pDN-Sth1Cas9
(Plasmid #221386)

• Purpose: Cas9 under control of AHT inducible TetR on integrating plasmid (KanR) using L5 integrase and attP for site-specific integration into attB
• Depositing Lab: Deborah Hung
• Publication: Neo et al J Bacteriol. 2024 Mar 21;206(3):e0033523. doi: 10.1128/jb.00335-23. Epub 2024 Feb 6.

Backbone

• Vector backbone: pJR962, colE1/phageL5 att/in origins
• Total vector size (bp): 8881
• Modifications to backbone: pJR962 (Addgene plasmid #115162; http://n2t.net/addgene:115162; RRID: Addgene_115162; gift from Jeremy Rock) was used as the template for the construction of the Cas9-containing plasmid (23). Site-directed mutagenesis was performed with the NEBuilder HiFi DNA Assembly Cloning Kit (New England Biolabs) to reintroduce A9D and A599H on the dCas9 allele. These two mutations restore the nuclease activity of the Cas9 allele to facilitate the formation of DSBs. The resulting plasmid pDN-Sth1Cas9 contains genes for the AHT-inducible Sth1Cas9, L5 integrase for integration into the attB site, tetracycline repressor TetR, and resistance selection marker KanR .
• Vector type: CRISPR

Growth in Bacteria

• Bacterial Resistance(s): Kanamycin, 50 μg/mL
• Growth Temperature: 37°C
• Growth Strain(s): DH5alpha
• Growth instructions: High copy in E. coli, single copy (integrated) in Mycobacterium
• Copy number: High Copy

Gene/Insert

• Gene/Insert name: Sth1 Cas9
• Species: Streptococcus thermophilus
• Insert Size (bp): 3366
• GenBank ID: WP_011680957.1

Resource Information

• Supplemental Documents:
  • pDN-Sth1Cas9.gb

Terms and Licenses

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