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Plasmid Files

pcDNA-dCas9-VP64

Mammalian cell plasmid for CRISPR activation by expressing catalytically inactive dCas9 fused to the VP64 transcriptional activation domain.

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pcDNA-dCas9-VP64 Sequence and MappcDNA-dCas9-VP64.dna
Map and Sequence File   
Sequence Author:  Gersbach Lab / Addgene #47107
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 SgrDI (9810) AmpR promoter PvuI (9259) AhdI (8889) PfoI (7423) RsrII (7164) PflFI - Tth111I (6766) MscI (6730) PstI (6701) PluTI (6651) SfoI (6649) NarI (6648) KasI (6647) SmaI (6461) BmeT110I (6460) AvaI - BsoBI - TspMI - XmaI (6459) AvrII (6438) StuI (6437) BseRI (6434) SexAI * (6205) DraIII (5915) PmeI (5391) AflII (5383) XbaI (5377) BspEI (5337) HA BsiWI (5327) PacI (5318) AscI (5160) SV40 NLS BglII (12) SpeI (249) SnaBI (590) T7 promoter ATG BspDI - ClaI (947) SV40 NLS SacII (1005) BsmBI (1201) BmgBI (1228) SwaI (1529) BsgI (1631) AleI (2255) Acc65I (2782) KpnI (2786) PmlI (3301) BstEII (5107) pcDNA-dCas9-VP64 9812 bp
SgrDI  (9810)
1 site
C G T C G A C G G C A G C T G C
PvuI  (9259)
1 site
C G A T C G G C T A G C
AhdI  (8889)
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  (7423)
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  (7164)
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  (6766)
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  (6766)
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  (6730)
1 site
T G G C C A A C C G G T
PstI  (6701)
1 site
C T G C A G G A C G T C
PluTI  (6651)
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  (6649)
1 site
G G C G C C C C G C G G
NarI  (6648)
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  (6647)
1 site
G G C G C C C C G C G G
SmaI  (6461)
1 site
C C C G G G G G G C C C

SmaI can be used at 37°C for brief incubations.
BmeT110I  (6460)
1 site
C Y C G R G G R G C Y C
AvaI  (6459)
1 site
C Y C G R G G R G C Y C

Sticky ends from different AvaI sites may not be compatible.
BsoBI  (6459)
1 site
C Y C G R G G R G C Y C

Sticky ends from different BsoBI sites may not be compatible.
BsoBI is typically used at 37°C, but can be used at temperatures
up to 65°C.
TspMI  (6459)
1 site
C C C G G G G G G C C C
XmaI  (6459)
1 site
C C C G G G G G G C C C

Efficient cleavage requires at least two copies of the XmaI
recognition sequence.
Full cleavage with XmaI may require a long incubation.
AvrII  (6438)
1 site
C C T A G G G G A T C C
StuI  (6437)
1 site
A G G C C T T C C G G A
BseRI  (6434)
1 site
G A G G A G ( N ) 8 N N C T C C T C ( N ) 8

Sticky ends from different BseRI sites may not be compatible.
BseRI quickly loses activity at 37°C.
Prolonged incubation with BseRI may lead to degradation of the
DNA.
SexAI  (6205)
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.
DraIII  (5915)
1 site
C A C N N N G T G G T G N N N C A C

Sticky ends from different DraIII sites may not be compatible.
PmeI  (5391)
1 site
G T T T A A A C C A A A T T T G
AflII  (5383)
1 site
C T T A A G G A A T T C

The sticky ends produced by AflII are hard to ligate.
XbaI  (5377)
1 site
T C T A G A A G A T C T
BspEI  (5337)
1 site
T C C G G A A G G C C T
BsiWI  (5327)
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.
PacI  (5318)
1 site
T T A A T T A A A A T T A A T T
AscI  (5160)
1 site
G G C G C G C C C C G C G C G G
BglII  (12)
1 site
A G A T C T T C T A G A
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.
BsgI  (1631)
1 site
G T G C A G ( N ) 14 N N C A C G T C ( N ) 14

Efficient cleavage requires at least two copies of the BsgI
recognition sequence.
Sticky ends from different BsgI sites may not be compatible.
For full activity, add fresh S-adenosylmethionine (SAM).
AleI  (2255)
1 site
C A C N N N N G T G G T G N N N N C A C
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.
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
AmpR
8816 .. 9676  =  861 bp
286 amino acids  =  31.6 kDa
   Segment 2:  
   8816 .. 9607  =  792 bp
   263 amino acids  =  28.9 kDa
Product: β-lactamase
confers resistance to ampicillin, carbenicillin, and
related antibiotics
AmpR
8816 .. 9676  =  861 bp
286 amino acids  =  31.6 kDa
   Segment 1:  signal sequence  
   9608 .. 9676  =  69 bp
   23 amino acids  =  2.6 kDa
Product: β-lactamase
confers resistance to ampicillin, carbenicillin, and
related antibiotics
AmpR
8816 .. 9676  =  861 bp
286 amino acids  =  31.6 kDa
2 segments
Product: β-lactamase
confers resistance to ampicillin, carbenicillin, and
related antibiotics
NeoR/KanR
6520 .. 7314  =  795 bp
264 amino acids  =  29.0 kDa
Product: aminoglycoside phosphotransferase from
Tn5
confers resistance to neomycin, kanamycin, and
G418 (Geneticin®)
NeoR/KanR
6520 .. 7314  =  795 bp
264 amino acids  =  29.0 kDa
Product: aminoglycoside phosphotransferase from
Tn5
confers resistance to neomycin, kanamycin, and
G418 (Geneticin®)
ori
8060 .. 8645  =  586 bp
high-copy-number ColE1/pMB1/pBR322/pUC origin
of replication
ori
8060 .. 8645  =  586 bp
high-copy-number ColE1/pMB1/pBR322/pUC origin
of replication
f1 ori
5682 .. 6110  =  429 bp
f1 bacteriophage origin of replication; arrow
indicates direction of (+) strand synthesis
f1 ori
5682 .. 6110  =  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
6124 .. 6453  =  330 bp
SV40 enhancer and early promoter
SV40 promoter
6124 .. 6453  =  330 bp
SV40 enhancer and early promoter
bGH poly(A) signal
5412 .. 5636  =  225 bp
bovine growth hormone polyadenylation signal
bGH poly(A) signal
5412 .. 5636  =  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
VP64
5167 .. 5316  =  150 bp
50 amino acids  =  5.5 kDa
Product: tetrameric repeat of the minimal activation
domain of herpes simplex virus VP16 (Beerli et al.,
1998)
VP64
5167 .. 5316  =  150 bp
50 amino acids  =  5.5 kDa
Product: tetrameric repeat of the minimal activation
domain of herpes simplex virus VP16 (Beerli et al.,
1998)
SV40 poly(A) signal
7488 .. 7609  =  122 bp
SV40 polyadenylation signal
SV40 poly(A) signal
7488 .. 7609  =  122 bp
SV40 polyadenylation signal
AmpR promoter
9677 .. 9781  =  105 bp
AmpR promoter
9677 .. 9781  =  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
5323 .. 5349  =  27 bp
9 amino acids  =  1.1 kDa
Product: HA (human influenza hemagglutinin)
epitope tag
HA
5323 .. 5349  =  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
6304 .. 6439  =  136 bp
SV40 origin of replication
SV40 ori
6304 .. 6439  =  136 bp
SV40 origin of replication
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