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Plasmid Files

SP-dCas9-VPR

Plasmid for mammalian cell expression of catalytically inactive Cas9m4 fused to the tripartite VP64-p65-Rta (VPR) transcriptional activator.

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SP-dCas9-VPR Sequence and MapSP-dCas9-VPR.dna
Map and Sequence File   
Sequence Author:  Church Lab / Addgene #63798
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 MfeI (11,295) SgrDI (11,132) AhdI (10,211) BstZ17I (8939) RsrII (8481) AvrII (7755) SexAI * (7522) DraIII (7232) PmeI (6687) KpnI (6600) Acc65I (6596) NotI (6588) attB2 BbvCI (6191) SpeI (64) SnaBI (405) SbfI (995) BlpI (1696) PmlI (3537) FspAI (3698) AarI (4008) PaeR7I - XhoI (4291) EcoRI (4570) AscI (4703) SV40 NLS PshAI (5898) SP-dCas9-VPR 11,319 bp
MfeI  (11,295)
1 site
C A A T T G G T T A A C
SgrDI  (11,132)
1 site
C G T C G A C G G C A G C T G C
AhdI  (10,211)
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.
BstZ17I  (8939)
1 site
G T A T A C C A T A T G
RsrII  (8481)
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.
AvrII  (7755)
1 site
C C T A G G G G A T C C
SexAI  (7522)
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  (7232)
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  (6687)
1 site
G T T T A A A C C A A A T T T G
KpnI  (6600)
1 site
G G T A C C C C A T G G
Acc65I  (6596)
1 site
G G T A C C C C A T G G
NotI  (6588)
1 site
G C G G C C G C C G C C G G C G
BbvCI  (6191)
1 site
C C T C A G C G G A G T C G
SpeI  (64)
1 site
A C T A G T T G A T C A
SnaBI  (405)
1 site
T A C G T A A T G C A T
SbfI  (995)
1 site
C C T G C A G G G G A C G T C C
BlpI  (1696)
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.
PmlI  (3537)
1 site
C A C G T G G T G C A C

PmlI gradually loses activity when stored at -20°C.
FspAI  (3698)
1 site
R T G C G C A Y Y A C G C G T R
AarI  (4008)
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.
PaeR7I  (4291)
1 site
C T C G A G G A G C T C

PaeR7I does not recognize the sequence CTCTCGAG.
XhoI  (4291)
1 site
C T C G A G G A G C T C
EcoRI  (4570)
1 site
G A A T T C C T T A A G
AscI  (4703)
1 site
G G C G C G C C C C G C G C G G
PshAI  (5898)
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.
Cas9m4
748 .. 4851  =  4104 bp
1368 amino acids  =  158.2 kDa
   Segment 1:  
   748 .. 774  =  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 mutations D10A,
D839A, H840A, and N863A (Mali et al., 2013)
Cas9m4
748 .. 4851  =  4104 bp
1368 amino acids  =  158.2 kDa
   Segment 2:  
   775 .. 777  =  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 mutations D10A,
D839A, H840A, and N863A (Mali et al., 2013)
Cas9m4
748 .. 4851  =  4104 bp
1368 amino acids  =  158.2 kDa
   Segment 3:  
   778 .. 3261  =  2484 bp
   828 amino acids  =  96.1 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 mutations D10A,
D839A, H840A, and N863A (Mali et al., 2013)
Cas9m4
748 .. 4851  =  4104 bp
1368 amino acids  =  158.2 kDa
   Segment 4:  
   3262 .. 3267  =  6 bp
   2 amino acids  =  160.2 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 mutations D10A,
D839A, H840A, and N863A (Mali et al., 2013)
Cas9m4
748 .. 4851  =  4104 bp
1368 amino acids  =  158.2 kDa
   Segment 5:  
   3268 .. 3333  =  66 bp
   22 amino acids  =  2.5 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 mutations D10A,
D839A, H840A, and N863A (Mali et al., 2013)
Cas9m4
748 .. 4851  =  4104 bp
1368 amino acids  =  158.2 kDa
   Segment 6:  
   3334 .. 3336  =  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 mutations D10A,
D839A, H840A, and N863A (Mali et al., 2013)
Cas9m4
748 .. 4851  =  4104 bp
1368 amino acids  =  158.2 kDa
   Segment 7:  
   3337 .. 4851  =  1515 bp
   505 amino acids  =  58.3 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 mutations D10A,
D839A, H840A, and N863A (Mali et al., 2013)
Cas9m4
748 .. 4851  =  4104 bp
1368 amino acids  =  158.2 kDa
7 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 mutations D10A,
D839A, H840A, and N863A (Mali et al., 2013)
AmpR
10,138 .. 10,998  =  861 bp
286 amino acids  =  31.6 kDa
   Segment 2:  
   10,138 .. 10,929  =  792 bp
   263 amino acids  =  28.9 kDa
Product: β-lactamase
confers resistance to ampicillin, carbenicillin, and
related antibiotics
AmpR
10,138 .. 10,998  =  861 bp
286 amino acids  =  31.6 kDa
   Segment 1:  signal sequence  
   10,930 .. 10,998  =  69 bp
   23 amino acids  =  2.6 kDa
Product: β-lactamase
confers resistance to ampicillin, carbenicillin, and
related antibiotics
AmpR
10,138 .. 10,998  =  861 bp
286 amino acids  =  31.6 kDa
2 segments
Product: β-lactamase
confers resistance to ampicillin, carbenicillin, and
related antibiotics
NeoR/KanR
7837 .. 8631  =  795 bp
264 amino acids  =  29.0 kDa
Product: aminoglycoside phosphotransferase from
Tn5
confers resistance to neomycin, kanamycin, and
G418 (Geneticin®)
NeoR/KanR
7837 .. 8631  =  795 bp
264 amino acids  =  29.0 kDa
Product: aminoglycoside phosphotransferase from
Tn5
confers resistance to neomycin, kanamycin, and
G418 (Geneticin®)
ori
9379 .. 9967  =  589 bp
high-copy-number ColE1/pMB1/pBR322/pUC origin
of replication
ori
9379 .. 9967  =  589 bp
high-copy-number ColE1/pMB1/pBR322/pUC origin
of replication
Rta AD
5974 .. 6543  =  570 bp
190 amino acids  =  20.6 kDa
Product: transcriptional activation domain from the
human herpesvirus 4 (Epstein-Barr virus) replication
and transcription activator Rta/BRLF1 (Hardwick et
al., 1992; Chavez et al., 2015)
Rta AD
5974 .. 6543  =  570 bp
190 amino acids  =  20.6 kDa
Product: transcriptional activation domain from the
human herpesvirus 4 (Epstein-Barr virus) replication
and transcription activator Rta/BRLF1 (Hardwick et
al., 1992; Chavez et al., 2015)
f1 ori
6999 .. 7427  =  429 bp
f1 bacteriophage origin of replication; arrow
indicates direction of (+) strand synthesis
f1 ori
6999 .. 7427  =  429 bp
f1 bacteriophage origin of replication; arrow
indicates direction of (+) strand synthesis
CMV enhancer
50 .. 429  =  380 bp
human cytomegalovirus immediate early enhancer
CMV enhancer
50 .. 429  =  380 bp
human cytomegalovirus immediate early enhancer
RelA (p65) AD
5599 .. 5955  =  357 bp
119 amino acids  =  12.4 kDa
Product: transcriptional activation domain of human
RelA, also known as p65 (O'Shea and Perkins, 2008)
RelA (p65) AD
5599 .. 5955  =  357 bp
119 amino acids  =  12.4 kDa
Product: transcriptional activation domain of human
RelA, also known as p65 (O'Shea and Perkins, 2008)
SV40 promoter
7441 .. 7770  =  330 bp
SV40 enhancer and early promoter
SV40 promoter
7441 .. 7770  =  330 bp
SV40 enhancer and early promoter
CMV promoter
430 .. 633  =  204 bp
human cytomegalovirus (CMV) immediate early
promoter
CMV promoter
430 .. 633  =  204 bp
human cytomegalovirus (CMV) immediate early
promoter
VP64
4975 .. 5124  =  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
4975 .. 5124  =  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
8807 .. 8928  =  122 bp
SV40 polyadenylation signal
SV40 poly(A) signal
8807 .. 8928  =  122 bp
SV40 polyadenylation signal
AmpR promoter
10,999 .. 11,103  =  105 bp
AmpR promoter
10,999 .. 11,103  =  105 bp
HSV TK poly(A) signal
6749 .. 6797  =  49 bp
herpes simplex virus thymidine kinase
polyadenylation signal (Cole and Stacy, 1985)
HSV TK poly(A) signal
6749 .. 6797  =  49 bp
herpes simplex virus thymidine kinase
polyadenylation signal (Cole and Stacy, 1985)
attB1
4921 .. 4945  =  25 bp
recombination site for the Gateway® BP reaction
attB1
4921 .. 4945  =  25 bp
recombination site for the Gateway® BP reaction
attB2
6548 .. 6572  =  25 bp
recombination site for the Gateway® BP reaction
attB2
6548 .. 6572  =  25 bp
recombination site for the Gateway® BP reaction
SV40 NLS
4864 .. 4884  =  21 bp
7 amino acids  =  883.1 Da
Product: nuclear localization signal of SV40 large T
antigen
SV40 NLS
4864 .. 4884  =  21 bp
7 amino acids  =  883.1 Da
Product: nuclear localization signal of SV40 large T
antigen
SV40 NLS
5149 .. 5169  =  21 bp
7 amino acids  =  883.1 Da
Product: nuclear localization signal of SV40 large T
antigen
SV40 NLS
5149 .. 5169  =  21 bp
7 amino acids  =  883.1 Da
Product: nuclear localization signal of SV40 large T
antigen
SV40 ori
7621 .. 7756  =  136 bp
SV40 origin of replication
SV40 ori
7621 .. 7756  =  136 bp
SV40 origin of replication
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