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pHR-SFFV-KRAB-dCas9-P2A-mCherry

Lentiviral CRISPR interference vector for expressing mCherry together with a KRAB domain fused to catalytically inactive dCas9.

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pHR-SFFV-KRAB-dCas9-P2A-mCherry Sequence and MappHR-SFFV-KRAB-dCas9-P2A-mCherry.dna
Map and Sequence File   
Sequence Author:  Weissman Lab / Addgene #60954
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 NotI (13,599) SV40 NLS SV40 NLS PasI (10,360) ATG MluI (8301) PflFI - Tth111I (8261) AscI (8239) cPPT/CTS AbsI - PspXI (14) AfeI (732) PvuI (1695) FspI (1841) SfiI (3409) AvrII (3456) AgeI (3946) PshAI (4286) HpaI (5244) NruI * (6380) FseI (6697) pHR-SFFV-KRAB-dCas9-P2A-mCherry 14,247 bp
NotI  (13,599)
1 site
G C G G C C G C C G C C G G C G
PasI  (10,360)
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.
MluI  (8301)
1 site
A C G C G T T G C G C A
PflFI  (8261)
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  (8261)
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.
AscI  (8239)
1 site
G G C G C G C C C C G C G C G G
AbsI  (14)
1 site
C C T C G A G G G G A G C T C C
PspXI  (14)
1 site
V C T C G A G B B G A G C T C V
AfeI  (732)
1 site
A G C G C T T C G C G A
PvuI  (1695)
1 site
C G A T C G G C T A G C
FspI  (1841)
1 site
T G C G C A A C G C G T
SfiI  (3409)
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.
AvrII  (3456)
1 site
C C T A G G G G A T C C
AgeI  (3946)
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.
PshAI  (4286)
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.
HpaI  (5244)
1 site
G T T A A C C A A T T G
NruI  (6380)
1 site
T C G C G A A G C G C T
* Blocked by Dam methylation.
FseI  (6697)
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.
dCas9
8591 .. 12,694  =  4104 bp
1368 amino acids  =  158.3 kDa
   Segment 1:  
   8591 .. 8617  =  27 bp
   9 amino acids  =  1.1 kDa
Product: catalytically dead mutant of the Cas9
endonuclease from the Streptococcus pyogenes
Type II CRISPR/Cas system
RNA-guided DNA-binding protein that lacks
endonuclease activity due to the D10A mutation in
the RuvC catalytic domain and the H840A mutation
in the HNH catalytic domain
dCas9
8591 .. 12,694  =  4104 bp
1368 amino acids  =  158.3 kDa
   Segment 2:  
   8618 .. 8620  =  3 bp
   1 amino acid  =  89.1 Da
Product: catalytically dead mutant of the Cas9
endonuclease from the Streptococcus pyogenes
Type II CRISPR/Cas system
RNA-guided DNA-binding protein that lacks
endonuclease activity due to the D10A mutation in
the RuvC catalytic domain and the H840A mutation
in the HNH catalytic domain
dCas9
8591 .. 12,694  =  4104 bp
1368 amino acids  =  158.3 kDa
   Segment 3:  
   8621 .. 11,107  =  2487 bp
   829 amino acids  =  96.3 kDa
Product: catalytically dead mutant of the Cas9
endonuclease from the Streptococcus pyogenes
Type II CRISPR/Cas system
RNA-guided DNA-binding protein that lacks
endonuclease activity due to the D10A mutation in
the RuvC catalytic domain and the H840A mutation
in the HNH catalytic domain
dCas9
8591 .. 12,694  =  4104 bp
1368 amino acids  =  158.3 kDa
   Segment 4:  
   11,108 .. 11,110  =  3 bp
   1 amino acid  =  89.1 Da
Product: catalytically dead mutant of the Cas9
endonuclease from the Streptococcus pyogenes
Type II CRISPR/Cas system
RNA-guided DNA-binding protein that lacks
endonuclease activity due to the D10A mutation in
the RuvC catalytic domain and the H840A mutation
in the HNH catalytic domain
dCas9
8591 .. 12,694  =  4104 bp
1368 amino acids  =  158.3 kDa
   Segment 5:  
   11,111 .. 12,694  =  1584 bp
   528 amino acids  =  60.9 kDa
Product: catalytically dead mutant of the Cas9
endonuclease from the Streptococcus pyogenes
Type II CRISPR/Cas system
RNA-guided DNA-binding protein that lacks
endonuclease activity due to the D10A mutation in
the RuvC catalytic domain and the H840A mutation
in the HNH catalytic domain
dCas9
8591 .. 12,694  =  4104 bp
1368 amino acids  =  158.3 kDa
5 segments
Product: catalytically dead mutant of the Cas9
endonuclease from the Streptococcus pyogenes
Type II CRISPR/Cas system
RNA-guided DNA-binding protein that lacks
endonuclease activity due to the D10A mutation in
the RuvC catalytic domain and the H840A mutation
in the HNH catalytic domain
AmpR
1277 .. 2137  =  861 bp
286 amino acids  =  31.6 kDa
   Segment 1:  signal sequence  
   1277 .. 1345  =  69 bp
   23 amino acids  =  2.6 kDa
Product: β-lactamase
confers resistance to ampicillin, carbenicillin, and
related antibiotics
AmpR
1277 .. 2137  =  861 bp
286 amino acids  =  31.6 kDa
   Segment 2:  
   1346 .. 2137  =  792 bp
   263 amino acids  =  28.9 kDa
Product: β-lactamase
confers resistance to ampicillin, carbenicillin, and
related antibiotics
AmpR
1277 .. 2137  =  861 bp
286 amino acids  =  31.6 kDa
2 segments
Product: β-lactamase
confers resistance to ampicillin, carbenicillin, and
related antibiotics
mCherry
12,887 .. 13,597  =  711 bp
236 amino acids  =  26.7 kDa
Product: monomeric derivative of DsRed fluorescent
protein (Shaner et al., 2004)
mammalian codon-optimized
mCherry
12,887 .. 13,597  =  711 bp
236 amino acids  =  26.7 kDa
Product: monomeric derivative of DsRed fluorescent
protein (Shaner et al., 2004)
mammalian codon-optimized
LTR
5548 .. 6181  =  634 bp
long terminal repeat (LTR) from HIV-1
LTR
5548 .. 6181  =  634 bp
long terminal repeat (LTR) from HIV-1
ori
2308 .. 2896  =  589 bp
high-copy-number ColE1/pMB1/pBR322/pUC origin
of replication
ori
2308 .. 2896  =  589 bp
high-copy-number ColE1/pMB1/pBR322/pUC origin
of replication
WPRE
13,657 .. 14,245  =  589 bp
woodchuck hepatitis virus posttranscriptional
regulatory element
WPRE
13,657 .. 14,245  =  589 bp
woodchuck hepatitis virus posttranscriptional
regulatory element
SFFV promoter
7866 .. 8273  =  408 bp
spleen focus-forming virus long terminal repeat
(LTR) promoter
SFFV promoter
7866 .. 8273  =  408 bp
spleen focus-forming virus long terminal repeat
(LTR) promoter
SV40 promoter
3142 .. 3471  =  330 bp
SV40 enhancer and early promoter
SV40 promoter
3142 .. 3471  =  330 bp
SV40 enhancer and early promoter
LTR (ΔU3)
108 .. 341  =  234 bp
self-inactivating long terminal repeat (LTR) from
HIV-1
LTR (ΔU3)
108 .. 341  =  234 bp
self-inactivating long terminal repeat (LTR) from
HIV-1
RRE
6850 .. 7082  =  233 bp
The Rev response element (RRE) of HIV-1 allows for
Rev-dependent mRNA export from the nucleus to
the cytoplasm.
RRE
6850 .. 7082  =  233 bp
The Rev response element (RRE) of HIV-1 allows for
Rev-dependent mRNA export from the nucleus to
the cytoplasm.
KRAB
8384 .. 8569  =  186 bp
62 amino acids  =  7.4 kDa
Product: Krüppel-associated box (KRAB)
transcriptional repression domain from the human
zinc finger protein ZNF10 (Margolin et al., 1994)
KRAB
8384 .. 8569  =  186 bp
62 amino acids  =  7.4 kDa
Product: Krüppel-associated box (KRAB)
transcriptional repression domain from the human
zinc finger protein ZNF10 (Margolin et al., 1994)
SV40 poly(A) signal
5245 .. 5379  =  135 bp
SV40 polyadenylation signal
SV40 poly(A) signal
5245 .. 5379  =  135 bp
SV40 polyadenylation signal
HIV-1 Ψ
6228 .. 6353  =  126 bp
packaging signal of human immunodeficiency virus
type 1
HIV-1 Ψ
6228 .. 6353  =  126 bp
packaging signal of human immunodeficiency virus
type 1
cPPT/CTS
7596 .. 7713  =  118 bp
central polypurine tract and central termination
sequence of HIV-1
cPPT/CTS
7596 .. 7713  =  118 bp
central polypurine tract and central termination
sequence of HIV-1
AmpR promoter
1172 .. 1276  =  105 bp
AmpR promoter
1172 .. 1276  =  105 bp
P2A
12,830 .. 12,886  =  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
12,830 .. 12,886  =  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.
HA
12,698 .. 12,724  =  27 bp
9 amino acids  =  1.1 kDa
Product: HA (human influenza hemagglutinin)
epitope tag
HA
12,698 .. 12,724  =  27 bp
9 amino acids  =  1.1 kDa
Product: HA (human influenza hemagglutinin)
epitope tag
SV40 NLS
12,743 .. 12,763  =  21 bp
7 amino acids  =  883.1 Da
Product: nuclear localization signal of SV40 large T
antigen
SV40 NLS
12,743 .. 12,763  =  21 bp
7 amino acids  =  883.1 Da
Product: nuclear localization signal of SV40 large T
antigen
SV40 NLS
12,770 .. 12,790  =  21 bp
7 amino acids  =  883.1 Da
Product: nuclear localization signal of SV40 large T
antigen
SV40 NLS
12,770 .. 12,790  =  21 bp
7 amino acids  =  883.1 Da
Product: nuclear localization signal of SV40 large T
antigen
ATG
8354 .. 8356  =  3 bp
1 amino acid  =  149.2 Da
Product: start codon
ATG
8354 .. 8356  =  3 bp
1 amino acid  =  149.2 Da
Product: start codon
SV40 ori
3322 .. 3457  =  136 bp
SV40 origin of replication
SV40 ori
3322 .. 3457  =  136 bp
SV40 origin of replication
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