pJEC726
(Plasmid #190814)

• Purpose: CRISPRi backbone plasmid harboring BbsI sites for easy cloning of sgRNA targeting regions via Golden gate.
• Depositing Lab: James Chappell
• Publication: Ameruoso et al Nucleic Acids Res. 2022 Jul 22;50(13):7751-7760. doi: 10.1093/nar/gkac556.

Backbone

• Vector backbone: pCRISPomyces-2
• Backbone manufacturer: Addgene
• Backbone size w/o insert (bp): 6573
• Total vector size (bp): 10680
• Modifications to backbone: Promoter driving dCas9 changed from rpsL(XC) to SP30. Immediately downstream of SP30, RiboJ and synthetic RBS R09 were added. Cas9 was mutated to dCas9.
• Vector type: CRISPR, Synthetic Biology

Growth in Bacteria

• Bacterial Resistance(s): Apramycin, 25 μg/mL
• Growth Temperature: 37°C
• Growth Strain(s): DH5alpha
• Copy number: High Copy

Gene/Insert

• Gene/Insert name: dCas9
• Species: Synthetic
• Mutation: S. pyogenes dCas9 codon optimized for Streptomyces expression. The D10A and H840A mutations were introduced to convert Cas9 to dCas9.
• Promoter: SP30

Cloning Information

• Cloning method: Unknown
• 5′ sequencing primer: ccttattcgcacctggcgg

Resource Information

• Supplemental Documents:
  • pJEC726.gb

Terms and Licenses

• Academic/Nonprofit Terms:
  • UBMTA
• Industry Terms:
  • Not Available to Industry