pKK-RNAi-nucEGFPmiR-mCherry-TEV
(Plasmid #105808)

• Purpose: (Empty Backbone) Functional studies using RNAi; rescue experiments; substituting protein for its different form. miRNA cassette with nuclear EGFP expression marker; your protein of interest with N-terminal mCherry.
• 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, RNAi ; Flp-In competent
• Selectable markers: Hygromycin
• Tag / Fusion Protein:
  • mCherry-TEV (N 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:
  • mCherry-TEV (N terminal on backbone)

Resource Information

• Supplemental Documents:
  • pKK-RNAi-nucEGFPmiR-mCherry-TEV.txt
• Addgene Notes:
  • Addgene Diagnostic Digest

Terms and Licenses

• Academic/Nonprofit Terms:
  • UBMTA
  • Takara Bio Limited Use Label License (formerly Clontech)
• Industry Terms:
  • Not Available to Industry

Depositor Comments

This vector is a member of the pKK-RNAi series which belongs to the pKK vector family. It enables concomitant expression of two genes: 1) a cassette encoding miRNAs that target the gene of interest, and 2) an allele of the gene of interest with the protein coding sequence harbouring silent mutations that make the mRNA insensitive to the miRNAs. As a result, the endogenous alleles of the gene of interest are silenced, whereas the exogenous copy is expressed. Furthermore, the miRNAs are cotranscriptionally expressed with a fluorescent protein reporter so that analysis can be narrowed down to cells that express miRNA, provided that applied assay can distinguish cells expressing reporter. The reporter of miRNA expression has 3 repeats of the nuclear localisation signal of SV40 large T antigen, thus, it localizes to nucleus. This vector is a derivate of the BI16 plasmid (PMID: 16212928) and is suitable for stable cell line generation using the Flp-In system. Transcription of cloned DNA is driven by a tetracycline responsive bidirectional CMV promoter (see Szczesny RJ et al. for detailed description and protocols; bioRxiv, doi: https://doi.org/10.1101/160101)