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Plasmid Files

pcDNA-dCas9-p300 Core

Mammalian cell plasmid for CRISPR activation by expressing catalytically inactive dCas9 fused to the p300 core histone acetyltransferase domain.

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pcDNA-dCas9-p300 Core Sequence and MappcDNA-dCas9-p300 Core.dna
Map and Sequence File   
Sequence Author:  Gersbach Lab / Addgene #61357
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 AmpR promoter PvuI (10,922) AhdI (10,552) PfoI (9086) RsrII (8827) PflFI - Tth111I (8429) MscI (8393) PluTI (8314) SfoI (8312) NarI (8311) KasI (8310) EagI (8217) AvrII (8101) StuI (8100) SexAI * (7868) PmeI (7054) BspEI (7032) HA BsiWI (7022) XcmI (6984) BstXI (6561) EcoNI (5975) XbaI (5928) SgrDI (11,473) SpeI (249) SnaBI (590) ATG BspDI - ClaI (947) SV40 NLS SacII (1005) BsmBI (1201) BmgBI (1228) SwaI (1529) EcoRI (2348) Acc65I (2782) KpnI (2786) PmlI (3301) BstEII (5107) AscI (5160) BbvCI (5267) BsrGI (5764) pcDNA-dCas9-p300 Core 11,475 bp
PvuI  (10,922)
1 site
C G A T C G G C T A G C
AhdI  (10,552)
1 site
G A C N N N N N G T C C T G N N N N N C A G

The 1-base overhangs produced by AhdI may be hard to ligate.
Sticky ends from different AhdI sites may not be compatible.
PfoI  (9086)
1 site
T C C N G G A A G G N C C T

Sticky ends from different PfoI sites may not be compatible.
RsrII  (8827)
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.
PflFI  (8429)
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  (8429)
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.
MscI  (8393)
1 site
T G G C C A A C C G G T
PluTI  (8314)
1 site
G G C G C C C C G C G G

Efficient cleavage requires at least two copies of the PluTI
recognition sequence.
SfoI  (8312)
1 site
G G C G C C C C G C G G
NarI  (8311)
1 site
G G C G C C C C G C G G

Efficient cleavage requires at least two copies of the NarI
recognition sequence.
KasI  (8310)
1 site
G G C G C C C C G C G G
EagI  (8217)
1 site
C G G C C G G C C G G C
AvrII  (8101)
1 site
C C T A G G G G A T C C
StuI  (8100)
1 site
A G G C C T T C C G G A
SexAI  (7868)
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.
PmeI  (7054)
1 site
G T T T A A A C C A A A T T T G
BspEI  (7032)
1 site
T C C G G A A G G C C T
BsiWI  (7022)
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.
XcmI  (6984)
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.
BstXI  (6561)
1 site
C C A N N N N N N T G G G G T N N N N N N A C C

Sticky ends from different BstXI sites may not be compatible.
EcoNI  (5975)
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.
XbaI  (5928)
1 site
T C T A G A A G A T C T
SgrDI  (11,473)
1 site
C G T C G A C G G C A G C T G C
SpeI  (249)
1 site
A C T A G T T G A T C A
SnaBI  (590)
1 site
T A C G T A A T G C A T
BspDI  (947)
1 site
A T C G A T T A G C T A
ClaI  (947)
1 site
A T C G A T T A G C T A
SacII  (1005)
1 site
C C G C G G G G C G C C

Efficient cleavage requires at least two copies of the SacII
recognition sequence.
BsmBI  (1201)
1 site
C G T C T C N G C A G A G N ( N ) 4

Sticky ends from different BsmBI sites may not be compatible.
BmgBI  (1228)
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.
SwaI  (1529)
1 site
A T T T A A A T T A A A T T T A

SwaI is typically used at 25°C, but is 50% active at 37°C.
EcoRI  (2348)
1 site
G A A T T C C T T A A G
Acc65I  (2782)
1 site
G G T A C C C C A T G G
KpnI  (2786)
1 site
G G T A C C C C A T G G
PmlI  (3301)
1 site
C A C G T G G T G C A C

PmlI gradually loses activity when stored at -20°C.
BstEII  (5107)
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.
AscI  (5160)
1 site
G G C G C G C C C C G C G C G G
BbvCI  (5267)
1 site
C C T C A G C G G A G T C G
BsrGI  (5764)
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.
dCas9
1009 .. 5112  =  4104 bp
1368 amino acids  =  158.3 kDa
   Segment 1:  
   1009 .. 1035  =  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
1009 .. 5112  =  4104 bp
1368 amino acids  =  158.3 kDa
   Segment 2:  
   1036 .. 1038  =  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
1009 .. 5112  =  4104 bp
1368 amino acids  =  158.3 kDa
   Segment 3:  
   1039 .. 3525  =  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
1009 .. 5112  =  4104 bp
1368 amino acids  =  158.3 kDa
   Segment 4:  
   3526 .. 3528  =  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
1009 .. 5112  =  4104 bp
1368 amino acids  =  158.3 kDa
   Segment 5:  
   3529 .. 5112  =  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
1009 .. 5112  =  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
p300 core
5167 .. 7017  =  1851 bp
617 amino acids  =  71.7 kDa
Product: catalytic histone acetyltransferase core of
the human E1A-associated protein p300 (Delvecchio
et al., 2013)
p300 core
5167 .. 7017  =  1851 bp
617 amino acids  =  71.7 kDa
Product: catalytic histone acetyltransferase core of
the human E1A-associated protein p300 (Delvecchio
et al., 2013)
AmpR
10,479 .. 11,339  =  861 bp
286 amino acids  =  31.6 kDa
   Segment 2:  
   10,479 .. 11,270  =  792 bp
   263 amino acids  =  28.9 kDa
Product: β-lactamase
confers resistance to ampicillin, carbenicillin, and
related antibiotics
AmpR
10,479 .. 11,339  =  861 bp
286 amino acids  =  31.6 kDa
   Segment 1:  signal sequence  
   11,271 .. 11,339  =  69 bp
   23 amino acids  =  2.6 kDa
Product: β-lactamase
confers resistance to ampicillin, carbenicillin, and
related antibiotics
AmpR
10,479 .. 11,339  =  861 bp
286 amino acids  =  31.6 kDa
2 segments
Product: β-lactamase
confers resistance to ampicillin, carbenicillin, and
related antibiotics
NeoR/KanR
8183 .. 8977  =  795 bp
264 amino acids  =  29.0 kDa
Product: aminoglycoside phosphotransferase from
Tn5
confers resistance to neomycin, kanamycin, and
G418 (Geneticin®)
NeoR/KanR
8183 .. 8977  =  795 bp
264 amino acids  =  29.0 kDa
Product: aminoglycoside phosphotransferase from
Tn5
confers resistance to neomycin, kanamycin, and
G418 (Geneticin®)
ori
9723 .. 10,308  =  586 bp
high-copy-number ColE1/pMB1/pBR322/pUC origin
of replication
ori
9723 .. 10,308  =  586 bp
high-copy-number ColE1/pMB1/pBR322/pUC origin
of replication
f1 ori
7345 .. 7773  =  429 bp
f1 bacteriophage origin of replication; arrow
indicates direction of (+) strand synthesis
f1 ori
7345 .. 7773  =  429 bp
f1 bacteriophage origin of replication; arrow
indicates direction of (+) strand synthesis
CMV enhancer
235 .. 614  =  380 bp
human cytomegalovirus immediate early enhancer
CMV enhancer
235 .. 614  =  380 bp
human cytomegalovirus immediate early enhancer
SV40 promoter
7787 .. 8116  =  330 bp
SV40 enhancer and early promoter
SV40 promoter
7787 .. 8116  =  330 bp
SV40 enhancer and early promoter
bGH poly(A) signal
7075 .. 7299  =  225 bp
bovine growth hormone polyadenylation signal
bGH poly(A) signal
7075 .. 7299  =  225 bp
bovine growth hormone polyadenylation signal
CMV promoter
615 .. 818  =  204 bp
human cytomegalovirus (CMV) immediate early
promoter
CMV promoter
615 .. 818  =  204 bp
human cytomegalovirus (CMV) immediate early
promoter
SV40 poly(A) signal
9151 .. 9272  =  122 bp
SV40 polyadenylation signal
SV40 poly(A) signal
9151 .. 9272  =  122 bp
SV40 polyadenylation signal
AmpR promoter
11,340 .. 11,444  =  105 bp
AmpR promoter
11,340 .. 11,444  =  105 bp
3xFLAG
907 .. 972  =  66 bp
22 amino acids  =  2.7 kDa
Product: three tandem FLAG® epitope tags,
followed by an enterokinase cleavage site
3xFLAG
907 .. 972  =  66 bp
22 amino acids  =  2.7 kDa
Product: three tandem FLAG® epitope tags,
followed by an enterokinase cleavage site
HA
7018 .. 7044  =  27 bp
9 amino acids  =  1.1 kDa
Product: HA (human influenza hemagglutinin)
epitope tag
HA
7018 .. 7044  =  27 bp
9 amino acids  =  1.1 kDa
Product: HA (human influenza hemagglutinin)
epitope tag
SV40 NLS
979 .. 999  =  21 bp
7 amino acids  =  883.1 Da
Product: nuclear localization signal of SV40 large T
antigen
SV40 NLS
979 .. 999  =  21 bp
7 amino acids  =  883.1 Da
Product: nuclear localization signal of SV40 large T
antigen
SV40 NLS
5137 .. 5157  =  21 bp
7 amino acids  =  883.1 Da
Product: nuclear localization signal of SV40 large T
antigen
SV40 NLS
5137 .. 5157  =  21 bp
7 amino acids  =  883.1 Da
Product: nuclear localization signal of SV40 large T
antigen
T7 promoter
863 .. 881  =  19 bp
promoter for bacteriophage T7 RNA polymerase
T7 promoter
863 .. 881  =  19 bp
promoter for bacteriophage T7 RNA polymerase
ATG
904 .. 906  =  3 bp
1 amino acid  =  149.2 Da
Product: start codon
ATG
904 .. 906  =  3 bp
1 amino acid  =  149.2 Da
Product: start codon
SV40 ori
7967 .. 8102  =  136 bp
SV40 origin of replication
SV40 ori
7967 .. 8102  =  136 bp
SV40 origin of replication
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