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Plasmid Files

p414-TEF1p-Cas9-CYC1t

Yeast centromeric plasmid for expressing human codon-optimized Cas9 with the constitutive TEF1 promoter.

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p414-TEF1p-Cas9-CYC1t Sequence and Mapp414-TEF1p-Cas9-CYC1t.dna
Map and Sequence File   
Sequence Author:  Church Lab / Addgene #43802
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 BstZ17I (9090) BstAPI (8981) MfeI (8913) DraIII (8062) KpnI (7630) Acc65I (7626) MluI (7546) BsrGI (7539) AscI (7177) EcoRI (7044) NruI (6462) FspAI (6172) BmgBI (5732) SnaBI (9205) SwaI (543) AhdI (1501) SpeI (3200) SbfI (3469) XcmI (3573) NdeI (3700) SalI (3762) BlpI (4170) p414-TEF1p-Cas9-CYC1t 9524 bp
BstZ17I  (9090)
1 site
G T A T A C C A T A T G
BstAPI  (8981)
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.
MfeI  (8913)
1 site
C A A T T G G T T A A C
DraIII  (8062)
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.
KpnI  (7630)
1 site
G G T A C C C C A T G G
Acc65I  (7626)
1 site
G G T A C C C C A T G G
MluI  (7546)
1 site
A C G C G T T G C G C A
BsrGI  (7539)
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.
AscI  (7177)
1 site
G G C G C G C C C C G C G C G G
EcoRI  (7044)
1 site
G A A T T C C T T A A G
NruI  (6462)
1 site
T C G C G A A G C G C T
FspAI  (6172)
1 site
R T G C G C A Y Y A C G C G T R
BmgBI  (5732)
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.
SnaBI  (9205)
1 site
T A C G T A A T G C A T
SwaI  (543)
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  (1501)
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.
SpeI  (3200)
1 site
A C T A G T T G A T C A
SbfI  (3469)
1 site
C C T G C A G G G G A C G T C C
XcmI  (3573)
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.
NdeI  (3700)
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  (3762)
1 site
G T C G A C C A G C T G
BlpI  (4170)
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.
Cas9
3222 .. 7325  =  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
3222 .. 7325  =  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
AmpR
714 .. 1574  =  861 bp
286 amino acids  =  31.6 kDa
   Segment 1:  signal sequence  
   714 .. 782  =  69 bp
   23 amino acids  =  2.6 kDa
Product: β-lactamase
confers resistance to ampicillin, carbenicillin, and
related antibiotics
AmpR
714 .. 1574  =  861 bp
286 amino acids  =  31.6 kDa
   Segment 2:  
   783 .. 1574  =  792 bp
   263 amino acids  =  28.9 kDa
Product: β-lactamase
confers resistance to ampicillin, carbenicillin, and
related antibiotics
AmpR
714 .. 1574  =  861 bp
286 amino acids  =  31.6 kDa
2 segments
Product: β-lactamase
confers resistance to ampicillin, carbenicillin, and
related antibiotics
TRP1
8383 .. 9057  =  675 bp
224 amino acids  =  24.1 kDa
Product: phosphoribosylanthranilate isomerase,
required for tryptophan biosynthesis
yeast auxotrophic marker
TRP1
8383 .. 9057  =  675 bp
224 amino acids  =  24.1 kDa
Product: phosphoribosylanthranilate isomerase,
required for tryptophan biosynthesis
yeast auxotrophic marker
ori
1745 .. 2333  =  589 bp
high-copy-number ColE1/pMB1/pBR322/pUC origin
of replication
ori
1745 .. 2333  =  589 bp
high-copy-number ColE1/pMB1/pBR322/pUC origin
of replication
CEN/ARS
69 .. 572  =  504 bp
S. cerevisiae CEN6 centromere fused to an
autonomously replicating sequence
CEN/ARS
69 .. 572  =  504 bp
S. cerevisiae CEN6 centromere fused to an
autonomously replicating sequence
f1 ori
7829 .. 8284  =  456 bp
f1 bacteriophage origin of replication; arrow
indicates direction of (+) strand synthesis
f1 ori
7829 .. 8284  =  456 bp
f1 bacteriophage origin of replication; arrow
indicates direction of (+) strand synthesis
TEF1 promoter
2792 .. 3190  =  399 bp
promoter for EF-1α
TEF1 promoter
2792 .. 3190  =  399 bp
promoter for EF-1α
TRP1 promoter
9058 .. 9338  =  281 bp
TRP1 promoter
9058 .. 9338  =  281 bp
CYC1 terminator
7374 .. 7621  =  248 bp
transcription terminator for CYC1
CYC1 terminator
7374 .. 7621  =  248 bp
transcription terminator for CYC1
AmpR promoter
609 .. 713  =  105 bp
AmpR promoter
609 .. 713  =  105 bp
SV40 NLS
7338 .. 7358  =  21 bp
7 amino acids  =  883.1 Da
Product: nuclear localization signal of SV40 large T
antigen
SV40 NLS
7338 .. 7358  =  21 bp
7 amino acids  =  883.1 Da
Product: nuclear localization signal of SV40 large T
antigen
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