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lenti dCAS-VP64_Blast

Zhang lab lentiviral vector encoding a blasticidin resistance marker plus catalytically inactive dCas9 fused to the VP64 transcriptional activation domain.

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lenti dCAS-VP64_Blast Sequence and Maplenti dCAS-VP64_Blast.dna
Map and Sequence File   
Sequence Author:  Zhang Lab / Addgene #61425
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 AmpR promoter SspI (13,969) FspI (13,387) BstZ17I (11,893) SgrAI (11,371) MauBI (11,293) EM7 promoter AvrII (11,128) PmeI (10,080) KpnI (9503) Acc65I (9499) Bsu36I (9495) BspDI - ClaI (9367) EcoRI (8753) BsrGI (8285) BamHI (8114) SV40 NLS AfeI (8032) SgrDI (1) MluI (231) SpeI (252) NdeI (487) SnaBI (593) HIV-1 Ψ AleI (1954) NheI (2601) BmtI (2605) AgeI (2765) BsiWI (3866) ATG lenti dCAS-VP64_Blast 14,085 bp
SspI  (13,969)
1 site
A A T A T T T T A T A A
FspI  (13,387)
1 site
T G C G C A A C G C G T
BstZ17I  (11,893)
1 site
G T A T A C C A T A T G
SgrAI  (11,371)
1 site
C R C C G G Y G G Y G G C C R C

Efficient cleavage requires at least two copies of the SgrAI
recognition sequence.
MauBI  (11,293)
1 site
C G C G C G C G G C G C G C G C
AvrII  (11,128)
1 site
C C T A G G G G A T C C
PmeI  (10,080)
1 site
G T T T A A A C C A A A T T T G
KpnI  (9503)
1 site
G G T A C C C C A T G G
Acc65I  (9499)
1 site
G G T A C C C C A T G G
Bsu36I  (9495)
1 site
C C T N A G G G G A N T C C

Sticky ends from different Bsu36I sites may not be compatible.
BspDI  (9367)
1 site
A T C G A T T A G C T A
ClaI  (9367)
1 site
A T C G A T T A G C T A
EcoRI  (8753)
1 site
G A A T T C C T T A A G
BsrGI  (8285)
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.
BamHI  (8114)
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.
AfeI  (8032)
1 site
A G C G C T T C G C G A
SgrDI  (1)
1 site
C G T C G A C G G C A G C T G C
MluI  (231)
1 site
A C G C G T T G C G C A
SpeI  (252)
1 site
A C T A G T T G A T C A
NdeI  (487)
1 site
C A T A T G G T A T A C

Prolonged incubation with NdeI may lead to removal of additional
nucleotides.
SnaBI  (593)
1 site
T A C G T A A T G C A T
AleI  (1954)
1 site
C A C N N N N G T G G T G N N N N C A C
NheI  (2601)
1 site
G C T A G C C G A T C G
BmtI  (2605)
1 site
G C T A G C C G A T C G
AgeI  (2765)
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.
BsiWI  (3866)
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.
dCas9
3929 .. 8029  =  4101 bp
1367 amino acids  =  158.2 kDa
   Segment 1:  
   3929 .. 3952  =  24 bp
   8 amino acids  =  923.1 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 N863A mutation
in the HNH catalytic domain
dCas9
3929 .. 8029  =  4101 bp
1367 amino acids  =  158.2 kDa
   Segment 2:  
   3953 .. 3955  =  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 N863A mutation
in the HNH catalytic domain
dCas9
3929 .. 8029  =  4101 bp
1367 amino acids  =  158.2 kDa
   Segment 3:  
   3956 .. 6511  =  2556 bp
   852 amino acids  =  98.9 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 N863A mutation
in the HNH catalytic domain
dCas9
3929 .. 8029  =  4101 bp
1367 amino acids  =  158.2 kDa
   Segment 4:  
   6512 .. 6514  =  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 N863A mutation
in the HNH catalytic domain
dCas9
3929 .. 8029  =  4101 bp
1367 amino acids  =  158.2 kDa
   Segment 5:  
   6515 .. 8029  =  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 D10A mutation in
the RuvC catalytic domain and the N863A mutation
in the HNH catalytic domain
dCas9
3929 .. 8029  =  4101 bp
1367 amino acids  =  158.2 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 N863A mutation
in the HNH catalytic domain
EF-1α promoter
2687 .. 3865  =  1179 bp
strong constitutive promoter for human elongation
factor EF-1α
EF-1α promoter
2687 .. 3865  =  1179 bp
strong constitutive promoter for human elongation
factor EF-1α
AmpR
13,092 .. 13,952  =  861 bp
286 amino acids  =  31.6 kDa
   Segment 2:  
   13,092 .. 13,883  =  792 bp
   263 amino acids  =  28.9 kDa
Product: β-lactamase
confers resistance to ampicillin, carbenicillin, and
related antibiotics
AmpR
13,092 .. 13,952  =  861 bp
286 amino acids  =  31.6 kDa
   Segment 1:  signal sequence  
   13,884 .. 13,952  =  69 bp
   23 amino acids  =  2.6 kDa
Product: β-lactamase
confers resistance to ampicillin, carbenicillin, and
related antibiotics
AmpR
13,092 .. 13,952  =  861 bp
286 amino acids  =  31.6 kDa
2 segments
Product: β-lactamase
confers resistance to ampicillin, carbenicillin, and
related antibiotics
WPRE
8777 .. 9365  =  589 bp
woodchuck hepatitis virus posttranscriptional
regulatory element
WPRE
8777 .. 9365  =  589 bp
woodchuck hepatitis virus posttranscriptional
regulatory element
ori
12,333 .. 12,921  =  589 bp
high-copy-number ColE1/pMB1/pBR322/pUC origin
of replication
ori
12,333 .. 12,921  =  589 bp
high-copy-number ColE1/pMB1/pBR322/pUC origin
of replication
f1 ori
10,372 .. 10,800  =  429 bp
f1 bacteriophage origin of replication; arrow
indicates direction of (+) strand synthesis
f1 ori
10,372 .. 10,800  =  429 bp
f1 bacteriophage origin of replication; arrow
indicates direction of (+) strand synthesis
BSD
8354 .. 8752  =  399 bp
132 amino acids  =  13.7 kDa
Product: blasticidin S deaminase
confers resistance to blasticidin
BSD
8354 .. 8752  =  399 bp
132 amino acids  =  13.7 kDa
Product: blasticidin S deaminase
confers resistance to blasticidin
CMV enhancer
238 .. 617  =  380 bp
human cytomegalovirus immediate early enhancer
CMV enhancer
238 .. 617  =  380 bp
human cytomegalovirus immediate early enhancer
BleoR
11,257 .. 11,631  =  375 bp
124 amino acids  =  13.8 kDa
Product: antibiotic-binding protein
confers resistance to bleomycin, phleomycin, and
Zeocin™
BleoR
11,257 .. 11,631  =  375 bp
124 amino acids  =  13.8 kDa
Product: antibiotic-binding protein
confers resistance to bleomycin, phleomycin, and
Zeocin™
SV40 promoter
10,814 .. 11,143  =  330 bp
SV40 enhancer and early promoter
SV40 promoter
10,814 .. 11,143  =  330 bp
SV40 enhancer and early promoter
RRE
1680 .. 1913  =  234 bp
The Rev response element (RRE) of HIV-1 allows for
Rev-dependent mRNA export from the nucleus to
the cytoplasm.
RRE
1680 .. 1913  =  234 bp
The Rev response element (RRE) of HIV-1 allows for
Rev-dependent mRNA export from the nucleus to
the cytoplasm.
bGH poly(A) signal
10,102 .. 10,326  =  225 bp
bovine growth hormone polyadenylation signal
bGH poly(A) signal
10,102 .. 10,326  =  225 bp
bovine growth hormone polyadenylation signal
CMV promoter
619 .. 817  =  199 bp
human cytomegalovirus (CMV) immediate early
promoter
CMV promoter
619 .. 817  =  199 bp
human cytomegalovirus (CMV) immediate early
promoter
LTR (truncated)
835 .. 1015  =  181 bp
truncated long terminal repeat (LTR) from HIV-1
LTR (truncated)
835 .. 1015  =  181 bp
truncated long terminal repeat (LTR) from HIV-1
LTR (truncated)
9890 .. 10,070  =  181 bp
truncated long terminal repeat (LTR) from HIV-1
LTR (truncated)
9890 .. 10,070  =  181 bp
truncated long terminal repeat (LTR) from HIV-1
VP64
8129 .. 8278  =  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
8129 .. 8278  =  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)
HIV-1 Ψ
1062 .. 1187  =  126 bp
packaging signal of human immunodeficiency virus
type 1
HIV-1 Ψ
1062 .. 1187  =  126 bp
packaging signal of human immunodeficiency virus
type 1
SV40 poly(A) signal
11,761 .. 11,882  =  122 bp
SV40 polyadenylation signal
SV40 poly(A) signal
11,761 .. 11,882  =  122 bp
SV40 polyadenylation signal
cPPT/CTS
2440 .. 2557  =  118 bp
central polypurine tract and central termination
sequence of HIV-1
cPPT/CTS
2440 .. 2557  =  118 bp
central polypurine tract and central termination
sequence of HIV-1
AmpR promoter
13,953 .. 14,057  =  105 bp
AmpR promoter
13,953 .. 14,057  =  105 bp
T2A
8300 .. 8353  =  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
8300 .. 8353  =  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
3881 .. 3928  =  48 bp
16 amino acids  =  1.7 kDa
Product: bipartite nuclear localization signal from
nucleoplasmin
nucleoplasmin NLS
3881 .. 3928  =  48 bp
16 amino acids  =  1.7 kDa
Product: bipartite nuclear localization signal from
nucleoplasmin
EM7 promoter
11,191 .. 11,238  =  48 bp
synthetic bacterial promoter
EM7 promoter
11,191 .. 11,238  =  48 bp
synthetic bacterial promoter
SV40 NLS
8084 .. 8104  =  21 bp
7 amino acids  =  883.1 Da
Product: nuclear localization signal of SV40 large T
antigen
SV40 NLS
8084 .. 8104  =  21 bp
7 amino acids  =  883.1 Da
Product: nuclear localization signal of SV40 large T
antigen
ATG
3878 .. 3880  =  3 bp
1 amino acid  =  149.2 Da
Product: start codon
ATG
3878 .. 3880  =  3 bp
1 amino acid  =  149.2 Da
Product: start codon
EF-1α intron A
2918 .. 3856  =  939 bp
intron upstream of the start codon of human EF-1α
EF-1α intron A
2918 .. 3856  =  939 bp
intron upstream of the start codon of human EF-1α
SV40 ori
10,994 .. 11,129  =  136 bp
SV40 origin of replication
SV40 ori
10,994 .. 11,129  =  136 bp
SV40 origin of replication
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