pKK-BI16-ORF1-3C-mRuby3_ORF2-TEV-mClover3
(Plasmid #105802)

• Purpose: (Empty Backbone) Concomitant expression of two proteins. One protein expressed with mRuby3, cleavable by 3C; second protein expressed with mClover3, cleavable by TEV protease; tags positions: C-termini.
• 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-mRuby3; ORF2: TEV-mClover3 (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-mRuby3; ORF2: TEV-mClover3 (C terminal on backbone)

Resource Information

• Supplemental Documents:
  • pKK-BI16-ORF1-C3-mRuby3_ORF2-TEV-mClover3.txt

Terms and Licenses

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

Depositor Comments

This vector is a member of the pKK-BI16 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. ORF2 is cloned with the universal SLIC protocol (just three universal PCR primers are required to clone a given coding sequence into all vectors from pKK family by a ligation-independent DNA cloning method), ORF1 can be cloned using SLIC with specifically designed primers (applicable enzymes: Bsp120I, ApaI, MluI, NotI, BspTI, Eco47III, MunI) or conventional restriction-ligase based cloning (see Szczesny RJ et al. for detailed description and protocols; bioRxiv, doi: https://doi.org/10.1101/160101) mClover3 is a brighter derivative of mEGFP. mRuby3 is brighter than mCherry and has slightly shifted excitation and emission spectra. The mClover3-mRuby3 pair is superior to EGFP-mCherry in FRET analysis (PMID: 26879144). Useful for experiments requiring co-expression of two proteins (e.g. FRET measurements).