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pMZ374

Fission yeast plasmid for expressing S. pyogenes Cas9 from the adh1 promoter plus a single guide RNA (sgRNA) from the rrk1 promoter/leader.

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pMZ374.dna
Map and Sequence File:    Download    Open   
Sequence Author:  Zaratiegui Lab / Addgene #59896
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CspCI (3567) CspCI (3601) pMZ374 11,384 bp
CspCI  (3567)
2 sites		 ( N ) 10-11 C A A N N N N N G T G G ( N ) 10-11 N N N N ( N ) 10-11 G T T N N N N N C A C C ( N ) 10-11
Efficient cleavage requires at least two copies of the CspCI recognition sequence.
CspCI makes two double-stranded cuts.
Sticky ends from different CspCI sites may not be compatible.
For full activity, add fresh S-adenosylmethionine (SAM).
CspCI  (3601)
2 sites		 ( N ) 10-11 C A A N N N N N G T G G ( N ) 10-11 N N N N ( N ) 10-11 G T T N N N N N C A C C ( N ) 10-11
Efficient cleavage requires at least two copies of the CspCI recognition sequence.
CspCI makes two double-stranded cuts.
Sticky ends from different CspCI sites may not be compatible.
For full activity, add fresh S-adenosylmethionine (SAM).
Cas9
6911 .. 11,014  =  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
6911 .. 11,014  =  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
5155 .. 6015  =  861 bp
286 amino acids  =  31.6 kDa
   Segment 2:  
   5155 .. 5946  =  792 bp
   263 amino acids  =  28.9 kDa
Product: β-lactamase
confers resistance to ampicillin, carbenicillin, and related antibiotics
AmpR
5155 .. 6015  =  861 bp
286 amino acids  =  31.6 kDa
   Segment 1:  signal sequence  
   5947 .. 6015  =  69 bp
   23 amino acids  =  2.6 kDa
Product: β-lactamase
confers resistance to ampicillin, carbenicillin, and related antibiotics
AmpR
5155 .. 6015  =  861 bp
286 amino acids  =  31.6 kDa
2 segments
Product: β-lactamase
confers resistance to ampicillin, carbenicillin, and related antibiotics
S. pombe ura4
1930 .. 2724  =  795 bp
264 amino acids  =  29.4 kDa
Product: orotidine 5'-phosphate decarboxylase, required for uracil biosynthesis
S. pombe ura4
1930 .. 2724  =  795 bp
264 amino acids  =  29.4 kDa
Product: orotidine 5'-phosphate decarboxylase, required for uracil biosynthesis
ars1
539 .. 1320  =  782 bp
Schizosaccharomyces pombe autonomously replicating sequence ars1
ars1
539 .. 1320  =  782 bp
Schizosaccharomyces pombe autonomously replicating sequence ars1
adh1 promoter
6149 .. 6890  =  742 bp
promoter for the Schizosaccharomyces pombe adh1 gene encoding alcohol dehydrogenase
adh1 promoter
6149 .. 6890  =  742 bp
promoter for the Schizosaccharomyces pombe adh1 gene encoding alcohol dehydrogenase
ori
4396 .. 4984  =  589 bp
high-copy-number ColE1/pMB1/pBR322/pUC origin of replication
ori
4396 .. 4984  =  589 bp
high-copy-number ColE1/pMB1/pBR322/pUC origin of replication
rrk1 promoter/leader
3601 .. 3957  =  357 bp
promoter and leader RNA for the Schizosaccharomyces pombe rrk1 gene encoding the K-RNA component of RNase P (Jacobs et al., 2014)
rrk1 promoter/leader
3601 .. 3957  =  357 bp
promoter and leader RNA for the Schizosaccharomyces pombe rrk1 gene encoding the K-RNA component of RNase P (Jacobs et al., 2014)
CYC1 terminator
11,063 .. 11,310  =  248 bp
transcription terminator for CYC1
CYC1 terminator
11,063 .. 11,310  =  248 bp
transcription terminator for CYC1
AmpR promoter
6016 .. 6120  =  105 bp
AmpR promoter
6016 .. 6120  =  105 bp
gRNA scaffold
3483 .. 3566  =  84 bp
guide RNA scaffold for the Streptococcus pyogenes CRISPR/Cas9 system
gRNA scaffold
3483 .. 3566  =  84 bp
guide RNA scaffold for the Streptococcus pyogenes CRISPR/Cas9 system
sTRSV HHRz
3431 .. 3482  =  52 bp
hammerhead ribozyme from the tobacco ringspot virus satellite RNA (Khvorova et al., 2003)
sTRSV HHRz
3431 .. 3482  =  52 bp
hammerhead ribozyme from the tobacco ringspot virus satellite RNA (Khvorova et al., 2003)
SV40 NLS
11,027 .. 11,047  =  21 bp
7 amino acids  =  883.1 Da
Product: nuclear localization signal of SV40 large T antigen
SV40 NLS
11,027 .. 11,047  =  21 bp
7 amino acids  =  883.1 Da
Product: nuclear localization signal of SV40 large T antigen
ORF:  1930 .. 2724  =  795 bp
ORF:  264 amino acids  =  29.4 kDa
ORF:  3172 .. 3543  =  372 bp
ORF:  123 amino acids  =  14.1 kDa
ORF:  6640 .. 6879  =  240 bp
ORF:  79 amino acids  =  8.9 kDa
ORF:  5285 .. 5551  =  267 bp
ORF:  88 amino acids  =  9.2 kDa
ORF:  6911 .. 11,050  =  4140 bp
ORF:  1379 amino acids  =  159.7 kDa
ORF:  6905 .. 7390  =  486 bp
ORF:  161 amino acids  =  18.0 kDa
ORF:  8315 .. 8833  =  519 bp
ORF:  172 amino acids  =  20.1 kDa
ORF:  8897 .. 9430  =  534 bp
ORF:  177 amino acids  =  20.0 kDa
ORF:  10,424 .. 10,957  =  534 bp
ORF:  177 amino acids  =  20.0 kDa
ORF:  2431 .. 2721  =  291 bp
ORF:  96 amino acids  =  10.7 kDa
ORF:  5155 .. 6015  =  861 bp
ORF:  286 amino acids  =  31.6 kDa
ORF:  6778 .. 7053  =  276 bp
ORF:  91 amino acids  =  10.4 kDa
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