pCRISPomyces-1

Streptomyces plasmid for expression of codon-optimized Cas9, tracrRNA, and custom crRNA.

Sequence Author: Zhao Lab / Addgene #61736

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XbaI (5961) DR BbsI (5882) lac operator rpsL promoter tracrRNA lambda t0 terminator DR BbsI (5421) pCRISPomyces-1 11,474 bp
XbaI  (5961)
1 site
T C T A G A A G A T C T
BbsI  (5882)
2 sites
G A A G A C N N C T T C T G N N ( N ) 4

Sticky ends from different BbsI sites may not be compatible.
BbsI gradually loses activity when stored at -20°C.
BbsI  (5421)
2 sites
G A A G A C N N C T T C T G N N ( N ) 4

Sticky ends from different BbsI sites may not be compatible.
BbsI gradually loses activity when stored at -20°C.
Cas9
303 .. 4409  =  4107 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
303 .. 4409  =  4107 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
pSG5 Rep
8534 .. 9976  =  1443 bp
480 amino acids  =  52.9 kDa
Product: replication initiator protein from the Streptomyces ghanaensis plasmid pSG5
pSG5 Rep
8534 .. 9976  =  1443 bp
480 amino acids  =  52.9 kDa
Product: replication initiator protein from the Streptomyces ghanaensis plasmid pSG5
ApmR
6795 .. 7595  =  801 bp
267 amino acids  =  28.8 kDa
Product: aminoglycoside 3-N-acetyltransferase type IV                
confers resistance to apramycin (Paget and Davies, 1996)
ApmR
6795 .. 7595  =  801 bp
267 amino acids  =  28.8 kDa
Product: aminoglycoside 3-N-acetyltransferase type IV                
confers resistance to apramycin (Paget and Davies, 1996)
ori
6003 .. 6591  =  589 bp
high-copy-number ColE1/pMB1/pBR322/pUC origin of replication
ori
6003 .. 6591  =  589 bp
high-copy-number ColE1/pMB1/pBR322/pUC origin of replication
rpsL promoter
4609 .. 4969  =  361 bp
promoter for the Cellulomonas flavigena ribosomal protein S12 gene
rpsL promoter
4609 .. 4969  =  361 bp
promoter for the Cellulomonas flavigena ribosomal protein S12 gene
rpsL promoter
1 .. 302  =  302 bp
promoter for the Xylanimonas cellulosilytica ribosomal protein S12 gene, mutated to remove the BbsI sites
rpsL promoter
1 .. 302  =  302 bp
promoter for the Xylanimonas cellulosilytica ribosomal protein S12 gene, mutated to remove the BbsI sites
lacZα
5434 .. 5730  =  297 bp
98 amino acids  =  11.0 kDa
Product: LacZα fragment of β-galactosidase
lacZα
5434 .. 5730  =  297 bp
98 amino acids  =  11.0 kDa
Product: LacZα fragment of β-galactosidase
gapdh promoter
5106 .. 5390  =  285 bp
glyceraldehyde-3-phosphate dehydrogenase promoter from Eggerthella lenta
gapdh promoter
5106 .. 5390  =  285 bp
glyceraldehyde-3-phosphate dehydrogenase promoter from Eggerthella lenta
oriT
11,144 .. 11,253  =  110 bp
incP origin of transfer
oriT
11,144 .. 11,253  =  110 bp
incP origin of transfer
tracrRNA
4970 .. 5048  =  79 bp
trans-activating CRISPR RNA for the Streptococcus pyogenes CRISPR/Cas9 system
tracrRNA
4970 .. 5048  =  79 bp
trans-activating CRISPR RNA for the Streptococcus pyogenes CRISPR/Cas9 system
T7 terminator
5913 .. 5960  =  48 bp
transcription terminator for bacteriophage T7 RNA polymerase
T7 terminator
5913 .. 5960  =  48 bp
transcription terminator for bacteriophage T7 RNA polymerase
fd terminator
4571 .. 4608  =  38 bp
central terminator from bacteriophage fd (Otsuka and Kunisawa, 1982)
fd terminator
4571 .. 4608  =  38 bp
central terminator from bacteriophage fd (Otsuka and Kunisawa, 1982)
DR
5393 .. 5427  =  35 bp
direct repeat for the Streptococcus pyogenes CRISPR/Cas system
DR
5393 .. 5427  =  35 bp
direct repeat for the Streptococcus pyogenes CRISPR/Cas system
lambda t0 terminator
5072 .. 5105  =  34 bp
minimal transcription terminator from phage lambda (Scholtissek and Grosse, 1987)
lambda t0 terminator
5072 .. 5105  =  34 bp
minimal transcription terminator from phage lambda (Scholtissek and Grosse, 1987)
lac promoter
5774 .. 5804  =  31 bp
3 segments
   Segment 3:  -10  
   5774 .. 5780  =  7 bp
promoter for the E. coli lac operon
lac promoter
5774 .. 5804  =  31 bp
3 segments
   Segment 2:  
   5781 .. 5798  =  18 bp
promoter for the E. coli lac operon
lac promoter
5774 .. 5804  =  31 bp
3 segments
   Segment 1:  -35  
   5799 .. 5804  =  6 bp
promoter for the E. coli lac operon
lac promoter
5774 .. 5804  =  31 bp
3 segments
promoter for the E. coli lac operon
DR
5882 .. 5912  =  31 bp
direct repeat for the Streptococcus pyogenes CRISPR/Cas system
DR
5882 .. 5912  =  31 bp
direct repeat for the Streptococcus pyogenes CRISPR/Cas system
lac operator
5750 .. 5766  =  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
5750 .. 5766  =  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).
ORF:  7859 .. 8371  =  513 bp
ORF:  170 amino acids  =  18.8 kDa
ORF:  8477 .. 8959  =  483 bp
ORF:  160 amino acids  =  17.1 kDa
ORF:  303 .. 4409  =  4107 bp
ORF:  1368 amino acids  =  158.4 kDa
ORF:  6795 .. 7598  =  804 bp
ORF:  267 amino acids  =  28.8 kDa
ORF:  8514 .. 8738  =  225 bp
ORF:  74 amino acids  =  7.9 kDa
ORF:  9663 .. 10,148  =  486 bp
ORF:  161 amino acids  =  17.9 kDa
ORF:  10,305 .. 10,541  =  237 bp
ORF:  78 amino acids  =  8.4 kDa
ORF:  5142 .. 5465  =  324 bp
ORF:  107 amino acids  =  11.4 kDa
ORF:  7011 .. 7604  =  594 bp
ORF:  197 amino acids  =  22.5 kDa
ORF:  17 .. 364  =  348 bp
ORF:  115 amino acids  =  12.2 kDa
ORF:  2216 .. 2665  =  450 bp
ORF:  149 amino acids  =  17.8 kDa
ORF:  6857 .. 7165  =  309 bp
ORF:  102 amino acids  =  10.5 kDa
ORF:  7601 .. 8515  =  915 bp
ORF:  304 amino acids  =  33.5 kDa
ORF:  8534 .. 10,495  =  1962 bp
ORF:  653 amino acids  =  71.0 kDa
ORF:  4483 .. 4977  =  495 bp
ORF:  164 amino acids  =  17.9 kDa
ORF:  5434 .. 5730  =  297 bp
ORF:  98 amino acids  =  11.0 kDa
ORF:  9136 .. 9393  =  258 bp
ORF:  85 amino acids  =  9.1 kDa
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