pKK-FRET-ORF1-3C-Cerulean_ORF2-TEV-Amber
(Plasmid #105806)

• Purpose: (Empty Backbone) Concomitant expression of two proteins. One protein expressed with Cerulean, cleavable by 3C; second protein expressed with Amber, cleavable by TEV. Control for protein interaction analysis by FRET.
• Depositing Lab: Andrzej Dziembowski
• Publication: Szczesny et al PLoS One. 2018 Mar 28;13(3):e0194887. doi: 10.1371/journal.pone.0194887. eCollection 2018.

Backbone

• Vector backbone: BI-16 (constructed based on pcDNA5/FRT/TO from Invitrogen)
• Backbone manufacturer: Sammarco & Grabczyk (PMID: 16212928)
• Vector type: Mammalian Expression ; Flp-In competent
• Selectable markers: Hygromycin
• Tag / Fusion Protein:
  • ORF1: 3C-Cerulean; ORF2: TEV-Amber (C terminal on backbone)

Growth in Bacteria

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

Gene/Insert

• Gene/Insert name: None
• Tag / Fusion Protein:
  • ORF1: 3C-Cerulean; ORF2: TEV-Amber (C terminal on backbone)

Resource Information

• Supplemental Documents:
  • pKK-FRET-ORF1-C3-mCerulean_ORF2-TEV-mAmber.txt

Terms and Licenses

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

Depositor Comments

This vector is a member of the pKK-FRET series which belongs to the pKK vector family. It enables concomitant expression of two genes, which transcription is driven by a tetracycline responsive bidirectional CMV promoter. This vector is a derivate of the BI16 plasmid (PMID: 16212928) and is suitable for stable cell line generation using the Flp-In system (see Szczesny RJ et al. for detailed description and protocols; bioRxiv, doi: https://doi.org/10.1101/160101) Negative control for FRET experiments. One protein expressed with Cerulean whereas second protein expressed with non-fluorescent Amber fusion tag. Amber is a mutant of Venus (Y67C) that retains the tertiary structure but has no fluorescent properties and does not absorb light. A no-FRET sample in which both putative interacting proteins are tagged can be superior to a donor-only sample (PMID: 17040988, 22264545). Amber coding sequence was cloned from Addgene ID: 27798.