G protein alpha-s-mCerulean
(Plasmid #66084)

• Purpose: G protein alpha-s internally tagged with mCerulean 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): 7103
• 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-mCerulean
• Alt name: Gnas
• Species: R. norvegicus (rat); Aequorea victoria
• Insert Size (bp): 2303
• 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 mCerulean was subcloned.
• Entrez Gene: Gnas (a.k.a. ALEX, G-alpha-8, Gnas1, Gnpas, Nesp55, SCG6)
• Promoter: CMV
• Tags / Fusion Proteins:
  • mCerulean 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. Monomeric Cerulean was obtained from David Piston, Vanderbilt University, Nashville, TN.

Terms and Licenses

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

Depositor Comments

Reference for alpha-s-EE: Wilson, P. T., and Bourne, H. R. (1995) Fatty acylation of αz. Effects of palmitoylation and myristoylation on αz signaling. J. Biol. Chem. 270, 9667-9675

Reference for mCerulean: Rizzo MA, Springer GH, Granada B, Piston DW. An improved cyan fluorescent protein variant useful for FRET. Nat Biotechnol. 2004;22(4):445-9. PubMed PMID: 14990965.