pRS424GFP-2xPH(Osh2)
(Plasmid #36095)

• Depositing Lab: Scott Emr
• Publication: Stefan et al Cell. 2011 Feb 4;144(3):389-401.

Backbone

• Vector backbone: pRS424
• Modifications to backbone: To create a yeast GFP expression vector (pGOGFP), the PRC1 promoter and GFP coding sequences were subcloned into the BamHI site of PRS426. To create a PtdIns(4)P-specific FLARE construct,pRS424GFP-2x(Osh2), we fused two tandem copies of the Osh2 PH domain to GFP. PCR was used to generate sequences encoding two Osh2 PH domains. These PCR products were cloned in-frame into pGOGFP (Burd and Emr, 1998), to create a plasmid harboring a GFP-2×PH(Osh2) fusion, pRS424GFP-2×PH(Osh2).
• Vector type: Yeast Expression
• Selectable markers: TRP1

Growth in Bacteria

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

Gene/Insert

• Gene/Insert name: 2xPH(Osh2) fusion
• Species: S. cerevisiae (budding yeast)
• Promoter: PRC1
• Tag / Fusion Protein:
  • GFP (N terminal on insert)

Cloning Information

• Cloning method: Restriction Enzyme
• 5′ cloning site: BamHI (destroyed during cloning)
• 3′ cloning site: SalI (destroyed during cloning)
• 5′ sequencing primer: T3
• 3′ sequencing primer: T7

Resource Information

• Article Citing this Plasmid:
  • 1 Reference

Terms and Licenses

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

Depositor Comments

Please note that the first Osh2 domain contains aa273-421 and the second Osh2 domains contains aa260-421 when compared to GenBank reference sequence NP_010265.1. According to this reference sequence the PH domain is aa 292–384. The additional residues surrounding the PH domain were intentionally cloned into this construct.