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Plasmid Files

pTPGI_dCas9_VP64

Yeast plasmid for inducible expression of catalytically inactive S. pyogenes dCas9 fused to the VP64 transcriptional activation domain.

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pTPGI_dCas9_VP64 Sequence and MappTPGI_dCas9_VP64.dna
Map and Sequence File   
Sequence Author:  Lu Lab / Addgene #49013
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 ZraI (10,920) AhdI (10,000) PciI (9107) KpnI (8713) Acc65I (8709) MluI (8628) BsrGI (8621) PaeR7I - PspXI - XhoI (8450) AvrII (8442) SalI (8273) KflI (7528) AatII (10,922) PfoI (46) BspEI * (332) PmlI (427) MfeI (607) Bsu36I (709) EcoRV (752) AarI (900) HindIII (980) NgoMIV (1566) NaeI (1568) PacI (2078) Eco53kI (2084) SacI (2086) AflII (2087) NdeI (2129) AfeI (2306) NsiI (2325) NruI (2430) EcoNI (2501) SpeI (2719) EagI - NotI (3134) NcoI (3157) PspOMI * (3326) ApaI * (3330) StuI (3388) EcoRI (3631) BseRI (3680) AgeI (3712) PstI (3914) BamHI (4094) ATG TspMI - XmaI (4148) SmaI (4150) SexAI * (4837) pTPGI_dCas9_VP64 10,987 bp
ZraI  (10,920)
1 site
G A C G T C C T G C A G
AhdI  (10,000)
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.
PciI  (9107)
1 site
A C A T G T T G T A C A

PciI is inhibited by nonionic detergents.
KpnI  (8713)
1 site
G G T A C C C C A T G G
Acc65I  (8709)
1 site
G G T A C C C C A T G G
MluI  (8628)
1 site
A C G C G T T G C G C A
BsrGI  (8621)
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.
PaeR7I  (8450)
1 site
C T C G A G G A G C T C

PaeR7I does not recognize the sequence CTCTCGAG.
PspXI  (8450)
1 site
V C T C G A G B B G A G C T C V
XhoI  (8450)
1 site
C T C G A G G A G C T C
AvrII  (8442)
1 site
C C T A G G G G A T C C
SalI  (8273)
1 site
G T C G A C C A G C T G
KflI  (7528)
1 site
G G G W C C C C C C W G G G

Sticky ends from different KflI sites may not be compatible.
AatII  (10,922)
1 site
G A C G T C C T G C A G
PfoI  (46)
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.
BspEI  (332)
1 site
T C C G G A A G G C C T
* Blocked by Dam methylation.
PmlI  (427)
1 site
C A C G T G G T G C A C

PmlI gradually loses activity when stored at -20°C.
MfeI  (607)
1 site
C A A T T G G T T A A C
Bsu36I  (709)
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.
EcoRV  (752)
1 site
G A T A T C C T A T A G

EcoRV is reportedly more prone than its isoschizomer Eco32I to
delete a base after cleavage.
AarI  (900)
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.
HindIII  (980)
1 site
A A G C T T T T C G A A
NgoMIV  (1566)
1 site
G C C G G C C G G C C G

Efficient cleavage requires at least two copies of the NgoMIV
recognition sequence.
NaeI  (1568)
1 site
G C C G G C C G G C C G

Efficient cleavage requires at least two copies of the NaeI
recognition sequence.
PacI  (2078)
1 site
T T A A T T A A A A T T A A T T
Eco53kI  (2084)
1 site
G A G C T C C T C G A G
SacI  (2086)
1 site
G A G C T C C T C G A G
AflII  (2087)
1 site
C T T A A G G A A T T C

The sticky ends produced by AflII are hard to ligate.
NdeI  (2129)
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.
AfeI  (2306)
1 site
A G C G C T T C G C G A
NsiI  (2325)
1 site
A T G C A T T A C G T A
NruI  (2430)
1 site
T C G C G A A G C G C T
EcoNI  (2501)
1 site
C C T N N N N N A G G G G A N N N N N T C C

The 1-base overhangs produced by EcoNI may be hard to ligate.
Sticky ends from different EcoNI sites may not be compatible.
SpeI  (2719)
1 site
A C T A G T T G A T C A
EagI  (3134)
1 site
C G G C C G G C C G G C
NotI  (3134)
1 site
G C G G C C G C C G C C G G C G
NcoI  (3157)
1 site
C C A T G G G G T A C C
PspOMI  (3326)
1 site
G G G C C C C C C G G G
* Blocked by Dcm methylation.
ApaI  (3330)
1 site
G G G C C C C C C G G G
* Blocked by Dcm methylation.
ApaI can be used between 25°C and 37°C.
StuI  (3388)
1 site
A G G C C T T C C G G A
EcoRI  (3631)
1 site
G A A T T C C T T A A G
BseRI  (3680)
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.
AgeI  (3712)
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.
PstI  (3914)
1 site
C T G C A G G A C G T C
BamHI  (4094)
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.
TspMI  (4148)
1 site
C C C G G G G G G C C C
XmaI  (4148)
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.
SmaI  (4150)
1 site
C C C G G G G G G C C C

SmaI can be used at 37°C for brief incubations.
SexAI  (4837)
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.
dCas9
4154 .. 8254  =  4101 bp
1367 amino acids  =  158.2 kDa
   Segment 1:  
   4154 .. 4177  =  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
4154 .. 8254  =  4101 bp
1367 amino acids  =  158.2 kDa
   Segment 2:  
   4178 .. 4180  =  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
4154 .. 8254  =  4101 bp
1367 amino acids  =  158.2 kDa
   Segment 3:  
   4181 .. 6667  =  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
4154 .. 8254  =  4101 bp
1367 amino acids  =  158.2 kDa
   Segment 4:  
   6668 .. 6670  =  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
4154 .. 8254  =  4101 bp
1367 amino acids  =  158.2 kDa
   Segment 5:  
   6671 .. 8254  =  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
4154 .. 8254  =  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
9927 .. 10,787  =  861 bp
286 amino acids  =  31.6 kDa
   Segment 2:  
   9927 .. 10,718  =  792 bp
   263 amino acids  =  28.9 kDa
Product: β-lactamase
confers resistance to ampicillin, carbenicillin, and
related antibiotics
AmpR
9927 .. 10,787  =  861 bp
286 amino acids  =  31.6 kDa
   Segment 1:  signal sequence  
   10,719 .. 10,787  =  69 bp
   23 amino acids  =  2.6 kDa
Product: β-lactamase
confers resistance to ampicillin, carbenicillin, and
related antibiotics
AmpR
9927 .. 10,787  =  861 bp
286 amino acids  =  31.6 kDa
2 segments
Product: β-lactamase
confers resistance to ampicillin, carbenicillin, and
related antibiotics
TRP1
468 .. 1142  =  675 bp
224 amino acids  =  24.1 kDa
Product: phosphoribosylanthranilate isomerase,
required for tryptophan biosynthesis
yeast auxotrophic marker
TRP1
468 .. 1142  =  675 bp
224 amino acids  =  24.1 kDa
Product: phosphoribosylanthranilate isomerase,
required for tryptophan biosynthesis
yeast auxotrophic marker
TetR
2088 .. 2711  =  624 bp
207 amino acids  =  23.4 kDa
Product: tetracycline repressor TetR
TetR binds to the tetracycline operator tetO to
inhibit transcription. This inhibition can be relieved
by adding tetracycline or doxycycline.
TetR
2088 .. 2711  =  624 bp
207 amino acids  =  23.4 kDa
Product: tetracycline repressor TetR
TetR binds to the tetracycline operator tetO to
inhibit transcription. This inhibition can be relieved
by adding tetracycline or doxycycline.
ori
9168 .. 9756  =  589 bp
high-copy-number ColE1/pMB1/pBR322/pUC origin
of replication
ori
9168 .. 9756  =  589 bp
high-copy-number ColE1/pMB1/pBR322/pUC origin
of replication
f1 ori
1241 .. 1696  =  456 bp
f1 bacteriophage origin of replication; arrow
indicates direction of (+) strand synthesis
f1 ori
1241 .. 1696  =  456 bp
f1 bacteriophage origin of replication; arrow
indicates direction of (+) strand synthesis
TPGI promoter
3643 .. 4084  =  442 bp
S. cerevisiae GAL1 promoter modified to contain two
copies of the tet operator (Ellis et al., 2009)
TPGI promoter
3643 .. 4084  =  442 bp
S. cerevisiae GAL1 promoter modified to contain two
copies of the tet operator (Ellis et al., 2009)
TEF1 promoter
2729 .. 3127  =  399 bp
promoter for EF-1α
TEF1 promoter
2729 .. 3127  =  399 bp
promoter for EF-1α
TRP1 promoter
187 .. 467  =  281 bp
TRP1 promoter
187 .. 467  =  281 bp
CYC1 terminator
8456 .. 8703  =  248 bp
transcription terminator for CYC1
CYC1 terminator
8456 .. 8703  =  248 bp
transcription terminator for CYC1
VP64
8291 .. 8440  =  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
8291 .. 8440  =  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)
AmpR promoter
10,788 .. 10,892  =  105 bp
AmpR promoter
10,788 .. 10,892  =  105 bp
SV40 NLS
4124 .. 4144  =  21 bp
7 amino acids  =  883.1 Da
Product: nuclear localization signal of SV40 large T
antigen
SV40 NLS
4124 .. 4144  =  21 bp
7 amino acids  =  883.1 Da
Product: nuclear localization signal of SV40 large T
antigen
ATG
4112 .. 4114  =  3 bp
1 amino acid  =  149.2 Da
Product: start codon
ATG
4112 .. 4114  =  3 bp
1 amino acid  =  149.2 Da
Product: start codon
UAS
3643 .. 3760  =  118 bp
upstream activating sequence mediating
Gal4-dependent induction
UAS
3643 .. 3760  =  118 bp
upstream activating sequence mediating
Gal4-dependent induction
tet operator
3968 .. 3986  =  19 bp
bacterial operator O2 for the tetR and tetA genes
tet operator
3968 .. 3986  =  19 bp
bacterial operator O2 for the tetR and tetA genes
tet operator
3989 .. 4007  =  19 bp
bacterial operator O2 for the tetR and tetA genes
tet operator
3989 .. 4007  =  19 bp
bacterial operator O2 for the tetR and tetA genes
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