pDuRCC-K
(Plasmid #81193)

• Purpose: Expression of Cas9 and two gRNA cassettes in S. cerevisiae; Kan Resistance
• Depositing Lab: Eckhard Boles
• Publication: Generoso et al J Microbiol Methods. 2016 Aug;127:203-5. doi: 10.1016/j.mimet.2016.06.020. Epub 2016 Jun 17.

Backbone

• Vector backbone: pRS42K
• Backbone manufacturer: Euroscarf
• Vector type: Yeast Expression, CRISPR
• Selectable markers: Neomycin (select with G418)

Growth in Bacteria

• Bacterial Resistance(s): Ampicillin, 100 μg/mL
• Growth Temperature: 37°C
• Growth Strain(s): DH5alpha
• Copy number: High Copy

Gene/Insert 1

• Gene/Insert name: Cas9
• Species: S. pyogenes
• Mutation: WT - codon optimized
• Promoter: ROX3
• Tag / Fusion Protein:
  • NTS (C terminal on backbone)

Cloning Information for Gene/Insert 1

• Cloning method: Gibson Cloning
• 5′ sequencing primer: unknown

Gene/Insert 2

• Gene/Insert name: empty gRNA cassette
• Species: Synthetic
• Mutation: Please see depositor comments below.
• Promoter: SNP52p

Cloning Information for Gene/Insert 2

• Cloning method: Gibson Cloning
• 5′ sequencing primer: unknown

Gene/Insert 3

• Gene/Insert name: empty gRNA cassette
• Species: Synthetic
• Mutation: Please view depositor comments below
• Promoter: SNP52p

Cloning Information for Gene/Insert 3

• Cloning method: Gibson Cloning
• 5′ sequencing primer: unknown

Resource Information

• Supplemental Documents:
  • Annotated GenBank file
• Addgene Notes:
  • Addgene Diagnostic Digest

Terms and Licenses

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

Depositor Comments

Please note: Addgene’s quality control sequences found the guanine nucleotide at the start of the structural guide-RNA sequence was missing. The Boles Lab has indicated that this is a “virtual guanine”. If you sequence this plasmid, you will notice this missing guanine. The Boles lab omitted the guanine in order to work with the empty vector in yeast, since once the structural gRNA has this G, it is active and could create problems in the cell. However, when primers are designed to create the CRISPR/Cas9 vector for a specific target, this G should be in the primer, so that the gRNA becomes functional again. If you do not include this G in your primer sequence, the system will not work.

Plasmid contains 2 gRNA cassettes together with the Cas9 overexpression cassette.