pWR63(YUM1)
(Plasmid #203336)

• Purpose: episomal Cas9 and YUM1-targeting sgRNA expression vector for S. stipitis
• Depositing Lab: J. Brian Robertson
• Publication: Reichard et al mSphere. 2023 Aug 24;8(4):e0022423. doi: 10.1128/msphere.00224-23. Epub 2023 Jun 22.

Backbone

• Vector backbone: pWR63
• Backbone manufacturer: Robertson
• Backbone size w/o insert (bp): 12119
• Total vector size (bp): 12117
• Modifications to backbone: Sequence encoding YUM-targeting sgRNA was added to pWR63
• Vector type: Yeast Expression, CRISPR
• Selectable markers: Hygromycin

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: TEF1 promoter (S. stipitis)
• Species: S. stipitis (yeast)
• Insert Size (bp): 726
• Entrez Gene: TEF1 (a.k.a. PICST_65303)

Gene/Insert 2

• Gene/Insert name: coHPH
• Species: Synthetic
• Insert Size (bp): 1029

Gene/Insert 3

• Gene/Insert name: ACT1 terminator (S. stipitis)
• Species: S. stipitis (yeast)
• Insert Size (bp): 400

Gene/Insert 4

• Gene/Insert name: CCW12 promoter (S. stipitis)
• Species: S. stipitis (yeast)
• Insert Size (bp): 749

Gene/Insert 5

• Gene/Insert name: CaCas9
• Species: Synthetic
• Insert Size (bp): 1629

Gene/Insert 6

• Gene/Insert name: ADH2 terminator (S. stipitis)
• Species: S. stipitis (yeast)
• Insert Size (bp): 374

Gene/Insert 7

• Gene/Insert name: ARS from S. stipitis chromosome 1
• Species: S. stipitis (yeast)
• Insert Size (bp): 1148

Gene/Insert 8

• Gene/Insert name: THD3 promoter (S. stipitis)
• Species: S. stipitis
• Insert Size (bp): 750

Gene/Insert 9

• Gene/Insert name: tRNA-sgRNA(SapI)-HDV
• Species: Synthetic
• Insert Size (bp): 242

Gene/Insert 10

• Gene/Insert name: ADH1 terminator (S. stipitis)
• Species: S. stipitis
• Insert Size (bp): 241

Gene/Insert 11

• Gene/Insert name: YUM1-targeting sequence
• Species: Synthetic

Cloning Information for Gene/Insert 11

• Cloning method: Restriction Enzyme
• 5′ cloning site: SapI (destroyed during cloning)
• 3′ cloning site: SapI (destroyed during cloning)
• 5′ sequencing primer: ctgcagccaatctgaaga

Resource Information

• Supplemental Documents:
  • WR63-YUM1.gb
  • WR63-YUM1.gb
• A portion of this plasmid was derived from a plasmid made by: CaCas9 that is in this plasmid was originally synthesized, cloned and published by Vyas VK, Barrasa MI, Fink GR. 2015. A Candida albicans CRISPR system permits genetic engineering of essential genes and gene families. Science advances. 1:e1500248.

Terms and Licenses

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