pLenti-Cas9-IRES-Puro

3rd-generation lentiviral vector for expressing Cas9 and a puromycin resistance marker under control of the CMV promoter.

Sequence Author: OriGene

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AmpR promoter PasI (11,134) ScaI (10,952) Bsu36I (9622) BsrGI (8782) StuI (8513) SmaI (8233) TspMI - XmaI (8231) BmgBI (8150) PaqCI (7946) PmlI (7923) PmeI (7577) FLAG PaeR7I - PspXI - XhoI (7499) BsiWI (7487) MluI (7484) SV40 NLS SV40 NLS XcmI (6868) SgrDI (1) FseI (1530) EcoNI (1550) BbvCI (1804) PstI (2417) cPPT/CTS XbaI (2613) EcoRI (3279) BamHI (3292) AsiSI (3324) BstZ17I (3543) BlpI (4008) SphI (4589) PshAI (4904) pLenti-Cas9-IRES-Puro 11,716 bp
PasI  (11,134)
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.
ScaI  (10,952)
1 site
A G T A C T T C A T G A
Bsu36I  (9622)
1 site
C C T N A G G G G A N T C C

Sticky ends from different Bsu36I sites may not be compatible.
BsrGI  (8782)
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.
StuI  (8513)
1 site
A G G C C T T C C G G A
SmaI  (8233)
1 site
C C C G G G G G G C C C

SmaI can be used at 37°C for brief incubations.
TspMI  (8231)
1 site
C C C G G G G G G C C C
XmaI  (8231)
1 site
C C C G G G G G G C C C

Cleavage may be enhanced when more than one copy of the XmaI recognition sequence is present.
BmgBI  (8150)
1 site
C A C G T C G T G C A G

This recognition sequence is asymmetric, so ligating blunt ends generated by BmgBI will not always regenerate a BmgBI site.
PaqCI  (7946)
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 PaqCI recognition sequence.
Sticky ends from different PaqCI sites may not be compatible.
Cleavage can be improved with PaqCI Activator.
PmlI  (7923)
1 site
C A C G T G G T G C A C
PmeI  (7577)
1 site
G T T T A A A C C A A A T T T G
PaeR7I  (7499)
1 site
C T C G A G G A G C T C

PaeR7I does not recognize the sequence CTCTCGAG.
PspXI  (7499)
1 site
V C T C G A G B B G A G C T C V
XhoI  (7499)
1 site
C T C G A G G A G C T C
BsiWI  (7487)
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.
MluI  (7484)
1 site
A C G C G T T G C G C A
XcmI  (6868)
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.
SgrDI  (1)
1 site
C G T C G A C G G C A G C T G C
FseI  (1530)
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.
EcoNI  (1550)
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.
BbvCI  (1804)
1 site
C C T C A G C G G A G T C G
PstI  (2417)
1 site
C T G C A G G A C G T C
XbaI  (2613)
1 site
T C T A G A A G A T C T
EcoRI  (3279)
1 site
G A A T T C C T T A A G
BamHI  (3292)
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.
AsiSI  (3324)
1 site
G C G A T C G C C G C T A G C G
BstZ17I  (3543)
1 site
G T A T A C C A T A T G
BlpI  (4008)
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.
SphI  (4589)
1 site
G C A T G C C G T A C G
PshAI  (4904)
1 site
G A C N N N N G T C C T G N N N N C A G

PshAI quickly loses activity at 37°C, but can be used at 25°C for long incubations.
Cas9
3329 .. 7432  =  4104 bp
1368 amino acids  =  158.4 kDa
Product: Cas9 (Csn1) endonuclease from the Streptococcus pyogenes Type II CRISPR/Cas system
generates RNA-guided double strand breaks in DNA
Cas9
3329 .. 7432  =  4104 bp
1368 amino acids  =  158.4 kDa
Product: Cas9 (Csn1) endonuclease from the Streptococcus pyogenes Type II CRISPR/Cas system
generates RNA-guided double strand breaks in DNA
SV40 NLS
7433 .. 7453  =  21 bp
7 amino acids  =  883.1 Da
Product: nuclear localization signal of SV40 large T antigen
SV40 NLS
7433 .. 7453  =  21 bp
7 amino acids  =  883.1 Da
Product: nuclear localization signal of SV40 large T antigen
SV40 NLS
7460 .. 7480  =  21 bp
7 amino acids  =  883.1 Da
Product: nuclear localization signal of SV40 large T antigen
SV40 NLS
7460 .. 7480  =  21 bp
7 amino acids  =  883.1 Da
Product: nuclear localization signal of SV40 large T antigen
Myc
7502 .. 7531  =  30 bp
10 amino acids  =  1.2 kDa
Product: Myc (human c-Myc oncogene) epitope tag
Myc
7502 .. 7531  =  30 bp
10 amino acids  =  1.2 kDa
Product: Myc (human c-Myc oncogene) epitope tag
FLAG
7550 .. 7573  =  24 bp
8 amino acids  =  1.0 kDa
Product: FLAG® epitope tag, followed by an enterokinase cleavage site
FLAG
7550 .. 7573  =  24 bp
8 amino acids  =  1.0 kDa
Product: FLAG® epitope tag, followed by an enterokinase cleavage site
CmR
10,924 .. 11,583  =  660 bp
219 amino acids  =  25.7 kDa
Product: chloramphenicol acetyltransferase
confers resistance to chloramphenicol
CmR
10,924 .. 11,583  =  660 bp
219 amino acids  =  25.7 kDa
Product: chloramphenicol acetyltransferase
confers resistance to chloramphenicol
PuroR
8182 .. 8781  =  600 bp
199 amino acids  =  21.5 kDa
Product: puromycin N-acetyltransferase
confers resistance to puromycin
PuroR
8182 .. 8781  =  600 bp
199 amino acids  =  21.5 kDa
Product: puromycin N-acetyltransferase
confers resistance to puromycin
WPRE
8900 .. 9488  =  589 bp
woodchuck hepatitis virus posttranscriptional regulatory element
WPRE
8900 .. 9488  =  589 bp
woodchuck hepatitis virus posttranscriptional regulatory element
ori
10,259 .. 10,847  =  589 bp
high-copy-number ColE1/pMB1/pBR322/pUC origin of replication
ori
10,259 .. 10,847  =  589 bp
high-copy-number ColE1/pMB1/pBR322/pUC origin of replication
IRES
7607 .. 8180  =  574 bp
internal ribosome entry site (IRES) of the encephalomyocarditis virus (EMCV)
IRES
7607 .. 8180  =  574 bp
internal ribosome entry site (IRES) of the encephalomyocarditis virus (EMCV)
CMV enhancer
237 .. 616  =  380 bp
human cytomegalovirus immediate early enhancer
CMV enhancer
237 .. 616  =  380 bp
human cytomegalovirus immediate early enhancer
CMV enhancer
2643 .. 3022  =  380 bp
human cytomegalovirus immediate early enhancer
CMV enhancer
2643 .. 3022  =  380 bp
human cytomegalovirus immediate early enhancer
RRE
1683 .. 1916  =  234 bp
The Rev response element (RRE) of HIV-1 allows for Rev-dependent mRNA export from the nucleus to the cytoplasm.
RRE
1683 .. 1916  =  234 bp
The Rev response element (RRE) of HIV-1 allows for Rev-dependent mRNA export from the nucleus to the cytoplasm.
CMV promoter
3023 .. 3226  =  204 bp
human cytomegalovirus (CMV) immediate early promoter
CMV promoter
3023 .. 3226  =  204 bp
human cytomegalovirus (CMV) immediate early promoter
CMV promoter
618 .. 816  =  199 bp
human cytomegalovirus (CMV) immediate early promoter
CMV promoter
618 .. 816  =  199 bp
human cytomegalovirus (CMV) immediate early promoter
5' LTR (truncated)
834 .. 1014  =  181 bp
truncated 5' long terminal repeat (LTR) from HIV-1
5' LTR (truncated)
834 .. 1014  =  181 bp
truncated 5' long terminal repeat (LTR) from HIV-1
5' LTR (truncated)
10,017 .. 10,197  =  181 bp
truncated 5' long terminal repeat (LTR) from HIV-1
5' LTR (truncated)
10,017 .. 10,197  =  181 bp
truncated 5' long terminal repeat (LTR) from HIV-1
HIV-1 Ψ
1061 .. 1186  =  126 bp
packaging signal of human immunodeficiency virus type 1
HIV-1 Ψ
1061 .. 1186  =  126 bp
packaging signal of human immunodeficiency virus type 1
cPPT/CTS
2443 .. 2560  =  118 bp
central polypurine tract and central termination sequence of HIV-1
cPPT/CTS
2443 .. 2560  =  118 bp
central polypurine tract and central termination sequence of HIV-1
AmpR promoter
11,584 .. 11,688  =  105 bp
AmpR promoter
11,584 .. 11,688  =  105 bp
T7 promoter
3252 .. 3270  =  19 bp
promoter for bacteriophage T7 RNA polymerase
T7 promoter
3252 .. 3270  =  19 bp
promoter for bacteriophage T7 RNA polymerase
ORF:  1561 .. 2448  =  888 bp
ORF:  295 amino acids  =  33.8 kDa
ORF:  8182 .. 8781  =  600 bp
ORF:  199 amino acids  =  21.5 kDa
ORF:  3182 .. 7579  =  4398 bp
ORF:  1465 amino acids  =  169.8 kDa
ORF:  8990 .. 9544  =  555 bp
ORF:  184 amino acids  =  20.5 kDa
ORF:  9003 .. 10,043  =  1041 bp
ORF:  346 amino acids  =  38.3 kDa
ORF:  3587 .. 3829  =  243 bp
ORF:  80 amino acids  =  9.6 kDa
ORF:  5381 .. 5725  =  345 bp
ORF:  114 amino acids  =  13.5 kDa
ORF:  5879 .. 6283  =  405 bp
ORF:  134 amino acids  =  15.6 kDa
ORF:  6752 .. 7375  =  624 bp
ORF:  207 amino acids  =  24.3 kDa
ORF:  10,924 .. 11,583  =  660 bp
ORF:  219 amino acids  =  25.7 kDa
ORF:  1395 .. 1856  =  462 bp
ORF:  153 amino acids  =  16.6 kDa
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