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Plasmid Files

pACT2-CAS9c

High-copy yeast plasmid for expressing S. pyogenes Cas9 under control of the ADH1 promoter and terminator.

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pACT2-CAS9c Sequence and MappACT2-CAS9c.dna
Map and Sequence File   
Sequence Author:  Zhao Lab / Addgene #51055
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 SpeI (11,278) XbaI (10,894) SnaBI (10,557) HpaI (9915) BstEII (8983) BspDI - ClaI (8868) KpnI (8775) Acc65I (8771) AgeI (8768) BsrGI (7897) DraIII (7458) AscI - BssHII (6573) EcoRI (6440) PaeR7I - XhoI (6161) FspAI (5568) BglI (800) AhdI (918) BspQI - SapI (1923) SbfI (2865) SalI (3158) Bsu36I (3884) TspMI - XmaI (3984) SmaI (3986) Eco53kI (4186) SacI (4188) BamHI (4623) NgoMIV (5357) NaeI (5359) PmlI (5407) pACT2-CAS9c 11,495 bp
SpeI  (11,278)
1 site
A C T A G T T G A T C A
XbaI  (10,894)
1 site
T C T A G A A G A T C T
SnaBI  (10,557)
1 site
T A C G T A A T G C A T
HpaI  (9915)
1 site
G T T A A C C A A T T G
BstEII  (8983)
1 site
G G T N A C C C C A N T G G

Sticky ends from different BstEII sites may not be compatible.
BstEII is typically used at 60°C, but is 50% active at 37°C.
BspDI  (8868)
1 site
A T C G A T T A G C T A
ClaI  (8868)
1 site
A T C G A T T A G C T A
KpnI  (8775)
1 site
G G T A C C C C A T G G
Acc65I  (8771)
1 site
G G T A C C C C A T G G
AgeI  (8768)
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.
BsrGI  (7897)
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.
DraIII  (7458)
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.
AscI  (6573)
1 site
G G C G C G C C C C G C G C G G
BssHII  (6573)
1 site
G C G C G C C G C G C G

BssHII is typically used at 50°C, but is 75% active at 37°C.
EcoRI  (6440)
1 site
G A A T T C C T T A A G
PaeR7I  (6161)
1 site
C T C G A G G A G C T C

PaeR7I does not recognize the sequence CTCTCGAG.
XhoI  (6161)
1 site
C T C G A G G A G C T C
FspAI  (5568)
1 site
R T G C G C A Y Y A C G C G T R
BglI  (800)
1 site
G C C N N N N N G G C C G G N N N N N C C G

Sticky ends from different BglI sites may not be compatible.
AhdI  (918)
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.
BspQI  (1923)
1 site
G C T C T T C N C G A G A A G N N N N

Sticky ends from different BspQI sites may not be compatible.
SapI  (1923)
1 site
G C T C T T C N C G A G A A G N N N N

Sticky ends from different SapI sites may not be compatible.
SapI gradually settles in solution, so a tube of SapI should be
mixed before removing an aliquot.
SbfI  (2865)
1 site
C C T G C A G G G G A C G T C C
SalI  (3158)
1 site
G T C G A C C A G C T G
Bsu36I  (3884)
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.
TspMI  (3984)
1 site
C C C G G G G G G C C C
XmaI  (3984)
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  (3986)
1 site
C C C G G G G G G C C C

SmaI can be used at 37°C for brief incubations.
Eco53kI  (4186)
1 site
G A G C T C C T C G A G
SacI  (4188)
1 site
G A G C T C C T C G A G
BamHI  (4623)
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.
NgoMIV  (5357)
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  (5359)
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.
PmlI  (5407)
1 site
C A C G T G G T G C A C

PmlI gradually loses activity when stored at -20°C.
Cas9
2618 .. 6721  =  4104 bp
1368 amino acids  =  158.4 kDa
Product: Cas9 (Csn1) endonuclease from the
Streptococcus pyogenes Type II CRISPR/Cas system
generates RNA-guided double strand breaks in DNA
Cas9
2618 .. 6721  =  4104 bp
1368 amino acids  =  158.4 kDa
Product: Cas9 (Csn1) endonuclease from the
Streptococcus pyogenes Type II CRISPR/Cas system
generates RNA-guided double strand breaks in DNA
2μ ori
9916 .. 11,258  =  1343 bp
yeast 2μ plasmid origin of replication
2μ ori
9916 .. 11,258  =  1343 bp
yeast 2μ plasmid origin of replication
LEU2
7928 .. 9022  =  1095 bp
364 amino acids  =  39.0 kDa
Product: 3-isopropylmalate dehydrogenase, required
for leucine biosynthesis
yeast auxotrophic marker
LEU2
7928 .. 9022  =  1095 bp
364 amino acids  =  39.0 kDa
Product: 3-isopropylmalate dehydrogenase, required
for leucine biosynthesis
yeast auxotrophic marker
AmpR
131 .. 991  =  861 bp
286 amino acids  =  31.5 kDa
   Segment 1:  signal sequence  
   131 .. 199  =  69 bp
   23 amino acids  =  2.6 kDa
Product: β-lactamase
confers resistance to ampicillin, carbenicillin, and
related antibiotics
AmpR
131 .. 991  =  861 bp
286 amino acids  =  31.5 kDa
   Segment 2:  
   200 .. 991  =  792 bp
   263 amino acids  =  28.9 kDa
Product: β-lactamase
confers resistance to ampicillin, carbenicillin, and
related antibiotics
AmpR
131 .. 991  =  861 bp
286 amino acids  =  31.5 kDa
2 segments
Product: β-lactamase
confers resistance to ampicillin, carbenicillin, and
related antibiotics
ori
1162 .. 1750  =  589 bp
high-copy-number ColE1/pMB1/pBR322/pUC origin
of replication
ori
1162 .. 1750  =  589 bp
high-copy-number ColE1/pMB1/pBR322/pUC origin
of replication
LEU2 promoter
9035 .. 9439  =  405 bp
LEU2 promoter
9035 .. 9439  =  405 bp
ADH1 promoter
2218 .. 2614  =  397 bp
promoter for alcohol dehydrogenase 1
ADH1 promoter
2218 .. 2614  =  397 bp
promoter for alcohol dehydrogenase 1
ADH1 terminator
6890 .. 7077  =  188 bp
transcription terminator for alcohol dehydrogenase 1
ADH1 terminator
6890 .. 7077  =  188 bp
transcription terminator for alcohol dehydrogenase 1
AmpR promoter
26 .. 130  =  105 bp
AmpR promoter
26 .. 130  =  105 bp
SV40 NLS
6734 .. 6754  =  21 bp
7 amino acids  =  883.1 Da
Product: nuclear localization signal of SV40 large T
antigen
SV40 NLS
6734 .. 6754  =  21 bp
7 amino acids  =  883.1 Da
Product: nuclear localization signal of SV40 large T
antigen
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