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pLenti7.3/V5-TOPO® (linearized)

Linearized lentiviral expression vector with 3'-T overhangs and bound topoisomerase, for TOPO® TA cloning of PCR products and co-expression with GFP.

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pLenti7.3 V5-TOPO (linearized) Sequence and MappLenti7.3 V5-TOPO (linearized).dna
Map and Sequence File   
Sequence Author:  Invitrogen (Life Technologies)
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 AflII (5666) PciI (4963) AhdI (4075) ScaI (3594) AanI (3060) DraIII (2935) SfiI (2447) AanI (2218) AanI (2162) AflII (2010) BsrGI (1812) BclI * (1088) KasI (5786) NarI (5787) SfoI (5788) BbeI (5790) NruI * (5982) EcoNI (6315) MfeI (6334) BbvCI (6569) PpuMI - SanDI (7079) HincII - HpaI (7173) BspDI - ClaI (7296) NdeI (7490) SnaBI (7596) BsmBI (7845) BamHI (7901) SpeI (7907) End (7935) Start (1) PstI (16) EcoRV (19) PaeR7I - PspXI - XhoI (40) PspOMI (52) ApaI (56) BstBI (63) MluI (107) PflMI (374) SexAI * (804) TspMI - XmaI (1058) SmaI (1060) pLenti7.3/V5-TOPO® 7934 bp
AflII  (5666)
2 sites
C T T A A G G A A T T C

The sticky ends produced by AflII are hard to ligate.
PciI  (4963)
1 site
A C A T G T T G T A C A

PciI is inhibited by nonionic detergents.
AhdI  (4075)
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.
ScaI  (3594)
1 site
A G T A C T T C A T G A
AanI  (3060)
3 sites
T T A T A A A A T A T T
DraIII  (2935)
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.
SfiI  (2447)
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.
AanI  (2218)
3 sites
T T A T A A A A T A T T
AanI  (2162)
3 sites
T T A T A A A A T A T T
AflII  (2010)
2 sites
C T T A A G G A A T T C

The sticky ends produced by AflII are hard to ligate.
BsrGI  (1812)
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.
BclI  (1088)
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.
KasI  (5786)
1 site
G G C G C C C C G C G G
NarI  (5787)
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.
SfoI  (5788)
1 site
G G C G C C C C G C G G
BbeI  (5790)
1 site
G G C G C C C C G C G G

Efficient cleavage requires at least two copies of the BbeI
recognition sequence.
NruI  (5982)
1 site
T C G C G A A G C G C T
* Blocked by Dam methylation.
EcoNI  (6315)
1 site
C C T N N N N N A G G G G A N N N N N T C C

The 1-base overhangs produced by EcoNI may be hard to ligate.
Sticky ends from different EcoNI sites may not be compatible.
MfeI  (6334)
1 site
C A A T T G G T T A A C
BbvCI  (6569)
1 site
C C T C A G C G G A G T C G
PpuMI  (7079)
1 site
R G G W C C Y Y C C W G G R

Sticky ends from different PpuMI sites may not be compatible.
SanDI  (7079)
1 site
G G G W C C C C C C W G G G

Sticky ends from different SanDI sites may not be compatible.
HincII  (7173)
1 site
G T Y R A C C A R Y T G
HpaI  (7173)
1 site
G T T A A C C A A T T G
BspDI  (7296)
1 site
A T C G A T T A G C T A
ClaI  (7296)
1 site
A T C G A T T A G C T A
NdeI  (7490)
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.
SnaBI  (7596)
1 site
T A C G T A A T G C A T
BsmBI  (7845)
1 site
C G T C T C N G C A G A G N ( N ) 4

Sticky ends from different BsmBI sites may not be compatible.
BamHI  (7901)
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  (7907)
1 site
A C T A G T T G A T C A
End  (7935)
0 sites
Start  (1)
0 sites
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.
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
PspOMI  (52)
1 site
G G G C C C C C C G G G
ApaI  (56)
1 site
G G G C C C C C C G G G

ApaI can be used between 25°C and 37°C.
BstBI  (63)
1 site
T T C G A A A A G C T T
MluI  (107)
1 site
A C G C G T T G C G C A
PflMI  (374)
1 site
C C A N N N N N T G G G G T N N N N N A C C

Sticky ends from different PflMI sites may not be compatible.
SexAI  (804)
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.
TspMI  (1058)
1 site
C C C G G G G G G C C C
XmaI  (1058)
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.
SmaI  (1060)
1 site
C C C G G G G G G C C C

SmaI can be used at 37°C for brief incubations.
AmpR
3288 .. 4148  =  861 bp
286 amino acids  =  31.6 kDa
   Segment 1:  signal sequence  
   3288 .. 3356  =  69 bp
   23 amino acids  =  2.6 kDa
Product: β-lactamase
confers resistance to ampicillin, carbenicillin, and
related antibiotics
AmpR
3288 .. 4148  =  861 bp
286 amino acids  =  31.6 kDa
   Segment 2:  
   3357 .. 4148  =  792 bp
   263 amino acids  =  28.9 kDa
Product: β-lactamase
confers resistance to ampicillin, carbenicillin, and
related antibiotics
AmpR
3288 .. 4148  =  861 bp
286 amino acids  =  31.6 kDa
2 segments
Product: β-lactamase
confers resistance to ampicillin, carbenicillin, and
related antibiotics
EmGFP
1103 .. 1822  =  720 bp
239 amino acids  =  26.9 kDa
   Segment 1:  
   1103 .. 1105  =  3 bp
   1 amino acid  =  149.2 Da
Product: Emerald GFP
mammalian codon-optimized
EmGFP
1103 .. 1822  =  720 bp
239 amino acids  =  26.9 kDa
   Segment 2:  1a  
   1106 .. 1108  =  3 bp
   1 amino acid  =  117.2 Da
Product: Emerald GFP
mammalian codon-optimized
EmGFP
1103 .. 1822  =  720 bp
239 amino acids  =  26.9 kDa
   Segment 3:  
   1109 .. 1822  =  714 bp
   237 amino acids  =  26.7 kDa
Product: Emerald GFP
mammalian codon-optimized
EmGFP
1103 .. 1822  =  720 bp
239 amino acids  =  26.9 kDa
3 segments
Product: Emerald GFP
mammalian codon-optimized
WPRE
133 .. 721  =  589 bp
woodchuck posttranscriptional regulatory element
WPRE
133 .. 721  =  589 bp
woodchuck posttranscriptional regulatory element
ori
4319 .. 4907  =  589 bp
high-copy-number colE1/pMB1/pBR322/pUC origin
of replication
ori
4319 .. 4907  =  589 bp
high-copy-number colE1/pMB1/pBR322/pUC origin
of replication
f1 ori
2702 .. 3157  =  456 bp
f1 bacteriophage origin of replication; arrow
indicates direction of (+) strand synthesis
f1 ori
2702 .. 3157  =  456 bp
f1 bacteriophage origin of replication; arrow
indicates direction of (+) strand synthesis
SV40 promoter
736 .. 1052  =  317 bp
SV40 enhancer and early promoter
SV40 promoter
736 .. 1052  =  317 bp
SV40 enhancer and early promoter
CMV enhancer
7317 .. 7620  =  304 bp
human cytomegalovirus immediate early enhancer
CMV enhancer
7317 .. 7620  =  304 bp
human cytomegalovirus immediate early enhancer
3'LTR (ΔU3)
1894 .. 2127  =  234 bp
self-inactivating 3' long terminal repeat (LTR) from
HIV-1
3'LTR (ΔU3)
1894 .. 2127  =  234 bp
self-inactivating 3' long terminal repeat (LTR) from
HIV-1
HIV-1 RRE
6448 .. 6681  =  234 bp
The Rev response element (RRE) of human
immunodeficiency virus type 1 allows for
Rev-dependent mRNA export from the nucleus to
the cytoplasm.
HIV-1 RRE
6448 .. 6681  =  234 bp
The Rev response element (RRE) of human
immunodeficiency virus type 1 allows for
Rev-dependent mRNA export from the nucleus to
the cytoplasm.
RSV promoter
5374 .. 5597  =  224 bp
Rous sarcoma virus promoter
RSV promoter
5374 .. 5597  =  224 bp
Rous sarcoma virus promoter
CMV promoter
7621 .. 7824  =  204 bp
human cytomegalovirus (CMV) immediate early
promoter
CMV promoter
7621 .. 7824  =  204 bp
human cytomegalovirus (CMV) immediate early
promoter
5' LTR
5603 .. 5783  =  181 bp
truncated 5' long terminal repeat (LTR) from HIV-1
5' LTR
5603 .. 5783  =  181 bp
truncated 5' long terminal repeat (LTR) from HIV-1
HIV-1 ψ
5830 .. 5955  =  126 bp
packaging signal of human immunodeficiency virus
type 1
HIV-1 ψ
5830 .. 5955  =  126 bp
packaging signal of human immunodeficiency virus
type 1
SV40 poly(A) signal
2199 .. 2320  =  122 bp
SV40 polyadenylation signal
SV40 poly(A) signal
2199 .. 2320  =  122 bp
SV40 polyadenylation signal
HIV-1 cPPT
7177 .. 7294  =  118 bp
central polypurine tract from pol gene of human
immunodeficiency virus type 1
HIV-1 cPPT
7177 .. 7294  =  118 bp
central polypurine tract from pol gene of human
immunodeficiency virus type 1
AmpR promoter
3183 .. 3287  =  105 bp
AmpR promoter
3183 .. 3287  =  105 bp
V5 tag
68 .. 109  =  42 bp
14 amino acids  =  1.4 kDa
Product: epitope tag from simian virus 5
V5 tag
68 .. 109  =  42 bp
14 amino acids  =  1.4 kDa
Product: epitope tag from simian virus 5
SV40 ori
903 .. 1038  =  136 bp
SV40 origin of replication
SV40 ori
903 .. 1038  =  136 bp
SV40 origin of replication
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