3xFlag-eGFP-Flag-SETX
(Plasmid #218838)

• Purpose: Mammalian expression of GFP-tagged SETX construct for subcellular localization studies with GFP, or immunoprecipitation with Flag tag.
• Depositing Lab: Albert La Spada
• Publication: Bennett et al Heliyon. 2020 Jun 13;6(6):e04165. doi: 10.1016/j.heliyon.2020.e04165. eCollection 2020 Jun.

Backbone

• Vector backbone: eGFP Clontech vector
• Backbone manufacturer: Clontech
• Backbone size w/o insert (bp): 5391
• Total vector size (bp): 14303
• Modifications to backbone: We PCR amplified eGFP from an eGFP Clontech vector. The upstream primers contained a 5’-prime extension that encoded FLAG tag. The downstream primer contained the Not I site. In a second amplification step, an extension primer is used to add the additional Flag tags and includes a Nhe I for ease of cloning. Finally, we took the re-derived 3xFlag-GFP containing eGFP vector and digested out the entire CMV 3xFlag-eGFP-Flag-SETX segment by Kpn I/Mlu I digestion and ligated this into the same restriction enzyme sites of our previously reported Flag-SETX construct.
• 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: Senataxin
• Alt name: SETX
• Species: H. sapiens (human)
• Insert Size (bp): 8913
• Entrez Gene: SETX (a.k.a. ALS4, AOA2, SCAN2, SCAR1, STEX, Sen1, bA479K20.2)
• Promoter: CMV
• Tag / Fusion Protein:
  • Flag and eGFP (N terminal on insert)

Cloning Information

• Cloning method: Restriction Enzyme
• 5′ cloning site: Nhe I (not destroyed)
• 3′ cloning site: Mlu I (not destroyed)
• 5′ sequencing primer: unknown

Terms and Licenses

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

Depositor Comments

For more details on cloning see the PubMed reference. There is also a second reference noted in the first, that describes the original cloning of the core SETX orf [Neurobiol Dis. 2006 Jul;23(1):97-108]: https://pubmed.ncbi.nlm.nih.gov/16644229/.