Starting from our original pHAGE-Ubc-NLS-MCP-YFP third generation lentiviral vector (Lionnet et al., 2011a (link)), we swapped the YFP with the HaloTag open reading frame (DeJesus-Hernandez et al., 2011 (link)) to generate the pHAGE-Ubc-NLS-MCP-HaloTag. To achieve this, a HaloTag ORF fragment was generated by PCR and inserted into the digested pHAGE-Ubc-NLS-MCP-YFP vector by ligation. We used the pHAGE-Ubc-NLS-MCP-HaloTag plasmid to create recombinant lentiviral particles (Mostoslavsky et al., 2006 (link)) generating expression of NLS-MCP-HaloTag driven from the human ubiquitin C promoter in target cells.
The alpha-Amanitin-resistant RPB1 fused to Dendra2 (Dendra2-RPB1Amr) ORF was excised from our original Dendra2-RPB1Amr expressing vector using HpaI and NheI (Cisse et al., 2013 (link)). We digested the PB53x EF1 Series Piggybac vector (System Biosciences, Palo Alto, CA) with EcoRI, generated blunt ends and performed a second digestion with NheI. The ORF insert was then ligated into the vector using the TaKaRa DNA ligation kit LONG (TaKaRa Bio-Clontech, Shiga, Japan) following the blunt end ligation protocol. Constructs were transformed into Stbl2 competent cells (Life Technologies, Carlsbad, CA), resistant colonies were then screened by restriction analysis and confirmed by sequencing.
The alpha-Amanitin-resistant RPB1 fused to Dendra2 (Dendra2-RPB1Amr) ORF was excised from our original Dendra2-RPB1Amr expressing vector using HpaI and NheI (Cisse et al., 2013 (link)). We digested the PB53x EF1 Series Piggybac vector (System Biosciences, Palo Alto, CA) with EcoRI, generated blunt ends and performed a second digestion with NheI. The ORF insert was then ligated into the vector using the TaKaRa DNA ligation kit LONG (TaKaRa Bio-Clontech, Shiga, Japan) following the blunt end ligation protocol. Constructs were transformed into Stbl2 competent cells (Life Technologies, Carlsbad, CA), resistant colonies were then screened by restriction analysis and confirmed by sequencing.
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