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Plasmid Files

pLKO.1

Stuffer-containing lentiviral vector for expression of shRNA. This plasmid is the official TRC cloning vector.

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pLKO.1 Sequence and MappLKO.1.dna
Map and Sequence File   
Sequence Author:  The RNAi Consortium
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 EcoRI (8691) BsaBI (8379) SwaI (8210) BstBI (7427) AgeI - SgrAI (6816) NdeI (6748) KflI - PpuMI (6441) BbvCI (5931) EcoNI (5677) NruI * (5344) PspFI (4025) XcmI (25) SpeI (253) BlpI (333) AscI (342) HincII (480) BamHI (510) PflFI - Tth111I (571) BsiWI (585) RsrII (645) BstEII (663) SexAI * (1096) Acc65I (1185) KpnI (1189) NcoI (1763) SfiI (1809) AvrII (1856) NgoMIV (2189) NaeI (2191) ScaI (2956) BseYI (4021) pLKO.1 8901 bp
EcoRI  (8691)
1 site
G A A T T C C T T A A G
BsaBI  (8379)
1 site
G A T N N N N A T C C T A N N N N T A G
SwaI  (8210)
1 site
A T T T A A A T T A A A T T T A

SwaI is typically used at 25°C, but is 50% active at 37°C.
BstBI  (7427)
1 site
T T C G A A A A G C T T
AgeI  (6816)
1 site
A C C G G T T G G C C A

AgeI quickly loses activity at 37°C, but can be used at 25°C for
long incubations.
SgrAI  (6816)
1 site
C R C C G G Y G G Y G G C C R C

Efficient cleavage requires at least two copies of the SgrAI
recognition sequence.
NdeI  (6748)
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.
KflI  (6441)
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.
PpuMI  (6441)
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.
BbvCI  (5931)
1 site
C C T C A G C G G A G T C G
EcoNI  (5677)
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.
NruI  (5344)
1 site
T C G C G A A G C G C T
* Blocked by Dam methylation.
PspFI  (4025)
1 site
C C C A G C G G G T C G
XcmI  (25)
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.
SpeI  (253)
1 site
A C T A G T T G A T C A
BlpI  (333)
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.
AscI  (342)
1 site
G G C G C G C C C C G C G C G G
HincII  (480)
1 site
G T Y R A C C A R Y T G
BamHI  (510)
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.
PflFI  (571)
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  (571)
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.
BsiWI  (585)
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.
RsrII  (645)
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.
BstEII  (663)
1 site
G G T N A C C C C A N T G G

Sticky ends from different BstEII sites may not be compatible.
BstEII is typically used at 60°C, but is 50% active at 37°C.
SexAI  (1096)
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.
Acc65I  (1185)
1 site
G G T A C C C C A T G G
KpnI  (1189)
1 site
G G T A C C C C A T G G
NcoI  (1763)
1 site
C C A T G G G G T A C C
SfiI  (1809)
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.
AvrII  (1856)
1 site
C C T A G G G G A T C C
NgoMIV  (2189)
1 site
G C C G G C C G G C C G

Efficient cleavage requires at least two copies of the NgoMIV
recognition sequence.
NaeI  (2191)
1 site
G C C G G C C G G C C G

Efficient cleavage requires at least two copies of the NaeI
recognition sequence.
ScaI  (2956)
1 site
A G T A C T T C A T G A
BseYI  (4021)
1 site
C C C A G C G G G T C G

After cleavage, BseYI can remain bound to DNA and alter its
electrophoretic mobility.
Stuffer
6816 .. 8696  =  1881 bp
can be removed with AgeI + EcoRI and replaced
with an shRNA
Stuffer
6816 .. 8696  =  1881 bp
can be removed with AgeI + EcoRI and replaced
with an shRNA
AmpR
2650 .. 3510  =  861 bp
286 amino acids  =  31.6 kDa
   Segment 1:  signal sequence  
   2650 .. 2718  =  69 bp
   23 amino acids  =  2.6 kDa
Product: β-lactamase
confers resistance to ampicillin, carbenicillin, and
related antibiotics
AmpR
2650 .. 3510  =  861 bp
286 amino acids  =  31.6 kDa
   Segment 2:  
   2719 .. 3510  =  792 bp
   263 amino acids  =  28.9 kDa
Product: β-lactamase
confers resistance to ampicillin, carbenicillin, and
related antibiotics
AmpR
2650 .. 3510  =  861 bp
286 amino acids  =  31.6 kDa
2 segments
Product: β-lactamase
confers resistance to ampicillin, carbenicillin, and
related antibiotics
PuroR
529 .. 1128  =  600 bp
199 amino acids  =  21.5 kDa
Product: puromycin N-acetyltransferase
confers resistance to puromycin
PuroR
529 .. 1128  =  600 bp
199 amino acids  =  21.5 kDa
Product: puromycin N-acetyltransferase
confers resistance to puromycin
ori
3681 .. 4269  =  589 bp
high-copy-number ColE1/pMB1/pBR322/pUC origin
of replication
ori
3681 .. 4269  =  589 bp
high-copy-number ColE1/pMB1/pBR322/pUC origin
of replication
hPGK promoter
8898 .. 507  =  511 bp
human phosphoglycerate kinase 1 promoter
hPGK promoter
8898 .. 507  =  511 bp
human phosphoglycerate kinase 1 promoter
f1 ori
2064 .. 2519  =  456 bp
f1 bacteriophage origin of replication; arrow
indicates direction of (+) strand synthesis
f1 ori
2064 .. 2519  =  456 bp
f1 bacteriophage origin of replication; arrow
indicates direction of (+) strand synthesis
U6 promoter
6570 .. 6810  =  241 bp
RNA polymerase III promoter for human U6 snRNA
U6 promoter
6570 .. 6810  =  241 bp
RNA polymerase III promoter for human U6 snRNA
3' LTR (ΔU3)
1256 .. 1489  =  234 bp
self-inactivating 3' long terminal repeat (LTR) from
HIV-1
3' LTR (ΔU3)
1256 .. 1489  =  234 bp
self-inactivating 3' long terminal repeat (LTR) from
HIV-1
RRE
5810 .. 6043  =  234 bp
The Rev response element (RRE) of HIV-1 allows for
Rev-dependent mRNA export from the nucleus to
the cytoplasm.
RRE
5810 .. 6043  =  234 bp
The Rev response element (RRE) of HIV-1 allows for
Rev-dependent mRNA export from the nucleus to
the cytoplasm.
5' LTR
4965 .. 5145  =  181 bp
truncated 5' long terminal repeat (LTR) from HIV-1
5' LTR
4965 .. 5145  =  181 bp
truncated 5' long terminal repeat (LTR) from HIV-1
SV40 ori
1722 .. 1857  =  136 bp
SV40 origin of replication
SV40 ori
1722 .. 1857  =  136 bp
SV40 origin of replication
HIV-1 ψ
5192 .. 5317  =  126 bp
packaging signal of human immunodeficiency virus
type 1
HIV-1 ψ
5192 .. 5317  =  126 bp
packaging signal of human immunodeficiency virus
type 1
SV40 poly(A) signal
1561 .. 1682  =  122 bp
SV40 polyadenylation signal
SV40 poly(A) signal
1561 .. 1682  =  122 bp
SV40 polyadenylation signal
cPPT/CTS
8732 .. 8849  =  118 bp
central polypurine tract and central termination
sequence of HIV-1
cPPT/CTS
8732 .. 8849  =  118 bp
central polypurine tract and central termination
sequence of HIV-1
AmpR promoter
2545 .. 2649  =  105 bp
AmpR promoter
2545 .. 2649  =  105 bp
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