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Plasmid Files

pCRISPomyces-2

Streptomyces plasmid for expression of codon-optimized Cas9 and a single guide RNA (sgRNA).

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pCRISPomyces-2 Sequence and MappCRISPomyces-2.dna
Map and Sequence File   
Sequence Author:  Zhao Lab / Addgene #61737
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 XbaI (5482) fd terminator BbsI (4889) lac operator BbsI (5350) lambda t0 terminator pCRISPomyces-2 10,995 bp
XbaI  (5482)
1 site
T C T A G A A G A T C T
BbsI  (4889)
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  (5350)
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
8055 .. 9497  =  1443 bp
480 amino acids  =  52.9 kDa
Product: replication initiator protein from the
Streptomyces ghanaensis plasmid pSG5
mutated to remove the BbsI site
pSG5 Rep
8055 .. 9497  =  1443 bp
480 amino acids  =  52.9 kDa
Product: replication initiator protein from the
Streptomyces ghanaensis plasmid pSG5
mutated to remove the BbsI site
ApmR
6316 .. 7116  =  801 bp
267 amino acids  =  28.8 kDa
Product: aminoglycoside 3-N-acetyltransferase type
IV                
confers resistance to apramycin (Paget and Davies,
1996)
ApmR
6316 .. 7116  =  801 bp
267 amino acids  =  28.8 kDa
Product: aminoglycoside 3-N-acetyltransferase type
IV                
confers resistance to apramycin (Paget and Davies,
1996)
ori
5524 .. 6112  =  589 bp
high-copy-number ColE1/pMB1/pBR322/pUC origin
of replication
ori
5524 .. 6112  =  589 bp
high-copy-number ColE1/pMB1/pBR322/pUC origin
of replication
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
4902 .. 5198  =  297 bp
98 amino acids  =  11.0 kDa
Product: LacZα fragment of β-galactosidase
lacZα
4902 .. 5198  =  297 bp
98 amino acids  =  11.0 kDa
Product: LacZα fragment of β-galactosidase
gapdh promoter
4609 .. 4893  =  285 bp
glyceraldehyde-3-phosphate dehydrogenase
promoter from Eggerthella lenta
gapdh promoter
4609 .. 4893  =  285 bp
glyceraldehyde-3-phosphate dehydrogenase
promoter from Eggerthella lenta
oriT
10,665 .. 10,774  =  110 bp
incP origin of transfer
oriT
10,665 .. 10,774  =  110 bp
incP origin of transfer
gRNA scaffold
5351 .. 5426  =  76 bp
guide RNA scaffold for the Streptococcus pyogenes
CRISPR/Cas9 system
gRNA scaffold
5351 .. 5426  =  76 bp
guide RNA scaffold for the Streptococcus pyogenes
CRISPR/Cas9 system
lambda t0 terminator
5432 .. 5482  =  51 bp
transcription terminator from phage lambda
lambda t0 terminator
5432 .. 5482  =  51 bp
transcription terminator from phage lambda
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)
lac promoter
5242 .. 5272  =  31 bp
   Segment 3:  -10  
   5242 .. 5248  =  7 bp
promoter for the E. coli lac operon
lac promoter
5242 .. 5272  =  31 bp
   Segment 2:  
   5249 .. 5266  =  18 bp
promoter for the E. coli lac operon
lac promoter
5242 .. 5272  =  31 bp
   Segment 1:  -35  
   5267 .. 5272  =  6 bp
promoter for the E. coli lac operon
lac promoter
5242 .. 5272  =  31 bp
3 segments
promoter for the E. coli lac operon
lac operator
5218 .. 5234  =  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
5218 .. 5234  =  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).
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