pDN-Cherry-sgRNA
(Plasmid #221387)

• Purpose: episomal (HygR), with constitutive mCherry (Psmyc) and AHT-inducible sgRNA cloning site, including Golden Gate for multiple sgRNAs
• 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: pCHERRY3 : pAL5000/colE1 origins
• Total vector size (bp): 6683
• Modifications to backbone: pCHERRY3 (Addgene plasmid #24659; http://n2t.net/addgene:24659; RRID: Addgene_24659; gift from Tanya Parish) was used as the template for the construction of the sgRNA-containing plasmid (25). The original mCherry promoter was replaced with the constitutive strong mycobacterial promoter Psmyc amplified from pJR962 to create pDN-Cherry. Next, the sgRNA cassette and Golden Gate cloning site from pJR962 were amplified and cloned into pDN-Cherry while also removing the native BsmBI site on pCHERRY3, which would interfere with subsequent cloning of targeting sgRNA sequences. The resulting plasmid pDN-Cherry-sgRNA contains genes for the AHT-inducible sgRNA cassette, Golden Gate sites for cloning multiple sgRNA guides, constitutively expressing mCherry, and resistance selection marker HygR
• Vector type: CRISPR

Growth in Bacteria

• Bacterial Resistance(s): Hygromycin, 200 μg/mL
• Growth Temperature: 37°C
• Growth Strain(s): DH5alpha
• Growth instructions: High copy in E. coli, lower I’m Mycobacterium
• Copy number: High Copy

Gene/Insert

• Gene/Insert name: none, cloning vector for introduction of targeting sequences
• gRNA/shRNA sequence: cloning vector for introduction of targeting sequences

Resource Information

• Supplemental Documents:
  • pDN-Cherry-sgRNA.gb

Terms and Licenses

• Academic/Nonprofit Terms:
  • UBMTA
  • Takara Bio Limited Use Label License (formerly Clontech)
• Industry Terms:
  • Not Available to Industry