BB3cH_pGAP_23*_pPFK300_Cas9
(Plasmid #104912)

• Purpose: hCas9 under control of pPFK300 for direct cloning of HH-sgRNA-HDV PCR products for episomal expression in P. pastoris and selection on Hyg
• Depositing Lab: Brigitte Gasser
• Publication: Gassler et al Methods Mol Biol. 2019;1923:211-225. doi: 10.1007/978-1-4939-9024-5_9.

Backbone

• Vector backbone: Episomal ARS/CEN Backbone
• Vector type: Yeast Expression, CRISPR
• Selectable markers: Hygromycin

Growth in Bacteria

• Bacterial Resistance(s): Hygromycin, 200 μg/mL
• Growth Temperature: 37°C
• Growth Strain(s): DH5alpha
• Copy number: High Copy

Gene/Insert 1

• Gene/Insert name: HH - FS23 linker - HDV

Cloning Information for Gene/Insert 1

• Cloning method: Restriction Enzyme
• 5′ cloning site: BbsI (not destroyed)
• 3′ cloning site: BbsI (not destroyed)
• 5′ sequencing primer: pGAP fw

Gene/Insert 2

• Gene/Insert name: human codon-optimized hcas9 sequence fused to a nuclear localization signal (NLS)
• Alt name: hcas9
• Species: H. sapiens (human); Streptococcus pyogenes, Pichia pastoris
• Insert Size (bp): 4140

Resource Information

• Supplemental Documents:
  • BB3_pGAP_Linker_BbsI(intern)_RPS25Att_pPFK300_Cas9_cycTT_Hyg.gbk
• A portion of this plasmid was derived from a plasmid made by: The original Cas9 sequence was taken from from DiCarlo et al., (Addgene ID 43802). Reference: DiCarlo JE, Norville JE, Mali P, Rios X, Aach J, Church GM. 2013. Genome engineering in Saccharomyces cerevisiae using CRISPR-Cas systems. Nucleic Acids Res 41:4336-43.

Terms and Licenses

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

Depositor Comments

Additional Publication: Prielhofer, R. etal (2017) BMC Syst Biol. 11,123 (PMID:29221460)