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pHAGE-TO-dCas9-3XGFP

Lentiviral vector for labeling chromosomal loci by tetracycline-regulatable expression of catalytically inactive dCas9 fused to three copies of GFP.

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pHAGE-TO-dCas9-3XGFP Sequence and MappHAGE-TO-dCas9-3XGFP.dna
Map and Sequence File   
Sequence Author:  Pederson Lab / Addgene #64107
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 StuI (12,535) SfiI (12,489) AhdI (11,369) PacI (10,354) SmaI (10,346) TspMI - XmaI (10,344) SacII (9879) XbaI (9369) PaeR7I - PspXI - XhoI (9355) BspDI - ClaI (8630) NotI (7896) BamHI (7165) SpeI (7132) AscI (6977) AvrII (12,536) FseI (1150) MfeI (1189) BbvCI (1424) BstAPI (1453) AleI (1577) NheI (2221) BmtI (2225) PspOMI (2233) ApaI (2237) SnaBI (2578) CMV promoter tet operator tet operator SgrDI (2850) ATG SV40 NLS EcoRI (3007) SbfI (3269) XcmI (3373) BlpI (3970) Bsu36I (4288) BmgBI (5532) BstXI (5811) FspAI (5972) AarI (6282) pHAGE-TO-dCas9-3XGFP 12,986 bp
StuI  (12,535)
1 site
A G G C C T T C C G G A
SfiI  (12,489)
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.
AhdI  (11,369)
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.
PacI  (10,354)
1 site
T T A A T T A A A A T T A A T T
SmaI  (10,346)
1 site
C C C G G G G G G C C C

SmaI can be used at 37°C for brief incubations.
TspMI  (10,344)
1 site
C C C G G G G G G C C C
XmaI  (10,344)
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.
SacII  (9879)
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.
XbaI  (9369)
1 site
T C T A G A A G A T C T
PaeR7I  (9355)
1 site
C T C G A G G A G C T C

PaeR7I does not recognize the sequence CTCTCGAG.
PspXI  (9355)
1 site
V C T C G A G B B G A G C T C V
XhoI  (9355)
1 site
C T C G A G G A G C T C
BspDI  (8630)
1 site
A T C G A T T A G C T A
ClaI  (8630)
1 site
A T C G A T T A G C T A
NotI  (7896)
1 site
G C G G C C G C C G C C G G C G
BamHI  (7165)
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.
SpeI  (7132)
1 site
A C T A G T T G A T C A
AscI  (6977)
1 site
G G C G C G C C C C G C G C G G
AvrII  (12,536)
1 site
C C T A G G G G A T C C
FseI  (1150)
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.
MfeI  (1189)
1 site
C A A T T G G T T A A C
BbvCI  (1424)
1 site
C C T C A G C G G A G T C G
BstAPI  (1453)
1 site
G C A N N N N N T G C C G T N N N N N A C G

Sticky ends from different BstAPI sites may not be compatible.
AleI  (1577)
1 site
C A C N N N N G T G G T G N N N N C A C
NheI  (2221)
1 site
G C T A G C C G A T C G
BmtI  (2225)
1 site
G C T A G C C G A T C G
PspOMI  (2233)
1 site
G G G C C C C C C G G G
ApaI  (2237)
1 site
G G G C C C C C C G G G

ApaI can be used between 25°C and 37°C.
SnaBI  (2578)
1 site
T A C G T A A T G C A T
SgrDI  (2850)
1 site
C G T C G A C G G C A G C T G C
EcoRI  (3007)
1 site
G A A T T C C T T A A G
SbfI  (3269)
1 site
C C T G C A G G G G A C G T C C
XcmI  (3373)
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.
BlpI  (3970)
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.
Bsu36I  (4288)
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.
BmgBI  (5532)
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.
BstXI  (5811)
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.
FspAI  (5972)
1 site
R T G C G C A Y Y A C G C G T R
AarI  (6282)
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.
dCas9
3025 .. 7125  =  4101 bp
1367 amino acids  =  158.2 kDa
   Segment 1:  
   3025 .. 3048  =  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 H840A mutation
in the HNH catalytic domain
dCas9
3025 .. 7125  =  4101 bp
1367 amino acids  =  158.2 kDa
   Segment 2:  
   3049 .. 3051  =  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
3025 .. 7125  =  4101 bp
1367 amino acids  =  158.2 kDa
   Segment 3:  
   3052 .. 5538  =  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
3025 .. 7125  =  4101 bp
1367 amino acids  =  158.2 kDa
   Segment 4:  
   5539 .. 5541  =  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
3025 .. 7125  =  4101 bp
1367 amino acids  =  158.2 kDa
   Segment 5:  
   5542 .. 7125  =  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
3025 .. 7125  =  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 H840A mutation
in the HNH catalytic domain
AmpR
10,582 .. 11,442  =  861 bp
286 amino acids  =  31.5 kDa
   Segment 1:  signal sequence  
   10,582 .. 10,650  =  69 bp
   23 amino acids  =  2.6 kDa
Product: β-lactamase
confers resistance to ampicillin, carbenicillin, and
related antibiotics
AmpR
10,582 .. 11,442  =  861 bp
286 amino acids  =  31.5 kDa
   Segment 2:  
   10,651 .. 11,442  =  792 bp
   263 amino acids  =  28.9 kDa
Product: β-lactamase
confers resistance to ampicillin, carbenicillin, and
related antibiotics
AmpR
10,582 .. 11,442  =  861 bp
286 amino acids  =  31.5 kDa
2 segments
Product: β-lactamase
confers resistance to ampicillin, carbenicillin, and
related antibiotics
superfolder GFP
7177 .. 7890  =  714 bp
238 amino acids  =  26.8 kDa
Product: GFP variant that folds robustly even when
fused to poorly folded proteins (Pédelacq et al.,
2006)
superfolder GFP
7177 .. 7890  =  714 bp
238 amino acids  =  26.8 kDa
Product: GFP variant that folds robustly even when
fused to poorly folded proteins (Pédelacq et al.,
2006)
superfolder GFP
7909 .. 8622  =  714 bp
238 amino acids  =  26.8 kDa
Product: GFP variant that folds robustly even when
fused to poorly folded proteins (Pédelacq et al.,
2006)
superfolder GFP
7909 .. 8622  =  714 bp
238 amino acids  =  26.8 kDa
Product: GFP variant that folds robustly even when
fused to poorly folded proteins (Pédelacq et al.,
2006)
superfolder GFP
8641 .. 9354  =  714 bp
238 amino acids  =  26.8 kDa
Product: GFP variant that folds robustly even when
fused to poorly folded proteins (Pédelacq et al.,
2006)
superfolder GFP
8641 .. 9354  =  714 bp
238 amino acids  =  26.8 kDa
Product: GFP variant that folds robustly even when
fused to poorly folded proteins (Pédelacq et al.,
2006)
3' LTR
1 .. 634  =  634 bp
3' long terminal repeat (LTR) from HIV-1
3' LTR
1 .. 634  =  634 bp
3' long terminal repeat (LTR) from HIV-1
WPRE
9376 .. 9964  =  589 bp
woodchuck hepatitis virus posttranscriptional
regulatory element
WPRE
9376 .. 9964  =  589 bp
woodchuck hepatitis virus posttranscriptional
regulatory element
ori
11,613 .. 12,201  =  589 bp
high-copy-number ColE1/pMB1/pBR322/pUC origin
of replication
ori
11,613 .. 12,201  =  589 bp
high-copy-number ColE1/pMB1/pBR322/pUC origin
of replication
CMV enhancer
2299 .. 2602  =  304 bp
human cytomegalovirus immediate early enhancer
CMV enhancer
2299 .. 2602  =  304 bp
human cytomegalovirus immediate early enhancer
RRE
1303 .. 1536  =  234 bp
The Rev response element (RRE) of HIV-1 allows for
Rev-dependent mRNA export from the nucleus to
the cytoplasm.
RRE
1303 .. 1536  =  234 bp
The Rev response element (RRE) of HIV-1 allows for
Rev-dependent mRNA export from the nucleus to
the cytoplasm.
3' LTR (ΔU3)
10,039 .. 10,272  =  234 bp
self-inactivating 3' long terminal repeat (LTR) from
HIV-1
3' LTR (ΔU3)
10,039 .. 10,272  =  234 bp
self-inactivating 3' long terminal repeat (LTR) from
HIV-1
CMV promoter
2603 .. 2806  =  204 bp
human cytomegalovirus (CMV) immediate early
promoter
CMV promoter
2603 .. 2806  =  204 bp
human cytomegalovirus (CMV) immediate early
promoter
HIV-1 Ψ
681 .. 806  =  126 bp
packaging signal of human immunodeficiency virus
type 1
HIV-1 Ψ
681 .. 806  =  126 bp
packaging signal of human immunodeficiency virus
type 1
cPPT/CTS
2058 .. 2175  =  118 bp
central polypurine tract and central termination
sequence of HIV-1
cPPT/CTS
2058 .. 2175  =  118 bp
central polypurine tract and central termination
sequence of HIV-1
AmpR promoter
10,477 .. 10,581  =  105 bp
AmpR promoter
10,477 .. 10,581  =  105 bp
SV40 NLS
2986 .. 3006  =  21 bp
7 amino acids  =  883.1 Da
Product: nuclear localization signal of SV40 large T
antigen
SV40 NLS
2986 .. 3006  =  21 bp
7 amino acids  =  883.1 Da
Product: nuclear localization signal of SV40 large T
antigen
SV40 NLS
7144 .. 7164  =  21 bp
7 amino acids  =  883.1 Da
Product: nuclear localization signal of SV40 large T
antigen
SV40 NLS
7144 .. 7164  =  21 bp
7 amino acids  =  883.1 Da
Product: nuclear localization signal of SV40 large T
antigen
tet operator
2808 .. 2826  =  19 bp
bacterial operator O2 for the tetR and tetA genes
tet operator
2808 .. 2826  =  19 bp
bacterial operator O2 for the tetR and tetA genes
tet operator
2829 .. 2847  =  19 bp
bacterial operator O2 for the tetR and tetA genes
tet operator
2829 .. 2847  =  19 bp
bacterial operator O2 for the tetR and tetA genes
ATG
2980 .. 2982  =  3 bp
1 amino acid  =  149.2 Da
Product: start codon
ATG
2980 .. 2982  =  3 bp
1 amino acid  =  149.2 Da
Product: start codon
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