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Plasmid Files

pLenti-EF1a-Cas9-IRES-Puro

3rd-generation lentiviral vector for expressing Cas9 and a puromycin resistance marker under control of the EF-1α promoter.

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pLenti-EF1a-Cas9-IRES-Puro.dna
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Sequence Author:  OriGene
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SgrDI (1) ScaI (11,492) Bsu36I (10,162) BsrGI (9322) RsrII (8838) AarI (8486) PmlI (8463) PmeI (8117) PspXI (8039) MluI (8024) XcmI (7408) SpeI (251) NdeI (486) SnaBI (592) XbaI (2614) AgeI (2703) EcoRI (3819) BamHI (3832) AsiSI (3864) BstZ17I (4083) BlpI (4548) SphI (5129) PshAI (5444) pLenti-EF1a-Cas9-IRES-Puro 12,256 bp
SgrDI  (1)
1 site
C G T C G A C G G C A G C T G C
ScaI  (11,492)
1 site
A G T A C T T C A T G A
Bsu36I  (10,162)
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  (9322)
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.
RsrII  (8838)
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.
AarI  (8486)
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.
PmlI  (8463)
1 site
C A C G T G G T G C A C

PmlI gradually loses activity when stored at -20°C.
PmeI  (8117)
1 site
G T T T A A A C C A A A T T T G
PspXI  (8039)
1 site
V C T C G A G B B G A G C T C V
MluI  (8024)
1 site
A C G C G T T G C G C A
XcmI  (7408)
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  (251)
1 site
A C T A G T T G A T C A
NdeI  (486)
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.
SnaBI  (592)
1 site
T A C G T A A T G C A T
XbaI  (2614)
1 site
T C T A G A A G A T C T
AgeI  (2703)
1 site
A C C G G T T G G C C A
EcoRI  (3819)
1 site
G A A T T C C T T A A G
BamHI  (3832)
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  (3864)
1 site
G C G A T C G C C G C T A G C G
BstZ17I  (4083)
1 site
G T A T A C C A T A T G
BlpI  (4548)
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  (5129)
1 site
G C A T G C C G T A C G
PshAI  (5444)
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
3869 .. 7972  =  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
3869 .. 7972  =  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
EF-1α promoter
2625 .. 3803  =  1179 bp
strong constitutive promoter for human elongation factor EF-1α
EF-1α promoter
2625 .. 3803  =  1179 bp
strong constitutive promoter for human elongation factor EF-1α
CmR
11,464 .. 12,123  =  660 bp
219 amino acids  =  25.7 kDa
Product: chloramphenicol acetyltransferase
confers resistance to chloramphenicol
CmR
11,464 .. 12,123  =  660 bp
219 amino acids  =  25.7 kDa
Product: chloramphenicol acetyltransferase
confers resistance to chloramphenicol
PuroR
8722 .. 9321  =  600 bp
199 amino acids  =  21.5 kDa
Product: puromycin N-acetyltransferase
confers resistance to puromycin
PuroR
8722 .. 9321  =  600 bp
199 amino acids  =  21.5 kDa
Product: puromycin N-acetyltransferase
confers resistance to puromycin
WPRE
9440 .. 10,028  =  589 bp
woodchuck hepatitis virus posttranscriptional regulatory element
WPRE
9440 .. 10,028  =  589 bp
woodchuck hepatitis virus posttranscriptional regulatory element
ori
10,799 .. 11,387  =  589 bp
high-copy-number ColE1/pMB1/pBR322/pUC origin of replication
ori
10,799 .. 11,387  =  589 bp
high-copy-number ColE1/pMB1/pBR322/pUC origin of replication
IRES
8147 .. 8720  =  574 bp
internal ribosome entry site (IRES) of the encephalomyocarditis virus (EMCV)
IRES
8147 .. 8720  =  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
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
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)
10,557 .. 10,737  =  181 bp
truncated 5' long terminal repeat (LTR) from HIV-1
5' LTR (truncated)
10,557 .. 10,737  =  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
12,124 .. 12,228  =  105 bp
AmpR promoter
12,124 .. 12,228  =  105 bp
Myc
8042 .. 8071  =  30 bp
10 amino acids  =  1.2 kDa
Product: Myc (human c-Myc oncogene) epitope tag
Myc
8042 .. 8071  =  30 bp
10 amino acids  =  1.2 kDa
Product: Myc (human c-Myc oncogene) epitope tag
FLAG
8090 .. 8113  =  24 bp
8 amino acids  =  1.0 kDa
Product: FLAG® epitope tag, followed by an enterokinase cleavage site
FLAG
8090 .. 8113  =  24 bp
8 amino acids  =  1.0 kDa
Product: FLAG® epitope tag, followed by an enterokinase cleavage site
SV40 NLS
7973 .. 7993  =  21 bp
7 amino acids  =  883.1 Da
Product: nuclear localization signal of SV40 large T antigen
SV40 NLS
7973 .. 7993  =  21 bp
7 amino acids  =  883.1 Da
Product: nuclear localization signal of SV40 large T antigen
SV40 NLS
8000 .. 8020  =  21 bp
7 amino acids  =  883.1 Da
Product: nuclear localization signal of SV40 large T antigen
SV40 NLS
8000 .. 8020  =  21 bp
7 amino acids  =  883.1 Da
Product: nuclear localization signal of SV40 large T antigen
EF-1α intron A
2856 .. 3794  =  939 bp
intron upstream of the start codon of human EF-1α
EF-1α intron A
2856 .. 3794  =  939 bp
intron upstream of the start codon of human EF-1α
ORF:  1561 .. 2448  =  888 bp
ORF:  295 amino acids  =  33.8 kDa
ORF:  3181 .. 3618  =  438 bp
ORF:  145 amino acids  =  15.9 kDa
ORF:  8722 .. 9321  =  600 bp
ORF:  199 amino acids  =  21.5 kDa
ORF:  2423 .. 2746  =  324 bp
ORF:  107 amino acids  =  12.1 kDa
ORF:  3260 .. 3493  =  234 bp
ORF:  77 amino acids  =  7.7 kDa
ORF:  3869 .. 8119  =  4251 bp
ORF:  1416 amino acids  =  164.1 kDa
ORF:  9530 .. 10,084  =  555 bp
ORF:  184 amino acids  =  20.5 kDa
ORF:  9543 .. 10,583  =  1041 bp
ORF:  346 amino acids  =  38.3 kDa
ORF:  4127 .. 4369  =  243 bp
ORF:  80 amino acids  =  9.6 kDa
ORF:  5921 .. 6265  =  345 bp
ORF:  114 amino acids  =  13.5 kDa
ORF:  6419 .. 6823  =  405 bp
ORF:  134 amino acids  =  15.6 kDa
ORF:  7292 .. 7915  =  624 bp
ORF:  207 amino acids  =  24.3 kDa
ORF:  2923 .. 3543  =  621 bp
ORF:  206 amino acids  =  23.0 kDa
ORF:  11,464 .. 12,123  =  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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