Workflow of ligation-independent cloning into the pDEP vectors. (A) Experimental steps, timeline, and hands-on time associated with each step of cloning a gene of interest into the pDEP vectors. From beginning to end, cloning take less than 5 days and ~6 hr of hands-on time. (B) Agarose separating gel visualized with ethidium bromide. Lane 1: digested vector pC-DEP, lane 2: DNA size marker, lane 3, PCR product of the HECT-domain of Rsp5 (Rsp5HECT, residues 446–809). (C) DNA fragments from Figure 3B was transformed into yeast and plated on selective media. Top plate (−), negative control containing only digested plasmid, and Bottom plate (+), successful recombination of PCR product and digested vector. (D) Western blot analysis for positive recombination events using α-GST antibodies. Lane 1, GST protein expression from pC-DEPC not containing an ORF, and lane 2, expression of GST-Rsp5HECT from recombination plasmid pC-DEPC- Rsp5HECT. (E) Schematic view of the plasmid insertion site and the PCR product designed to integrate into the pDEP plasmids via homologous recombination in yeast. The flanking ends can be generated as part of the PCR primers and will place the open reading frame of interest in frame with the 5’ GST open reading frame.