. 4030  =  861 bp
286 amino acids  =  31.6 kDa
2 segments
Product: β-lactamase
confers resistance to ampicillin, carbenicillin, and
related antibiotics
ori
2411 .. 2999  =  589 bp
high-copy-number ColE1/pMB1/pBR322/pUC origin
of replication
ori
2411 .. 2999  =  589 bp
high-copy-number ColE1/pMB1/pBR322/pUC origin
of replication
CMV enhancer
4401 .. 4780  =  380 bp
human cytomegalovirus immediate early enhancer
CMV enhancer
4401 .. 4780  =  380 bp
human cytomegalovirus immediate early enhancer
bGH poly(A) signal
124 .. 348  =  225 bp
bovine growth hormone polyadenylation signal
bGH poly(A) signal
124 .. 348  =  225 bp
bovine growth hormone polyadenylation signal
CMV promoter
4781 .. 4984  =  204 bp
human cytomegalovirus (CMV) immediate early
promoter
CMV promoter
4781 .. 4984  =  204 bp
human cytomegalovirus (CMV) immediate early
promoter
SV40 poly(A) signal
1839 .. 1960  =  122 bp
SV40 polyadenylation signal
SV40 poly(A) signal
1839 .. 1960  =  122 bp
SV40 polyadenylation signal
AmpR promoter
4031 .. 4135  =  105 bp
AmpR promoter
4031 .. 4135  =  105 bp
FRT
632 .. 679  =  48 bp
FLP-mediated recombination occurs in the 8-bp core
sequence TCTAGAAA (Turan and Bode, 2011).
FRT
632 .. 679  =  48 bp
FLP-mediated recombination occurs in the 8-bp core
sequence TCTAGAAA (Turan and Bode, 2011).
tet operator
4986 .. 5004  =  19 bp
bacterial operator O2 for the tetR and tetA genes
tet operator
4986 .. 5004  =  19 bp
bacterial operator O2 for the tetR and tetA genes
tet operator
5007 .. 5025  =  19 bp
bacterial operator O2 for the tetR and tetA genes
tet operator
5007 .. 5025  =  19 bp
bacterial operator O2 for the tetR and tetA genes
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