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pCRCT

High-copy yeast plasmid for disrupting genes by expressing S. pyogenes Cas9 together with a tracrRNA and one or more CRISPR RNAs (crRNAs).

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pCRCT.dna
Map and Sequence File:    Download    Open   
Sequence Author:  Zhao Lab / Addgene #60621
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BsaI (1245) BsaI (1704) pCRCT 10,805 bp
BsaI  (1245)
2 sites		 G G T C T C N C C A G A G N ( N ) 4
Sticky ends from different BsaI sites may not be compatible.
BsaI can be used between 37°C and 50°C.
BsaI  (1704)
2 sites		 G G T C T C N C C A G A G N ( N ) 4
Sticky ends from different BsaI sites may not be compatible.
BsaI can be used between 37°C and 50°C.
iCas9
2215 .. 6315  =  4101 bp
1367 amino acids  =  158.4 kDa
Product: improved Cas9 (Csn1) endonuclease from the Streptococcus pyogenes Type II CRISPR/Cas system; includes the accidental D147Y and P411T mutations
generates RNA-guided double strand breaks in DNA
iCas9
2215 .. 6315  =  4101 bp
1367 amino acids  =  158.4 kDa
Product: improved Cas9 (Csn1) endonuclease from the Streptococcus pyogenes Type II CRISPR/Cas system; includes the accidental D147Y and P411T mutations
generates RNA-guided double strand breaks in DNA
2μ ori
9396 .. 10,738  =  1343 bp
yeast 2μ plasmid origin of replication
2μ ori
9396 .. 10,738  =  1343 bp
yeast 2μ plasmid origin of replication
AmpR
8404 .. 9264  =  861 bp
286 amino acids  =  31.6 kDa
   Segment 2:  
   8404 .. 9195  =  792 bp
   263 amino acids  =  28.9 kDa
Product: β-lactamase
confers resistance to ampicillin, carbenicillin, and related antibiotics
AmpR
8404 .. 9264  =  861 bp
286 amino acids  =  31.6 kDa
   Segment 1:  signal sequence  
   9196 .. 9264  =  69 bp
   23 amino acids  =  2.6 kDa
Product: β-lactamase
confers resistance to ampicillin, carbenicillin, and related antibiotics
AmpR
8404 .. 9264  =  861 bp
286 amino acids  =  31.6 kDa
2 segments
Product: β-lactamase
confers resistance to ampicillin, carbenicillin, and related antibiotics
URA3
54 .. 857  =  804 bp
267 amino acids  =  29.3 kDa
Product: orotidine-5'-phosphate decarboxylase, required for uracil biosynthesis
yeast auxotrophic marker, counterselectable with 5-fluoroorotic acid (5-FOA)
URA3
54 .. 857  =  804 bp
267 amino acids  =  29.3 kDa
Product: orotidine-5'-phosphate decarboxylase, required for uracil biosynthesis
yeast auxotrophic marker, counterselectable with 5-fluoroorotic acid (5-FOA)
ori
7645 .. 8233  =  589 bp
high-copy-number ColE1/pMB1/pBR322/pUC origin of replication
ori
7645 .. 8233  =  589 bp
high-copy-number ColE1/pMB1/pBR322/pUC origin of replication
RPR1 promoter
6655 .. 7061  =  407 bp
promoter for the S. cerevisiae RNase P RNA gene
RPR1 promoter
6655 .. 7061  =  407 bp
promoter for the S. cerevisiae RNase P RNA gene
TEF1 promoter
1757 .. 2155  =  399 bp
promoter for EF-1α
TEF1 promoter
1757 .. 2155  =  399 bp
promoter for EF-1α
ADH2 terminator
6343 .. 6642  =  300 bp
transcription terminator for the S. cerevisiae alcohol dehydrogenase 2 (ADH2) gene
ADH2 terminator
6343 .. 6642  =  300 bp
transcription terminator for the S. cerevisiae alcohol dehydrogenase 2 (ADH2) gene
lacZα
1257 .. 1553  =  297 bp
98 amino acids  =  11.0 kDa
Product: LacZα fragment of β-galactosidase
lacZα
1257 .. 1553  =  297 bp
98 amino acids  =  11.0 kDa
Product: LacZα fragment of β-galactosidase
SNR52 promoter
946 .. 1214  =  269 bp
promoter for the S. cerevisiae small nucleolar RNA gene SNR52
SNR52 promoter
946 .. 1214  =  269 bp
promoter for the S. cerevisiae small nucleolar RNA gene SNR52
AmpR promoter
9265 .. 9369  =  105 bp
AmpR promoter
9265 .. 9369  =  105 bp
tracrRNA
7062 .. 7140  =  79 bp
trans-activating CRISPR RNA for the Streptococcus pyogenes CRISPR/Cas9 system
tracrRNA
7062 .. 7140  =  79 bp
trans-activating CRISPR RNA for the Streptococcus pyogenes CRISPR/Cas9 system
RPR1 terminator
7148 .. 7210  =  63 bp
transcription terminator for the S. cerevisiae RNase P RNA gene
RPR1 terminator
7148 .. 7210  =  63 bp
transcription terminator for the S. cerevisiae RNase P RNA gene
truncated URA3 promoter
8 .. 53  =  46 bp
Truncating this promoter forces yeast cells to maintain the plasmid at very high copy number.
truncated URA3 promoter
8 .. 53  =  46 bp
Truncating this promoter forces yeast cells to maintain the plasmid at very high copy number.
5' DR
1217 .. 1249  =  33 bp
5' portion of the direct repeat for the Streptococcus pyogenes CRISPR/Cas system
5' DR
1217 .. 1249  =  33 bp
5' portion of the direct repeat for the Streptococcus pyogenes CRISPR/Cas system
lac promoter
1597 .. 1627  =  31 bp
   Segment 3:  -10  
   1597 .. 1603  =  7 bp
promoter for the E. coli lac operon
lac promoter
1597 .. 1627  =  31 bp
   Segment 2:  
   1604 .. 1621  =  18 bp
promoter for the E. coli lac operon
lac promoter
1597 .. 1627  =  31 bp
   Segment 1:  -35  
   1622 .. 1627  =  6 bp
promoter for the E. coli lac operon
lac promoter
1597 .. 1627  =  31 bp
3 segments
promoter for the E. coli lac operon
3' DR
1705 .. 1735  =  31 bp
3' portion of the direct repeat for the Streptococcus pyogenes CRISPR/Cas system
3' DR
1705 .. 1735  =  31 bp
3' portion of the direct repeat for the Streptococcus pyogenes CRISPR/Cas system
FLAG
2167 .. 2190  =  24 bp
8 amino acids  =  1.0 kDa
Product: FLAG® epitope tag, followed by an enterokinase cleavage site
FLAG
2167 .. 2190  =  24 bp
8 amino acids  =  1.0 kDa
Product: FLAG® epitope tag, followed by an enterokinase cleavage site
SV40 NLS
2194 .. 2214  =  21 bp
7 amino acids  =  883.1 Da
Product: nuclear localization signal of SV40 large T antigen
SV40 NLS
2194 .. 2214  =  21 bp
7 amino acids  =  883.1 Da
Product: nuclear localization signal of SV40 large T antigen
SV40 NLS
6319 .. 6339  =  21 bp
7 amino acids  =  883.1 Da
Product: nuclear localization signal of SV40 large T antigen
SV40 NLS
6319 .. 6339  =  21 bp
7 amino acids  =  883.1 Da
Product: nuclear localization signal of SV40 large T antigen
SUP4 terminator
1736 .. 1755  =  20 bp
transcription terminator for the S. cerevisiae SUP4 tRNA gene
SUP4 terminator
1736 .. 1755  =  20 bp
transcription terminator for the S. cerevisiae SUP4 tRNA gene
lac operator
1573 .. 1589  =  17 bp
The lac repressor binds to the lac operator to inhibit transcription in E. coli. This inhibition can be relieved by adding lactose or isopropyl-β-D-thiogalactopyranoside (IPTG).
lac operator
1573 .. 1589  =  17 bp
The lac repressor binds to the lac operator to inhibit transcription in E. coli. This inhibition can be relieved by adding lactose or isopropyl-β-D-thiogalactopyranoside (IPTG).
ATG
2164 .. 2166  =  3 bp
1 amino acid  =  149.2 Da
Product: start codon
ATG
2164 .. 2166  =  3 bp
1 amino acid  =  149.2 Da
Product: start codon
ORF:  2164 .. 6342  =  4179 bp
ORF:  1392 amino acids  =  161.4 kDa
ORF:  8534 .. 8800  =  267 bp
ORF:  88 amino acids  =  9.2 kDa
ORF:  9431 .. 9721  =  291 bp
ORF:  96 amino acids  =  11.5 kDa
ORF:  54 .. 857  =  804 bp
ORF:  267 amino acids  =  29.3 kDa
ORF:  1257 .. 1553  =  297 bp
ORF:  98 amino acids  =  11.0 kDa
ORF:  10,197 .. 10,517  =  321 bp
ORF:  106 amino acids  =  12.9 kDa
ORF:  89 .. 565  =  477 bp
ORF:  158 amino acids  =  16.8 kDa
ORF:  8404 .. 9264  =  861 bp
ORF:  286 amino acids  =  31.6 kDa
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