pGG442
(Plasmid #165616)

• Purpose: Vector for expression of SpCas9-Zif268 in yeast after assembly with PID fragments: TEF1p-SpCas9::KanR-1xNLS-3xHA-1xNLS-Zif268-1xNLS-CYC1t (KanR cassette in place of the PID)
• Depositing Lab: Marcus Noyes
• Publication: Goldberg et al Nat Commun. 2021 Jan 13;12(1):349. doi: 10.1038/s41467-020-20650-x.

Backbone

• Vector backbone: pGG211
• Backbone manufacturer: Marcus Noyes Lab
• Vector type: Yeast Expression, CRISPR
• Selectable markers: LEU2

Growth in Bacteria

• Bacterial Resistance(s): Ampicillin and Kanamycin, 100 & 50 μg/mL
• Growth Temperature: 37°C
• Growth Strain(s): XL1 Blue
• Copy number: High Copy

Gene/Insert

• Gene/Insert name: TEF1p-SpCas9::KanR(in place of PID)-1xNLS-3xHA-1xNLS-Zif268-1xNLS-CYC1t
• Mutation: Corrected homology relative to wild type SpCas9 (immediately adjacent to the NheI site upstream of KanR cassette) and inserted Zif268 immediately upstream of the C-terminal NLS (with a linker fragment between SpCas9 and Zif268)
• Promoter: TEF1
• Tags / Fusion Proteins:
  • SV40 NLS (C terminal on insert)
  • 3x HA (C terminal on insert)
  • c-Myc-like NLS (C terminal on insert)
  • Zif268 (C terminal on insert)
  • SV40 NLS (C terminal on insert)

Cloning Information

• Cloning method: Gibson Cloning
• 5′ sequencing primer: n/a (multiple primers used)
• 3′ sequencing primer: n/a (multiple primers used)

Resource Information

• Supplemental Documents:
  • pGG442.gbk

Terms and Licenses

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