TRIPZ-bioGEF-UBnc-PURO
(Plasmid #208044)

• Purpose: Enables inducible expression of low-affinity Avitag-UBnc; to perform bioE3; nc = non-cleavable mutation; selection with puromycin
• Depositing Labs: Rosa Barrio, James Sutherland
• Publication: Barroso-Gomila et al Nat Commun. 2023 Nov 23;14(1):7656. doi: 10.1038/s41467-023-43326-8.

Backbone

• Vector backbone: TRIPZ
• Backbone manufacturer: Open Biosystems
• Vector type: Lentiviral
• Selectable markers: Puromycin

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: UBnc
• Species: H. sapiens (human)
• Mutation: L73P Mutation near C-terminus suppresses cleavage by SENPs/DUBs
• Entrez Gene: UBC (a.k.a. HMG20)
• Promoter: tetO/UBC
• Tag / Fusion Protein:
  • Low-affinity Avitag (N terminal on insert)

Cloning Information

• Cloning method: Gibson Cloning

Terms and Licenses

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

Depositor Comments

Lentiviral packaging of TRIPZ is more efficient with TAT expression (e.g using pcDNA1-TatHIV Addgene ID: 138478) and 2nd generation gag/pol/env combination (e.g. using PSPAX2 Addgene ID: 12260 and pMD2.G Addgene ID: 12259); Use of bioGEF-UBwt may be necessary for certain E3 ligases (e.g. NEDD4 HECT ligase); Use of "non-cleavable" versions may reduce recycling of biotinylated UB and may lead to higher yields of biotinylated protein for proteomic analysis, but induction times should be minimized to avoid toxicity.