pRS426GFP-2×PH(PLCδ)
(Plasmid #36092)

• Depositing Lab: Scott Emr
• Publication: Stefan et al Mol Biol Cell. 2002 Feb;13(2):542-57.

Backbone

• Vector backbone: pRS426
• 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(4,5)P2-specific FLARE, we fused two tandem copies of the PLCδ1 PH domain (Kavran et al., 1998) to GFP. PCR was used to generate sequences encoding two PLCδ1 PH domains flanked by either BamHI/EcoRI orEcoRI/SalI restriction sites. These PCR products were digested with BamHI/EcoRI orEcoRI/SalI and cloned in-frame into pGO35 (Burd and Emr, 1998), which had been cut with BglII and SalI to create a plasmid harboring a GFP-2×PH(PLCδ) fusion, pRS426GFP-2×PH(PLCδ).
• Vector type: Yeast Expression
• Selectable markers: URA3

Growth in Bacteria

• Bacterial Resistance(s): Ampicillin, 100 μg/mL
• Growth Temperature: 37°C
• Growth Strain(s): XL1 Blue
• Copy number: Unknown

Gene/Insert

• Gene/Insert name: 2xPH(PLCdelta) fusion
• Species: R. norvegicus (rat)
• Mutation: contains aa11-140 of PLCδ (including the PH domain)
• Promoter: PRC1
• Tag / Fusion Protein:
  • GFP (N terminal on insert)

Cloning Information

• Cloning method: Restriction Enzyme
• 5′ cloning site: BglII (destroyed during cloning)
• 3′ cloning site: SalI (not destroyed)
• 5′ sequencing primer: T3
• 3′ sequencing primer: T7

Resource Information

• A portion of this plasmid was derived from a plasmid made by: Dr. Mark Lemmon, University of Pennsylvania School of Medicine

Terms and Licenses

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

Depositor Comments

Please note that this plasmid contains aa11-140 when compared to GenBank reference sequence NP_058731.1. According to this reference sequence the PH domain is aa17-128. The additional residues surrounding the PH domain were intentionally cloned into this construct.