pTetON-DHFRdd-SpvB; CMV-mCherry
(Plasmid #89463)

• Purpose: Tet/Dox-inducible expression of DHRFdd-SpvB (Salmonella SpvB 375-591) plus constitutive expression of mCherry in mammalian cells (Dual-promoter)
• Depositing Lab: Brad Zuchero
• Publication: Harterink et al Nat Methods. 2017 Apr 10. doi: 10.1038/nmeth.4257.

Backbone

• Vector backbone: pTRE3G-luciferase
• Backbone manufacturer: Clontech
• Backbone size w/o insert (bp): 3383
• Total vector size (bp): 4100
• Modifications to backbone: Removed luciferase, replaced with EGFP
• Vector type: Mammalian Expression

Growth in Bacteria

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

Gene/Insert

• Gene/Insert name: DeAct-SpvB
• Alt name: Salmonella SpvB(375-591)
• Species: Salmonella enterica
• Insert Size (bp): 648
• Mutation: Truncation spanning SpvB amino acids 375-591 (mono(ADP-ribosyl)transferase domain)
• GenBank ID: D14490.1
• Entrez Gene: spvB (a.k.a. pOU1113_03)
• Promoter: TRE3G
• Tags / Fusion Proteins:
  • E. coli DHFRdd destabilization domain (N terminal on insert)
  • mCherry (opposite strand) (N terminal on backbone)

Cloning Information

• Cloning method: Ligation Independent Cloning
• 5′ sequencing primer: gcagagctcgtttagtgaa
• 3′ sequencing primer: CATCAGTTCCATAGGTTG

Resource Information

• Supplemental Documents:
  • pTetON-DHFRdd-SpvB; CMV-mCherry.gb
• Article Citing this Plasmid:
  • 1 Reference

Terms and Licenses

• Academic/Nonprofit Terms:
  • UBMTA
  • Takara Bio Limited Use Label License (formerly Clontech)
  • Tet Systems Limited Use Label License
• Industry Terms:
  • Not Available to Industry

Depositor Comments

Uses Clontech 3rd generation Tet-responsive promoter; also requires Tet-On 3G transactivator protein.
E. coli dihydrofolate reductase destabilization domain (DHFRdd) with R12Y, G67S, Y100I mutations was a kind gift of Tom Wandless (Stanford): Iwamoto, M., Björklund, T., Lundberg, C., Kirik, D. & Wandless, T. J. A general chemical method to regulate protein stability in the mammalian central nervous system. Chem. Biol. 17, 981–988 (2010).