pFLAG-74.84S Tetherin
(Plasmid #41071)

• Depositing Lab: Paul Spearman
• Publication: Hammonds et al J Virol. 2012 Feb;86(4):2259-72. Epub 2011 Nov 30.

Backbone

• Vector backbone: pFLAG-Hsp90α
• Backbone manufacturer: Len Neckers Laboratory (NCI, Bethesda, MD) (Koga et al. 2006, PMID: 16844778)
• Backbone size w/o insert (bp): 5400
• Total vector size (bp): 6000
• Modifications to backbone: From pcDNA3: in-frame N-terminal FLAG epitope added, Hsp90α cds added. From pFLAG-Hsp90α: Hsp90α cds replaced with Tetherin cds by BamHI and XhoI digestion.
• Vector type: Mammalian Expression
• Selectable markers: Neomycin (select with G418)

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: Tetherin
• Alt name: BST-2
• Alt name: bone marrow stromal cell antigen 2
• Alt name: CD317
• Species: H. sapiens (human)
• Insert Size (bp): 550
• Mutation: 74S, 84S
• GenBank ID: NM_004335.2 NP_004326.1
• Entrez Gene: BST2 (a.k.a. CD317, HM1.24, TETHERIN)
• Promoter: CMV
• Tag / Fusion Protein:
  • FLAG (N terminal on backbone)

Cloning Information

• Cloning method: Restriction Enzyme
• 5′ cloning site: BamHI (not destroyed)
• 3′ cloning site: XhoI (not destroyed)
• 5′ sequencing primer: CMV-F
• 3′ sequencing primer: BGH-rev

Resource Information

• A portion of this plasmid was derived from a plasmid made by: Plasmid pFLAG-Hsp90α from the laboratory of Len Neckers (NCI, Bethesda, MD) (Koga et al. 2006, PMID: 16844778)

Terms and Licenses

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

Depositor Comments

This plasmid was generated by cloning of tetherin from plasmid pCMV-HA.Tetherin (Addgene plasmid #41068) into the BamHI and XhoI sites of pFLAG-Hsp90α to generate pFLAG-Tetherin (Addgene plasmid #41070). Serine substitutions at positions aa74 and aa84 were then generated by Quik Change II Site-directed Mutagenesis Kit (Strategene).