pCRISPR-Cas3
(Plasmid #209427)

• Purpose: Plasmid for CASCADE-Cas3 based genome engineering of streptomycetes
• Depositing Lab: Tilmann Weber
• Publication: Whitford et al bioRxiv 2023.05.09.539971

Backbone

• Vector backbone: pGM1190
• Vector type: CRISPR, Synthetic Biology

Growth in Bacteria

• Bacterial Resistance(s): Apramycin, 25 μg/mL
• Growth Temperature: 37°C
• Growth Strain(s): DH5alpha
• Growth instructions: Cultivation of streptomycetes at 30 C. Temperature sensitive pSG5 replicon, only stable below 37 C
• Copy number: Unknown

Gene/Insert

• Gene/Insert name: codon optimized minimal type I-C CASCADE-Cas3 from Pseudomonas aeruginosa
• Alt name: CASCADE-Cas3
• Species: Synthetic; Pseudomonas aeruginosa
• Insert Size (bp): 5601
• Promoter: PtipA; crRNA under control of PermE*

Cloning Information

• Cloning method: Gibson Cloning
• 5′ sequencing primer: -
• 3′ sequencing primer: -

Resource Information

• Supplemental Documents:
  • p096_pCRISPR-Cas3_addgene.gb

Terms and Licenses

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

Depositor Comments

Please visit https://doi.org/10.1101/2023.05.09.539971 for bioRxiv preprint.

The used type I-C CRISPR system was first described by:
Csörgő, B., León, L.M., Chau-Ly, I.J. et al. A compact Cascade–Cas3 system for targeted genome engineering. Nat Methods 17, 1183–1190 (2020). https://doi.org/10.1038/s41592-020-00980-w

Plasmid contains an IS prokaryotic transposable element that does not affect plasmid function.

The sequences used in pCRISPR-Cas3 were obtained via gene synthesis following codon optimization for S. coelicolor.