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Plasmid Files

p415-GalL-Cas9-CYC1t

Yeast centromeric plasmid for expressing human codon-optimized Cas9 with the inducible GAL1 promoter.

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p415-GalL-Cas9-CYC1t Sequence and Mapp415-GalL-Cas9-CYC1t.dna
Map and Sequence File   
Sequence Author:  Church Lab / Addgene #43804
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 BtgI (9897) BspDI - ClaI (9188) BstEII (9070) HpaI (8619) DraIII (8098) MluI (7582) AscI (7213) AarI (6518) NruI (6498) FspAI (6208) BmgBI (5768) SwaI (544) AhdI (1502) BstAPI (3093) XbaI (3228) SpeI (3234) SbfI (3505) NdeI (3736) SalI (3798) BlpI (4206) Bsu36I (4524) p415-GalL-Cas9-CYC1t 10,793 bp
BtgI  (9897)
1 site
C C R Y G G G G Y R C C

Sticky ends from different BtgI sites may not be compatible.
BspDI  (9188)
1 site
A T C G A T T A G C T A
ClaI  (9188)
1 site
A T C G A T T A G C T A
BstEII  (9070)
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.
HpaI  (8619)
1 site
G T T A A C C A A T T G
DraIII  (8098)
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.
MluI  (7582)
1 site
A C G C G T T G C G C A
AscI  (7213)
1 site
G G C G C G C C C C G C G C G G
AarI  (6518)
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.
NruI  (6498)
1 site
T C G C G A A G C G C T
FspAI  (6208)
1 site
R T G C G C A Y Y A C G C G T R
BmgBI  (5768)
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.
SwaI  (544)
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.
AhdI  (1502)
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.
BstAPI  (3093)
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.
XbaI  (3228)
1 site
T C T A G A A G A T C T
SpeI  (3234)
1 site
A C T A G T T G A T C A
SbfI  (3505)
1 site
C C T G C A G G G G A C G T C C
NdeI  (3736)
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.
SalI  (3798)
1 site
G T C G A C C A G C T G
BlpI  (4206)
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  (4524)
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.
Cas9
3258 .. 7361  =  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
3258 .. 7361  =  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
LEU2
9037 .. 10,131  =  1095 bp
364 amino acids  =  39.0 kDa
Product: 3-isopropylmalate dehydrogenase, required
for leucine biosynthesis
yeast auxotrophic marker
LEU2
9037 .. 10,131  =  1095 bp
364 amino acids  =  39.0 kDa
Product: 3-isopropylmalate dehydrogenase, required
for leucine biosynthesis
yeast auxotrophic marker
AmpR
715 .. 1575  =  861 bp
286 amino acids  =  31.6 kDa
   Segment 1:  signal sequence  
   715 .. 783  =  69 bp
   23 amino acids  =  2.6 kDa
Product: β-lactamase
confers resistance to ampicillin, carbenicillin, and
related antibiotics
AmpR
715 .. 1575  =  861 bp
286 amino acids  =  31.6 kDa
   Segment 2:  
   784 .. 1575  =  792 bp
   263 amino acids  =  28.9 kDa
Product: β-lactamase
confers resistance to ampicillin, carbenicillin, and
related antibiotics
AmpR
715 .. 1575  =  861 bp
286 amino acids  =  31.6 kDa
2 segments
Product: β-lactamase
confers resistance to ampicillin, carbenicillin, and
related antibiotics
ori
1746 .. 2334  =  589 bp
high-copy-number ColE1/pMB1/pBR322/pUC origin
of replication
ori
1746 .. 2334  =  589 bp
high-copy-number ColE1/pMB1/pBR322/pUC origin
of replication
CEN/ARS
70 .. 573  =  504 bp
S. cerevisiae CEN6 centromere fused to an
autonomously replicating sequence
CEN/ARS
70 .. 573  =  504 bp
S. cerevisiae CEN6 centromere fused to an
autonomously replicating sequence
f1 ori
7865 .. 8320  =  456 bp
f1 bacteriophage origin of replication; arrow
indicates direction of (+) strand synthesis
f1 ori
7865 .. 8320  =  456 bp
f1 bacteriophage origin of replication; arrow
indicates direction of (+) strand synthesis
GAL1 promoter
2794 .. 3220  =  427 bp
inducible promoter, regulated by Gal4
GAL1 promoter
2794 .. 3220  =  427 bp
inducible promoter, regulated by Gal4
LEU2 promoter
8620 .. 9024  =  405 bp
LEU2 promoter
8620 .. 9024  =  405 bp
AmpR promoter
610 .. 714  =  105 bp
AmpR promoter
610 .. 714  =  105 bp
SV40 NLS
7374 .. 7394  =  21 bp
7 amino acids  =  883.1 Da
Product: nuclear localization signal of SV40 large T
antigen
SV40 NLS
7374 .. 7394  =  21 bp
7 amino acids  =  883.1 Da
Product: nuclear localization signal of SV40 large T
antigen
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