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Plasmid Files

pTDH3-dCas9

Yeast centromeric vector for expressing catalytically inactive dCas9 with the TDH3 (glyceraldehyde 3-phosphate dehydrogenase) promoter.

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pTDH3-dCas9 Sequence and MappTDH3-dCas9.dna
Map and Sequence File   
Sequence Author:  Qi & Weissman Labs / Addgene #46920
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 EcoRI (106) HpaI (10,202) BspDI - ClaI (9640) AgeI (9540) SwaI (7493) PmlI (7469) AhdI (6536) PstI (5238) SpeI (117) SnaBI (166) NcoI (835) SgrDI (1203) BlpI (1516) BstAPI (1617) AfeI (2297) MluI (4670) BamHI (4980) PaeR7I - XhoI (4992) BstZ17I (5057) SphI (5189) XbaI (5223) pTDH3-dCas9 11,057 bp
EcoRI  (106)
1 site
G A A T T C C T T A A G
HpaI  (10,202)
1 site
G T T A A C C A A T T G
BspDI  (9640)
1 site
A T C G A T T A G C T A
ClaI  (9640)
1 site
A T C G A T T A G C T A
AgeI  (9540)
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.
SwaI  (7493)
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.
PmlI  (7469)
1 site
C A C G T G G T G C A C

PmlI gradually loses activity when stored at -20°C.
AhdI  (6536)
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.
PstI  (5238)
1 site
C T G C A G G A C G T C
SpeI  (117)
1 site
A C T A G T T G A T C A
SnaBI  (166)
1 site
T A C G T A A T G C A T
NcoI  (835)
1 site
C C A T G G G G T A C C
SgrDI  (1203)
1 site
C G T C G A C G G C A G C T G C
BlpI  (1516)
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.
BstAPI  (1617)
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.
AfeI  (2297)
1 site
A G C G C T T C G C G A
MluI  (4670)
1 site
A C G C G T T G C G C A
BamHI  (4980)
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.
PaeR7I  (4992)
1 site
C T C G A G G A G C T C

PaeR7I does not recognize the sequence CTCTCGAG.
XhoI  (4992)
1 site
C T C G A G G A G C T C
BstZ17I  (5057)
1 site
G T A T A C C A T A T G
SphI  (5189)
1 site
G C A T G C C G T A C G
XbaI  (5223)
1 site
T C T A G A A G A T C T
dCas9
837 .. 4940  =  4104 bp
1368 amino acids  =  158.3 kDa
   Segment 1:  
   837 .. 863  =  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
837 .. 4940  =  4104 bp
1368 amino acids  =  158.3 kDa
   Segment 2:  
   864 .. 866  =  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
837 .. 4940  =  4104 bp
1368 amino acids  =  158.3 kDa
   Segment 3:  
   867 .. 3353  =  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
837 .. 4940  =  4104 bp
1368 amino acids  =  158.3 kDa
   Segment 4:  
   3354 .. 3356  =  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
837 .. 4940  =  4104 bp
1368 amino acids  =  158.3 kDa
   Segment 5:  
   3357 .. 4940  =  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
837 .. 4940  =  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
LEU2
8700 .. 9794  =  1095 bp
364 amino acids  =  39.0 kDa
Product: 3-isopropylmalate dehydrogenase, required
for leucine biosynthesis
yeast auxotrophic marker
LEU2
8700 .. 9794  =  1095 bp
364 amino acids  =  39.0 kDa
Product: 3-isopropylmalate dehydrogenase, required
for leucine biosynthesis
yeast auxotrophic marker
AmpR
6463 .. 7323  =  861 bp
286 amino acids  =  31.6 kDa
   Segment 2:  
   6463 .. 7254  =  792 bp
   263 amino acids  =  29.0 kDa
Product: β-lactamase
confers resistance to ampicillin, carbenicillin, and
related antibiotics
AmpR
6463 .. 7323  =  861 bp
286 amino acids  =  31.6 kDa
   Segment 1:  signal sequence  
   7255 .. 7323  =  69 bp
   23 amino acids  =  2.6 kDa
Product: β-lactamase
confers resistance to ampicillin, carbenicillin, and
related antibiotics
AmpR
6463 .. 7323  =  861 bp
286 amino acids  =  31.6 kDa
2 segments
Product: β-lactamase
confers resistance to ampicillin, carbenicillin, and
related antibiotics
GAP promoter
136 .. 802  =  667 bp
promoter for glyceraldehyde-3-phosphate
dehydrogenase
GAP promoter
136 .. 802  =  667 bp
promoter for glyceraldehyde-3-phosphate
dehydrogenase
ori
5704 .. 6292  =  589 bp
high-copy-number ColE1/pMB1/pBR322/pUC origin
of replication
ori
5704 .. 6292  =  589 bp
high-copy-number ColE1/pMB1/pBR322/pUC origin
of replication
CEN/ARS
7465 .. 7968  =  504 bp
S. cerevisiae CEN6 centromere fused to an
autonomously replicating sequence
CEN/ARS
7465 .. 7968  =  504 bp
S. cerevisiae CEN6 centromere fused to an
autonomously replicating sequence
f1 ori
10,502 .. 10,957  =  456 bp
f1 bacteriophage origin of replication; arrow
indicates direction of (+) strand synthesis
f1 ori
10,502 .. 10,957  =  456 bp
f1 bacteriophage origin of replication; arrow
indicates direction of (+) strand synthesis
LEU2 promoter
9795 .. 10,202  =  408 bp
LEU2 promoter
9795 .. 10,202  =  408 bp
ADH1 terminator
4998 .. 5185  =  188 bp
transcription terminator for alcohol dehydrogenase 1
ADH1 terminator
4998 .. 5185  =  188 bp
transcription terminator for alcohol dehydrogenase 1
AmpR promoter
7324 .. 7428  =  105 bp
AmpR promoter
7324 .. 7428  =  105 bp
SV40 NLS
4950 .. 4970  =  21 bp
7 amino acids  =  883.1 Da
Product: nuclear localization signal of SV40 large T
antigen
SV40 NLS
4950 .. 4970  =  21 bp
7 amino acids  =  883.1 Da
Product: nuclear localization signal of SV40 large T
antigen
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