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pLenti-Cas9-P2A-tGFP

3rd-generation lentiviral vector for expressing Cas9 and TurboGFP under control of the CMV promoter.

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pLenti-Cas9-P2A-tGFP.dna
Map and Sequence File:    Download    Open   
Sequence Author:  OriGene
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PasI (10,717) ScaI (10,535) Bsu36I (9205) BsrGI (8365) AarI (8097) BmgBI (7624) PaeR7I - PspXI - XhoI (7499) NotI (7493) BsiWI (7487) MluI (7484) BstEII (7427) XcmI (6868) PflFI - Tth111I (5902) SgrDI (1) BssHII (1091) EcoNI (1550) BbvCI (1804) XbaI (2613) EcoRI (3279) BamHI (3292) AsiSI (3324) BstZ17I (3543) SphI (4589) PshAI (4904) SexAI * (5900) pLenti-Cas9-P2A-tGFP 11,299 bp
PasI  (10,717)
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  (10,535)
1 site
A G T A C T T C A T G A
Bsu36I  (9205)
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  (8365)
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.
AarI  (8097)
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.
BmgBI  (7624)
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.
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
NotI  (7493)
1 site
G C G G C C G C C G C C G G C G
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
BstEII  (7427)
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.
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.
PflFI  (5902)
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  (5902)
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.
SgrDI  (1)
1 site
C G T C G A C G G C A G C T G C
BssHII  (1091)
1 site
G C G C G C C G C G C G

BssHII is typically used at 50°C, but is 75% active at 37°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
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
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.
SexAI  (5900)
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.
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
TurboGFP
7646 .. 8341  =  696 bp
232 amino acids  =  25.7 kDa
Product: improved green fluorescent protein from Pontellina plumata (Evdokimov et al., 2006)
mammalian codon-optimized
TurboGFP
7646 .. 8341  =  696 bp
232 amino acids  =  25.7 kDa
Product: improved green fluorescent protein from Pontellina plumata (Evdokimov et al., 2006)
mammalian codon-optimized
CmR
10,507 .. 11,166  =  660 bp
219 amino acids  =  25.7 kDa
Product: chloramphenicol acetyltransferase
confers resistance to chloramphenicol
CmR
10,507 .. 11,166  =  660 bp
219 amino acids  =  25.7 kDa
Product: chloramphenicol acetyltransferase
confers resistance to chloramphenicol
WPRE
8483 .. 9071  =  589 bp
woodchuck hepatitis virus posttranscriptional regulatory element
WPRE
8483 .. 9071  =  589 bp
woodchuck hepatitis virus posttranscriptional regulatory element
ori
9842 .. 10,430  =  589 bp
high-copy-number ColE1/pMB1/pBR322/pUC origin of replication
ori
9842 .. 10,430  =  589 bp
high-copy-number ColE1/pMB1/pBR322/pUC origin of replication
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)
9600 .. 9780  =  181 bp
truncated 5' long terminal repeat (LTR) from HIV-1
5' LTR (truncated)
9600 .. 9780  =  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
11,167 .. 11,271  =  105 bp
AmpR promoter
11,167 .. 11,271  =  105 bp
P2A
7589 .. 7645  =  57 bp
19 amino acids  =  2.0 kDa
Product: 2A peptide from porcine teschovirus-1 polyprotein
Eukaryotic ribosomes fail to insert a peptide bond between the Gly and Pro residues, yielding separate polypeptides.
P2A
7589 .. 7645  =  57 bp
19 amino acids  =  2.0 kDa
Product: 2A peptide from porcine teschovirus-1 polyprotein
Eukaryotic ribosomes fail to insert a peptide bond between the Gly and Pro residues, yielding separate polypeptides.
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 ..&#