G protein alpha-s-mCherry
(Plasmid #66968)

• Purpose: G protein alpha-s internally tagged with mCherry and EE epitope
• Depositing Lab: Catherine Berlot
• Publication: Hynes et al J Biol Chem. 2004 Oct 15;279(42):44101-12. Epub 2004 Aug 5.

Backbone

• Vector backbone: pcDNAI/Amp
• Backbone manufacturer: Invitrogen
• Backbone size w/o insert (bp): 4800
• Total vector size (bp): 7094
• Vector type: Mammalian Expression

Growth in Bacteria

• Bacterial Resistance(s): Ampicillin, 100 μg/mL
• Growth Temperature: 37°C
• Growth Strain(s): DH10B
• Copy number: High Copy

Gene/Insert

• Gene/Insert name: G-alpha-s-EE-mCherry
• Alt name: Gnas
• Species: R. norvegicus (rat); Discosoma sp.
• Insert Size (bp): 2284
• Mutation: BamHI sites in the alpha-s cDNA were removed and alpha-s residues 73-84 were replaced with Gly-Ser adding a unique BamHI site into which mCherry was subcloned.
• Entrez Gene: Gnas (a.k.a. ALEX, G-alpha-8, Gnas1, Gnpas, Nesp55, SCG6)
• Promoter: CMV
• Tags / Fusion Proteins:
  • mCherry flanked by SGGGS on each side was inserted in place of alpha-s residues 73- 84.
  • internal EE epitope (residues 189-194 in alpha- s sequence are EYMPTE)

Cloning Information

• Cloning method: Restriction Enzyme
• 5′ cloning site: HindIII (not destroyed)
• 3′ cloning site: HindIII (not destroyed)
• 5′ sequencing primer: T7
• 3′ sequencing primer: SP6

Resource Information

• A portion of this plasmid was derived from a plasmid made by: Alpha-s-EE was produced in the laboratory of Henry Bourne, University of California, San Francisco, CA. mCherry was obtained from Roger Tsien, University of California, San Diego, CA.

Terms and Licenses

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

Depositor Comments

Reference for alpha-s-EE:
Wilson PT, Bourne HR. Fatty acylation of αz. Effects of palmitoylation and myristoylation on αz signaling. J Biol Chem. 1995;270:9667-75.

Reference for mCherry:
Shaner NC, Campbell RE, Steinbach PA, Giepmans BN, Palmer AE, Tsien RY. Improved monomeric red, orange and yellow fluorescent proteins derived from Discosoma sp. red fluorescent protein. Nat Biotechnol. 2004;22(12):1567-72. PubMed PMID: 15558047.