pLKO.1 puro

Empty lentiviral vector for expression of shRNA. See also pLKO.1.

Sequence Author: Weinberg Lab / Addgene

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EcoRI (6840) MluI (6834) AgeI (6816) BfuAI - BspMI (6815) NdeI (6748) BspDI * - ClaI * (6530) KflI - PpuMI (6441) AleI (6084) BbvCI - Bpu10I (5931) MfeI (5696) EcoNI (5677) NruI * (5344) SphI (4828) T3 promoter PciI (4325) PspFI (4025) BseYI (4021) XcmI (25) SpeI (253) BlpI (333) AscI (342) HincII (480) BamHI (510) PflFI - Tth111I (571) BsiWI (585) BstEII (663) CsiI - SexAI * (1096) NsiI (1182) Acc65I (1185) KpnI (1189) BbsI (1287) NcoI (1763) SfiI (1809) AvrII (1856) NgoMIV (2189) NaeI (2191) ScaI (2956) pLKO.1 puro 7050 bp
EcoRI  (6840)
1 site
G A A T T C C T T A A G
MluI  (6834)
1 site
A C G C G T T G C G C A
AgeI  (6816)
1 site
A C C G G T T G G C C A
BfuAI  (6815)
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  (6815)
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.
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.
BspDI  (6530)
1 site
A T C G A T T A G C T A
* Blocked by Dam methylation.
ClaI  (6530)
1 site
A T C G A T T A G C T A
* Blocked by Dam methylation.
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.
AleI  (6084)
1 site
C A C N N N N G T G G T G N N N N C A C
BbvCI  (5931)
1 site
C C T C A G C G G A G T C G
Bpu10I  (5931)
1 site
C C T N A G C G G A N T C G

Cleavage may be enhanced when more than one copy of the Bpu10I recognition sequence is present.
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.
MfeI  (5696)
1 site
C A A T T G G T T A A C
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.
SphI  (4828)
1 site
G C A T G C C G T A C G
PciI  (4325)
1 site
A C A T G T T G T A C A

PciI is inhibited by nonionic detergents.
PspFI  (4025)
1 site
C C C A G C G G G T C G
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.
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.
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.
CsiI  (1096)
1 site
A C C W G G T T G G W C C A

Sticky ends from different CsiI sites may not be compatible.
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.
NsiI  (1182)
1 site
A T G C A T T A C G T A
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
BbsI  (1287)
1 site
G A A G A C N N C T T C T G N N ( N ) 4

Sticky ends from different BbsI sites may not be compatible.
BbsI gradually loses activity when stored at -20°C.
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
AmpR
2650 .. 3510  =  861 bp
286 amino acids  =  31.6 kDa
2 segments
   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
2 segments
   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
7047 .. 507  =  511 bp
human phosphoglycerate kinase 1 promoter
hPGK promoter
7047 .. 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 .. 6818  =  249 bp
RNA polymerase III promoter for human U6 snRNA (Domitrovich & Kunkel, 2003)
U6 promoter
6570 .. 6818  =  249 bp
RNA polymerase III promoter for human U6 snRNA (Domitrovich & Kunkel, 2003)
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.
RSV promoter
4736 .. 4964  =  229 bp
Rous sarcoma virus enhancer/promoter
RSV promoter
4736 .. 4964  =  229 bp
Rous sarcoma virus enhancer/promoter
5' LTR (truncated)
4965 .. 5145  =  181 bp
truncated 5' long terminal repeat (LTR) from HIV-1
5' LTR (truncated)
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
6881 .. 6998  =  118 bp
central polypurine tract and central termination sequence of HIV-1
cPPT/CTS
6881 .. 6998  =  118 bp
central polypurine tract and central termination sequence of HIV-1
AmpR promoter
2545 .. 2649  =  105 bp
AmpR promoter
2545 .. 2649  =  105 bp
T3 promoter
4692 .. 4710  =  19 bp
promoter for bacteriophage T3 RNA polymerase
T3 promoter
4692 .. 4710  =  19 bp
promoter for bacteriophage T3 RNA polymerase
ORF:  289 .. 1128  =  840 bp
ORF:  279 amino acids  =  29.8 kDa
ORF:  2650 .. 3510  =  861 bp
ORF:  286 amino acids  =  31.6 kDa
ORF:  1583 .. 1819  =  237 bp
ORF:  78 amino acids  =  8.6 kDa
ORF:  4667 .. 4912  =  246 bp
ORF:  81 amino acids  =  8.9 kDa
ORF:  5688 .. 6545  =  858 bp
ORF:  285 amino acids  =  32.9 kDa
ORF:  6861 .. 257  =  447 bp
ORF:  148 amino acids  =  17.0 kDa
ORF:  334 .. 1179  =  846 bp
ORF:  281 amino acids  =  29.2 kDa
ORF:  4594 .. 4893  =  300 bp
ORF:  99 amino acids  =  10.9 kDa
ORF:  6966 .. 530  =  615 bp
ORF:  204 amino acids  =  22.2 kDa
ORF:  3114 .. 3380  =  267 bp
ORF:  88 amino acids  =  9.2 kDa
ORF:  5603 .. 5983  =  381 bp
ORF:  126 amino acids  =  13.3 kDa
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