PurposeGateway destination clone of GSK3B (human) tagged with N-terminal miniTurbo-V5 for generating protein-proximity networks using proximity-dependent biotinylation proteomics
|Item||Catalog #||Description||Quantity||Price (USD)|
|Plasmid||158165||Standard format: Plasmid sent in bacteria as agar stab||1||$75|
This material is available to academics and nonprofits only.
- Backbone size w/o insert (bp) 9019
Vector typeMammalian Expression, Lentiviral ; Gateway Destination
Growth in Bacteria
Growth Strain(s)NEB Stable
Copy numberLow Copy
SpeciesH. sapiens (human)
- Promoter Ubiquitin
/ Fusion Protein
- miniTurbo-V5 (N terminal on insert)
- Cloning method Gateway Cloning
- 5′ sequencing primer GCCCGAAGGAATAGAAGAAGAA
- 3′ sequencing primer CATAGCGTAAAAGGAGCAACA (Common Sequencing Primers)
These plasmids were generated as part of the Illuminating the Druggable Genome (IDG) program sponsored by the NIH Common Fund. The goal of this program is to identify, gather, and distribute information and resources for proteins that currently are not well-studied yet belong to commonly drug-targeted protein families: protein kinases, non-olfactory G-protein coupled receptors (GPCRs), and ion channels. The IDG program is designed to develop fundamental research tools for understudied proteins, elucidate their function, and disseminate the IDG-related resources and data to the greater scientific community. These lentiviral gateway destination plasmids were generated, as part of the Kinase-Data and Resource Generating Center (DRGC), using the single fragment or multisite gateway cloning technology. They were used for generating proteomics-based protein-interaction and protein-proximity networks that can be accessed through the kinase-DRGC website https://darkkinome.org/. Each plasmid has either an N- or C-terminal fusion protein (Flag/ V5/V5-miniTurbo/V5-TurboID/miniTurbo-V5/TurboID-V5) tagged understudied kinase driven under either a CMV or Ubiquitin (UBC) promoter. All inserts in the pENTR plasmids used to generate the deposited destination plasmids were end-sequenced and insert size validated prior to multi-site gateway cloning. The combined insert size of the single fragment or three fragment destination plasmids were confirmed by restriction digestion (BsrGI, and EcoRV for pDest667; BsrGI, EcoRV, and BstZ17I for pDest663; and BsrGI for pHAGE) and all plasmids were partially sequenced to ensure in-frame ORFs and fusion tags.
These plasmids were created by your colleagues. Please acknowledge the Principal Investigator, cite the article in which the plasmids were described, and include Addgene in the Materials and Methods of your future publications.
For your Materials & Methods section:p663-UBC-miniTurbo-V5-GSK3B_IDG-K was a gift from Ben Major (Addgene plasmid # 158165 ; http://n2t.net/addgene:158165 ; RRID:Addgene_158165)