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pLenti6.4/R4R2/V5-DEST

Lentiviral Gateway® destination vector for a micro-RNA (miRNA) expression cassette.

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pLenti6.4 R4R2 V5-DEST Sequence and MappLenti6.4 R4R2 V5-DEST.dna
Map and Sequence File   
Sequence Author:  Invitrogen (Life Technologies)
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 PciI (8512) AhdI (7624) DraIII (6484) AvrII (6043) SfiI (5996) PmlI (4889) SpeI (4624) EcoNI (942) MfeI (961) BstAPI (1225) AleI (1349) KflI (1706) HpaI (1936) EcoRI (2396) PasI (2628) BstZ17I (2931) TspMI - XmaI (3265) SmaI - SrfI (3267) BfuAI - BspMI (3511) PstI (3522) PaeR7I - PspXI - XhoI (3679) PspOMI (3691) ApaI (3695) BstBI (3702) MluI (3746) PfoI (4045) pLenti6.4/R4R2/V5-DEST 8922 bp
PciI  (8512)
1 site
A C A T G T T G T A C A

PciI is inhibited by nonionic detergents.
AhdI  (7624)
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.
DraIII  (6484)
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.
AvrII  (6043)
1 site
C C T A G G G G A T C C
SfiI  (5996)
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.
PmlI  (4889)
1 site
C A C G T G G T G C A C

PmlI gradually loses activity when stored at -20°C.
SpeI  (4624)
1 site
A C T A G T T G A T C A
EcoNI  (942)
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  (961)
1 site
C A A T T G G T T A A C
BstAPI  (1225)
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.
AleI  (1349)
1 site
C A C N N N N G T G G T G N N N N C A C
KflI  (1706)
1 site
G G G W C C C C C C W G G G

Sticky ends from different KflI sites may not be compatible.
HpaI  (1936)
1 site
G T T A A C C A A T T G
EcoRI  (2396)
1 site
G A A T T C C T T A A G
PasI  (2628)
1 site
C C C W G G G G G G W C C C

Sticky ends from different PasI sites may not be compatible.
BstZ17I  (2931)
1 site
G T A T A C C A T A T G
TspMI  (3265)
1 site
C C C G G G G G G C C C
XmaI  (3265)
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  (3267)
1 site
C C C G G G G G G C C C

SmaI can be used at 37°C for brief incubations.
SrfI  (3267)
1 site
G C C C G G G C C G G G C C C G
BfuAI  (3511)
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  (3511)
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.
PstI  (3522)
1 site
C T G C A G G A C G T C
PaeR7I  (3679)
1 site
C T C G A G G A G C T C

PaeR7I does not recognize the sequence CTCTCGAG.
PspXI  (3679)
1 site
V C T C G A G B B G A G C T C V
XhoI  (3679)
1 site
C T C G A G G A G C T C
PspOMI  (3691)
1 site
G G G C C C C C C G G G
ApaI  (3695)
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  (3702)
1 site
T T C G A A A A G C T T
MluI  (3746)
1 site
A C G C G T T G C G C A
PfoI  (4045)
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.
AmpR
6837 .. 7697  =  861 bp
286 amino acids  =  31.6 kDa
   Segment 1:  signal sequence  
   6837 .. 6905  =  69 bp
   23 amino acids  =  2.6 kDa
Product: β-lactamase
confers resistance to ampicillin, carbenicillin, and
related antibiotics
AmpR
6837 .. 7697  =  861 bp
286 amino acids  =  31.6 kDa
   Segment 2:  
   6906 .. 7697  =  792 bp
   263 amino acids  =  28.9 kDa
Product: β-lactamase
confers resistance to ampicillin, carbenicillin, and
related antibiotics
AmpR
6837 .. 7697  =  861 bp
286 amino acids  =  31.6 kDa
2 segments
Product: β-lactamase
confers resistance to ampicillin, carbenicillin, and
related antibiotics
CmR
2183 .. 2842  =  660 bp
219 amino acids  =  25.7 kDa
Product: chloramphenicol acetyltransferase
confers resistance to chloramphenicol
CmR
2183 .. 2842  =  660 bp
219 amino acids  =  25.7 kDa
Product: chloramphenicol acetyltransferase
confers resistance to chloramphenicol
WPRE
3772 .. 4360  =  589 bp
woodchuck posttranscriptional regulatory element
WPRE
3772 .. 4360  =  589 bp
woodchuck posttranscriptional regulatory element
ori
7868 .. 8456  =  589 bp
high-copy-number ColE1/pMB1/pBR322/pUC origin
of replication
ori
7868 .. 8456  =  589 bp
high-copy-number ColE1/pMB1/pBR322/pUC origin
of replication
PGK promoter
4379 .. 4878  =  500 bp
mouse phosphoglycerate kinase 1 promoter
PGK promoter
4379 .. 4878  =  500 bp
mouse phosphoglycerate kinase 1 promoter
f1 ori
6251 .. 6706  =  456 bp
f1 bacteriophage origin of replication; arrow
indicates direction of (+) strand synthesis
f1 ori
6251 .. 6706  =  456 bp
f1 bacteriophage origin of replication; arrow
indicates direction of (+) strand synthesis
BSD
4958 .. 5356  =  399 bp
132 amino acids  =  13.7 kDa
Product: blasticidin S deaminase
confers resistance to blasticidin
BSD
4958 .. 5356  =  399 bp
132 amino acids  =  13.7 kDa
Product: blasticidin S deaminase
confers resistance to blasticidin
ccdB
3184 .. 3489  =  306 bp
101 amino acids  =  11.7 kDa
Product: CcdB, a bacterial toxin that poisons DNA
gyrase
Plasmids containing the ccdB gene cannot be
propagated in standard E. coli strains.
ccdB
3184 .. 3489  =  306 bp
101 amino acids  =  11.7 kDa
Product: CcdB, a bacterial toxin that poisons DNA
gyrase
Plasmids containing the ccdB gene cannot be
propagated in standard E. coli strains.
RRE
1075 .. 1308  =  234 bp
The Rev response element (RRE) of HIV-1 allows for
Rev-dependent mRNA export from the nucleus to
the cytoplasm.
RRE
1075 .. 1308  =  234 bp
The Rev response element (RRE) of HIV-1 allows for
Rev-dependent mRNA export from the nucleus to
the cytoplasm.
3' LTR (ΔU3)
5443 .. 5676  =  234 bp
self-inactivating 3' long terminal repeat (LTR) from
HIV-1
3' LTR (ΔU3)
5443 .. 5676  =  234 bp
self-inactivating 3' long terminal repeat (LTR) from
HIV-1
RSV promoter
1 .. 229  =  229 bp
Rous Sarcoma Virus enhancer/promoter
RSV promoter
1 .. 229  =  229 bp
Rous Sarcoma Virus enhancer/promoter
5' LTR (truncated)
230 .. 410  =  181 bp
truncated 5' long terminal repeat (LTR) from HIV-1
5' LTR (truncated)
230 .. 410  =  181 bp
truncated 5' long terminal repeat (LTR) from HIV-1
SV40 ori
5909 .. 6044  =  136 bp
SV40 origin of replication
SV40 ori
5909 .. 6044  =  136 bp
SV40 origin of replication
HIV-1 Ψ
457 .. 582  =  126 bp
packaging signal of human immunodeficiency virus
type 1
HIV-1 Ψ
457 .. 582  =  126 bp
packaging signal of human immunodeficiency virus
type 1
attR2
3530 .. 3654  =  125 bp
recombination site for the Gateway® LR reaction
attR2
3530 .. 3654  =  125 bp
recombination site for the Gateway® LR reaction
attR4
1951 .. 2074  =  124 bp
recombination site for the Gateway® LR reaction
attR4
1951 .. 2074  =  124 bp
recombination site for the Gateway® LR reaction
SV40 poly(A) signal
5748 .. 5869  =  122 bp
SV40 polyadenylation signal
SV40 poly(A) signal
5748 .. 5869  =  122 bp
SV40 polyadenylation signal
cPPT/CTS
1804 .. 1921  =  118 bp
central polypurine tract and central termination
sequence of HIV-1
cPPT/CTS
1804 .. 1921  =  118 bp
central polypurine tract and central termination
sequence of HIV-1
AmpR promoter
6732 .. 6836  =  105 bp
AmpR promoter
6732 .. 6836  =  105 bp
EM7 promoter
4892 .. 4939  =  48 bp
synthetic bacterial promoter
EM7 promoter
4892 .. 4939  =  48 bp
synthetic bacterial promoter
V5 tag
3707 .. 3748  =  42 bp
14 amino acids  =  1.4 kDa
Product: epitope tag from simian virus 5
V5 tag
3707 .. 3748  =  42 bp
14 amino acids  =  1.4 kDa
Product: epitope tag from simian virus 5
lac UV5 promoter
2099 .. 2129  =  31 bp
   Segment 1:  -35  
   2099 .. 2104  =  6 bp
E. coli lac promoter with an "up" mutation
lac UV5 promoter
2099 .. 2129  =  31 bp
   Segment 2:  
   2105 .. 2122  =  18 bp
E. coli lac promoter with an "up" mutation
lac UV5 promoter
2099 .. 2129  =  31 bp
   Segment 3:  -10  
   2123 .. 2129  =  7 bp
E. coli lac promoter with an "up" mutation
lac UV5 promoter
2099 .. 2129  =  31 bp
3 segments
E. coli lac promoter with an "up" mutation
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