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Plasmid Files

pLVX-puro-Cas9

Lentiviral Cas9 expression vector with the constitutive CMV promoter and a puromycin resistance marker.

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pLVX-puro-Cas9.dna
Map and Sequence File:    Download    Open   
Sequence Author:  Clontech (TaKaRa)
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SgrDI (11,937) AhdI (11,016) KpnI (8956) Acc65I (8952) SexAI * (8181) BstEII (7748) RsrII (7730) BsiWI (7670) SpeI (7339) AgeI (7204) SphI (7133) XbaI (7078) AscI (6893) EcoRI (6760) AarI (6198) FseI (1150) MfeI (1189) BbvCI (1424) AleI (1577) BspDI - ClaI (2180) SnaBI (2480) SbfI (3185) TspMI - XmaI (4304) SmaI (4306) BamHI (4943) BstXI (5727) FspAI (5888) pLVX-puro-Cas9 12,305 bp
SgrDI  (11,937)
1 site
C G T C G A C G G C A G C T G C
AhdI  (11,016)
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.
KpnI  (8956)
1 site
G G T A C C C C A T G G
Acc65I  (8952)
1 site
G G T A C C C C A T G G
SexAI  (8181)
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.
BstEII  (7748)
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.
RsrII  (7730)
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.
BsiWI  (7670)
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.
SpeI  (7339)
1 site
A C T A G T T G A T C A
AgeI  (7204)
1 site
A C C G G T T G G C C A
SphI  (7133)
1 site
G C A T G C C G T A C G
XbaI  (7078)
1 site
T C T A G A A G A T C T
AscI  (6893)
1 site
G G C G C G C C C C G C G C G G
EcoRI  (6760)
1 site
G A A T T C C T T A A G
AarI  (6198)
1 site
C A C C T G C ( N ) 4 G T G G A C G ( N ) 4 ( N ) 4

Efficient cleavage requires at least two copies of the AarI recognition sequence.
Sticky ends from different AarI sites may not be compatible.
After cleavage, AarI can remain bound to DNA and alter its electrophoretic mobility.
FseI  (1150)
1 site
G G C C G G C C C C G G C C G G

FseI gradually loses activity when stored at -20°C.
MfeI  (1189)
1 site
C A A T T G G T T A A C
BbvCI  (1424)
1 site
C C T C A G C G G A G T C G
AleI  (1577)
1 site
C A C N N N N G T G G T G N N N N C A C
BspDI  (2180)
1 site
A T C G A T T A G C T A
ClaI  (2180)
1 site
A T C G A T T A G C T A
SnaBI  (2480)
1 site
T A C G T A A T G C A T
SbfI  (3185)
1 site
C C T G C A G G G G A C G T C C
TspMI  (4304)
1 site
C C C G G G G G G C C C
XmaI  (4304)
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  (4306)
1 site
C C C G G G G G G C C C

SmaI can be used at 37°C for brief incubations.
BamHI  (4943)
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  (5727)
1 site
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.
FspAI  (5888)
1 site
R T G C G C A Y Y A C G C G T R
Cas9
2941 .. 7041  =  4101 bp
1367 amino acids  =  158.3 kDa
Product: Cas9 (Csn1) endonuclease from the Streptococcus pyogenes Type II CRISPR/Cas system
generates RNA-guided double strand breaks in DNA
Cas9
2941 .. 7041  =  4101 bp
1367 amino acids  =  158.3 kDa
Product: Cas9 (Csn1) endonuclease from the Streptococcus pyogenes Type II CRISPR/Cas system
generates RNA-guided double strand breaks in DNA
AmpR
10,943 .. 11,803  =  861 bp
286 amino acids  =  31.6 kDa
   Segment 2:  
   10,943 .. 11,734  =  792 bp
   263 amino acids  =  28.9 kDa
Product: β-lactamase
confers resistance to ampicillin, carbenicillin, and related antibiotics
AmpR
10,943 .. 11,803  =  861 bp
286 amino acids  =  31.6 kDa
   Segment 1:  signal sequence  
   11,735 .. 11,803  =  69 bp
   23 amino acids  =  2.6 kDa
Product: β-lactamase
confers resistance to ampicillin, carbenicillin, and related antibiotics
AmpR
10,943 .. 11,803  =  861 bp
286 amino acids  =  31.6 kDa
2 segments
Product: β-lactamase
confers resistance to ampicillin, carbenicillin, and related antibiotics
3' LTR
1 .. 634  =  634 bp
3' long terminal repeat (LTR) from HIV-1
3' LTR
1 .. 634  =  634 bp
3' long terminal repeat (LTR) from HIV-1
3' LTR
9023 .. 9656  =  634 bp
3' long terminal repeat (LTR) from HIV-1
3' LTR
9023 .. 9656  =  634 bp
3' long terminal repeat (LTR) from HIV-1
PuroR
7614 .. 8213  =  600 bp
199 amino acids  =  21.5 kDa
Product: puromycin N-acetyltransferase
confers resistance to puromycin
PuroR
7614 .. 8213  =  600 bp
199 amino acids  =  21.5 kDa
Product: puromycin N-acetyltransferase
confers resistance to puromycin
WPRE
8227 .. 8815  =  589 bp
woodchuck hepatitis virus posttranscriptional regulatory element
WPRE
8227 .. 8815  =  589 bp
woodchuck hepatitis virus posttranscriptional regulatory element
ori
10,187 .. 10,772  =  586 bp
high-copy-number ColE1/pMB1/pBR322/pUC origin of replication
ori
10,187 .. 10,772  =  586 bp
high-copy-number ColE1/pMB1/pBR322/pUC origin of replication
PGK promoter
7094 .. 7593  =  500 bp
mouse phosphoglycerate kinase 1 promoter
PGK promoter
7094 .. 7593  =  500 bp
mouse phosphoglycerate kinase 1 promoter
CMV enhancer
2201 .. 2504  =  304 bp
human cytomegalovirus immediate early enhancer
CMV enhancer
2201 .. 2504  =  304 bp
human cytomegalovirus immediate early enhancer
RRE
1303 .. 1536  =  234 bp
The Rev response element (RRE) of HIV-1 allows for Rev-dependent mRNA export from the nucleus to the cytoplasm.
RRE
1303 .. 1536  =  234 bp
The Rev response element (RRE) of HIV-1 allows for Rev-dependent mRNA export from the nucleus to the cytoplasm.
CMV promoter
2505 .. 2708  =  204 bp
human cytomegalovirus (CMV) immediate early promoter
CMV promoter
2505 .. 2708  =  204 bp
human cytomegalovirus (CMV) immediate early promoter
HIV-1 Ψ
681 .. 806  =  126 bp
packaging signal of human immunodeficiency virus type 1
HIV-1 Ψ
681 .. 806  =  126 bp
packaging signal of human immunodeficiency virus type 1
cPPT/CTS
2028 .. 2143  =  116 bp
central polypurine tract and central termination sequence of HIV-1 (lacking the first T)
cPPT/CTS
2028 .. 2143  =  116 bp
central polypurine tract and central termination sequence of HIV-1 (lacking the first T)
AmpR promoter
11,804 .. 11,908  =  105 bp
AmpR promoter
11,804 .. 11,908  =  105 bp
3xFLAG
2824 .. 2889  =  66 bp
22 amino acids  =  2.7 kDa
Product: three tandem FLAG® epitope tags, followed by an enterokinase cleavage site
3xFLAG
2824 .. 2889  =  66 bp
22 amino acids  =  2.7 kDa
Product: three tandem FLAG® epitope tags, followed by an enterokinase cleavage site
SV40 NLS
2896 .. 2916  =  21 bp
7 amino acids  =  883.1 Da
Product: nuclear localization signal of SV40 large T antigen
SV40 NLS
2896 .. 2916  =  21 bp
7 amino acids  =  883.1 Da
Product: nuclear localization signal of SV40 large T antigen
SV40 NLS
7054 .. 7074  =  21 bp
7 amino acids  =  883.1 Da
Product: nuclear localization signal of SV40 large T antigen
SV40 NLS
7054 .. 7074  =  21 bp
7 amino acids  =  883.1 Da
Product: nuclear localization signal of SV40 large T antigen
ATG
2821 .. 2823  =  3 bp
1 amino acid  =  149.2 Da
Product: start codon
ATG
2821 .. 2823  =  3 bp
1 amino acid  =  149.2 Da
Product: start codon
ORF:  2821 .. 7077  =  4257 bp
ORF:  1418 amino acids  =  164.2 kDa
ORF:  8317 .. 8844  =  528 bp
ORF:  175 amino acids  =  19.0 kDa
ORF:  1181 .. 2032  =  852 bp
ORF:  283 amino acids  =  32.7 kDa
ORF:  8330 .. 8626  =  297 bp
ORF:  98 amino acids  =  10.2 kDa
ORF:  7374 .. 8213  =  840 bp
ORF:  279 amino acids  =  30.1 kDa
ORF:  11,073 .. 11,339  =  267 bp
ORF:  88 amino acids  =  9.2 kDa
ORF:  10,943 .. 11,803  =  861 bp
ORF:  286 amino acids  =  31.6 kDa
ORF:  1015 .. 1476  =  462 bp
ORF:  153 amino acids  =  16.6 kDa
ORF:  2809 .. 3417  =  609 bp
ORF:  202 amino acids  =  22.5 kDa
ORF:  4342 .. 4860  =  519 bp
ORF:  172 amino acids  =  20.1 kDa
ORF:  4924 .. 5457  =  534 bp
ORF:  177 amino acids  =  19.9 kDa
ORF:  6451 .. 6984  =  534 bp
ORF:  177 amino acids  =  20.0 kDa
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