pDD427
(Plasmid #202080)

• Purpose: Frame +2/-1 selector for NHEJ knock-in
• Depositing Lab: Daniel Dickinson
• Publication: Shi et al Development. 2023 May 15;150(10):dev201367. doi: 10.1242/dev.201367. Epub 2023 May 25.

Backbone

• Vector backbone: pCRISPAINT
• Vector type: CRISPR

Growth in Bacteria

• Bacterial Resistance(s): Ampicillin, 100 μg/mL
• Growth Temperature: 30°C
• Growth Strain(s): NEB Stable
• Copy number: High Copy

Gene/Insert

• Gene/Insert name: pEF1a-mCherry-Cas9 + Frame +2/-1 sgRNA
• gRNA/shRNA sequence: Gggccagtacccaaaaagcg
• Species: S. pyogenes (Cas9)
• Promoter: EF1a

Cloning Information

• Cloning method: Gibson Cloning

Terms and Licenses

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

Depositor Comments

This construct is known to be prone to recombination due to the LTR repeats. We’ve found that an mCherry sequence, which is present in the plasmid, is expressed in the E. coli host but lost upon recombination. Looking for red fluorescent colonies is a reliable way to confirm that the plasmid is intact, and picking a single red clone has been a reliable way for us to re-isolate the correct plasmid from stocks that have undergone partial recombination.