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pRSGC1-U6-sg-HTS6-CMV-Cas9-2A-Puro

Lentiviral vector for coexpressing Cas9 from the CMV promoter together with a single guide RNA (sgRNA), for permanently knocking out a target gene.

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pRSGC1-U6-sg-HTS6-CMV-Cas9-2A-Puro Sequence and MappRSGC1-U6-sg-HTS6-CMV-Cas9-2A-Puro.dna
Map and Sequence File   
Sequence Author:  Cellecta
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 SspI (10,918) PciI (9221) SfiI (8799) AanI (8570) AanI (8514) KpnI (8179) Acc65I (8175) HincII (7580) SalI (7578) SexAI * (7539) BstEII (7106) RsrII (7088) BsiWI (7028) PflFI - Tth111I (7014) BamHI (6906) NotI (6898) FseI (6877) BsaBI * (6243) MluI (1) NruI * (612) MfeI (964) BbvCI (1199) AleI (1352) BspDI - ClaI (1798) BbsI - BpiI (2064) cloning site BbsI - BpiI (2090) PaeR7I - PspXI - XhoI (2113) EcoRI (2280) AgeI (2289) BstBI (2298) SnaBI (2361) SV40 NLS SV40 NLS SV40 NLS PspOMI (4100) ApaI (4104) EcoRV (4598) PmlI (5534) pRSGC1-U6-sg-HTS6-CMV-Cas9-2A-Puro 11,505 bp
SspI  (10,918)
1 site
A A T A T T T T A T A A
PciI  (9221)
1 site
A C A T G T T G T A C A

PciI is inhibited by nonionic detergents.
SfiI  (8799)
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.
AanI  (8570)
2 sites
T T A T A A A A T A T T
AanI  (8514)
2 sites
T T A T A A A A T A T T
KpnI  (8179)
1 site
G G T A C C C C A T G G
Acc65I  (8175)
1 site
G G T A C C C C A T G G
HincII  (7580)
1 site
G T Y R A C C A R Y T G
SalI  (7578)
1 site
G T C G A C C A G C T G
SexAI  (7539)
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  (7106)
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  (7088)
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  (7028)
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.
PflFI  (7014)
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  (7014)
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.
BamHI  (6906)
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.
NotI  (6898)
1 site
G C G G C C G C C G C C G G C G
FseI  (6877)
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.
BsaBI  (6243)
1 site
G A T N N N N A T C C T A N N N N T A G
* Blocked by Dam methylation.
MluI  (1)
1 site
A C G C G T T G C G C A
NruI  (612)
1 site
T C G C G A A G C G C T
* Blocked by Dam methylation.
MfeI  (964)
1 site
C A A T T G G T T A A C
BbvCI  (1199)
1 site
C C T C A G C G G A G T C G
AleI  (1352)
1 site
C A C N N N N G T G G T G N N N N C A C
BspDI  (1798)
1 site
A T C G A T T A G C T A
ClaI  (1798)
1 site
A T C G A T T A G C T A
BbsI  (2064)
2 sites
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.
BpiI  (2064)
2 sites
G A A G A C N N C T T C T G N N ( N ) 4

Sticky ends from different BpiI sites may not be compatible.
BbsI  (2090)
2 sites
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.
BpiI  (2090)
2 sites
G A A G A C N N C T T C T G N N ( N ) 4

Sticky ends from different BpiI sites may not be compatible.
PaeR7I  (2113)
1 site
C T C G A G G A G C T C

PaeR7I does not recognize the sequence CTCTCGAG.
PspXI  (2113)
1 site
V C T C G A G B B G A G C T C V
XhoI  (2113)
1 site
C T C G A G G A G C T C
EcoRI  (2280)
1 site
G A A T T C C T T A A G
AgeI  (2289)
1 site
A C C G G T T G G C C A

AgeI quickly loses activity at 37°C, but can be used at 25°C for
long incubations.
BstBI  (2298)
1 site
T T C G A A A A G C T T
SnaBI  (2361)
1 site
T A C G T A A T G C A T
PspOMI  (4100)
1 site
G G G C C C C C C G G G
ApaI  (4104)
1 site
G G G C C C C C C G G G

ApaI can be used between 25°C and 37°C.
EcoRV  (4598)
1 site
G A T A T C C T A T A G

EcoRV is reportedly more prone than its isoschizomer Eco32I to
delete a base after cleavage.
PmlI  (5534)
1 site
C A C G T G G T G C A C

PmlI gradually loses activity when stored at -20°C.
Cas9
2748 .. 6848  =  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
2748 .. 6848  =  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
10,041 .. 10,901  =  861 bp
286 amino acids  =  31.6 kDa
   Segment 2:  
   10,041 .. 10,832  =  792 bp
   263 amino acids  =  28.9 kDa
Product: β-lactamase
confers resistance to ampicillin, carbenicillin, and
related antibiotics
AmpR
10,041 .. 10,901  =  861 bp
286 amino acids  =  31.6 kDa
   Segment 1:  signal sequence  
   10,833 .. 10,901  =  69 bp
   23 amino acids  =  2.6 kDa
Product: β-lactamase
confers resistance to ampicillin, carbenicillin, and
related antibiotics
AmpR
10,041 .. 10,901  =  861 bp
286 amino acids  =  31.6 kDa
2 segments
Product: β-lactamase
confers resistance to ampicillin, carbenicillin, and
related antibiotics
PuroR
6972 .. 7571  =  600 bp
199 amino acids  =  21.5 kDa
Product: puromycin N-acetyltransferase
confers resistance to puromycin
PuroR
6972 .. 7571  =  600 bp
199 amino acids  =  21.5 kDa
Product: puromycin N-acetyltransferase
confers resistance to puromycin
WPRE
7584 .. 8172  =  589 bp
woodchuck hepatitis virus posttranscriptional
regulatory element
WPRE
7584 .. 8172  =  589 bp
woodchuck hepatitis virus posttranscriptional
regulatory element
ori
9282 .. 9870  =  589 bp
high-copy-number ColE1/pMB1/pBR322/pUC origin
of replication
ori
9282 .. 9870  =  589 bp
high-copy-number ColE1/pMB1/pBR322/pUC origin
of replication
U6 promoter
1819 .. 2059  =  241 bp
RNA polymerase III promoter for human U6 snRNA
U6 promoter
1819 .. 2059  =  241 bp
RNA polymerase III promoter for human U6 snRNA
RRE
1078 .. 1311  =  234 bp
The Rev response element (RRE) of HIV-1 allows for
Rev-dependent mRNA export from the nucleus to
the cytoplasm.
RRE
1078 .. 1311  =  234 bp
The Rev response element (RRE) of HIV-1 allows for
Rev-dependent mRNA export from the nucleus to
the cytoplasm.
3' LTR (ΔU3)
8246 .. 8479  =  234 bp
self-inactivating 3' long terminal repeat (LTR) from
HIV-1
3' LTR (ΔU3)
8246 .. 8479  =  234 bp
self-inactivating 3' long terminal repeat (LTR) from
HIV-1
RSV promoter
6 .. 232  =  227 bp
Rous sarcoma virus enhancer/promoter
RSV promoter
6 .. 232  =  227 bp
Rous sarcoma virus enhancer/promoter
CMV promoter
2386 .. 2589  =  204 bp
human cytomegalovirus (CMV) immediate early
promoter
CMV promoter
2386 .. 2589  =  204 bp
human cytomegalovirus (CMV) immediate early
promoter
5' LTR (truncated)
233 .. 413  =  181 bp
truncated 5' long terminal repeat (LTR) from HIV-1
5' LTR (truncated)
233 .. 413  =  181 bp
truncated 5' long terminal repeat (LTR) from HIV-1
SV40 ori
8712 .. 8847  =  136 bp
SV40 origin of replication
SV40 ori
8712 .. 8847  =  136 bp
SV40 origin of replication
HIV-1 Ψ
460 .. 585  =  126 bp
packaging signal of human immunodeficiency virus
type 1
HIV-1 Ψ
460 .. 585  =  126 bp
packaging signal of human immunodeficiency virus
type 1
SV40 poly(A) signal
8551 .. 8672  =  122 bp
SV40 polyadenylation signal
SV40 poly(A) signal
8551 .. 8672  =  122 bp
SV40 polyadenylation signal
cPPT/CTS
2122 .. 2237  =  116 bp
central polypurine tract and central termination
sequence of HIV-1 (lacking the first T)
cPPT/CTS
2122 .. 2237  =  116 bp
central polypurine tract and central termination
sequence of HIV-1 (lacking the first T)
AmpR promoter
10,902 .. 11,006  =  105 bp
AmpR promoter
10,902 .. 11,006  =  105 bp
T2A
6912 .. 6965  =  54 bp
18 amino acids  =  1.8 kDa
Product: 2A peptide from Thosea asigna virus capsid
protein
Eukaryotic ribosomes fail to insert a peptide bond
between the Gly and Pro residues, yielding separate
polypeptides.
T2A
6912 .. 6965  =  54 bp
18 amino acids  =  1.8 kDa
Product: 2A peptide from Thosea asigna virus capsid
protein
Eukaryotic ribosomes fail to insert a peptide bond
between the Gly and Pro residues, yielding separate
polypeptides.
nucleoplasmin NLS
6849 .. 6896  =  48 bp
16 amino acids  =  1.7 kDa
Product: bipartite nuclear localization signal from
nucleoplasmin
nucleoplasmin NLS
6849 .. 6896  =  48 bp
16 amino acids  =  1.7 kDa
Product: bipartite nuclear localization signal from
nucleoplasmin
cloning site
2065 .. 2094  =  30 bp
sgRNA cloning site
cloning site
2065 .. 2094  =  30 bp
sgRNA cloning site
SV40 NLS
2673 .. 2693  =  21 bp
7 amino acids  =  883.1 Da
Product: nuclear localization signal of SV40 large T
antigen
SV40 NLS
2673 .. 2693  =  21 bp
7 amino acids  =  883.1 Da
Product: nuclear localization signal of SV40 large T
antigen
SV40 NLS
2697 .. 2717  =  21 bp
7 amino acids  =  883.1 Da
Product: nuclear localization signal of SV40 large T
antigen
SV40 NLS
2697 .. 2717  =  21 bp
7 amino acids  =  883.1 Da
Product: nuclear localization signal of SV40 large T
antigen
SV40 NLS
2721 .. 2741  =  21 bp
7 amino acids  =  883.1 Da
Product: nuclear localization signal of SV40 large T
antigen
SV40 NLS
2721 .. 2741  =  21 bp
7 amino acids  =  883.1 Da
Product: nuclear localization signal of SV40 large T
antigen
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