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URL: http://www.addgene.org/105809
Proper Citation: RRID:Addgene_105809
Bacterial Resistance: Ampicillin
Defining Citation: PMID:29590189
Vector Backbone Description: Backbone Marker:Sammarco & Grabczyk (PMID: 16212928); Vector Backbone:BI-16 (constructed based on pcDNA5/FRT/TO from Invitrogen); Vector Types:Mammalian Expression, RNAi, Flp-In competent; Bacterial Resistance:Ampicillin
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).
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Source: Addgene
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