Desalted primers

PCR amplification with Phusion Hot Start II high-fidelity polymerase (Thermo Scientific, Waltham, MA) was performed according to the manufacturer’s manual using high-performance liquid chromatography (HPLC)- or PAGE-purified, custom-synthesized oligonucleotide primers (Sigma-Aldrich). Diagnostic PCR was done with DreamTaq (Thermo Scientific) and desalted primers (Sigma-Aldrich). DNA fragments obtained by PCR were loaded on gels containing 1% or 2% (wt/vol) agarose (Thermo Scientific) and 1× Tris-acetate-EDTA buffer (Thermo Scientific), excised, and purified (Zymoclean, D2004; Zymo Research, Irvine, CA). Alternatively, fragments were purified using the GenElute PCR Cleanup kit (Sigma-Aldrich). Plasmids were isolated from E. coli with the Sigma GenElute Plasmid kit (Sigma-Aldrich) according to the supplier’s manual. Yeast plasmids were isolated according to the methods described in reference 50 (link). Yeast genomic DNA was isolated using a YeaStar genomic DNA kit (Zymo Research). E. coli DH5α (18258-012; Invitrogen) was transformed chemically (T3001; Zymo Research) or by electroporation. Chemical transformation was done according to the supplier’s instructions. Electroporation was done in a 2-mm cuvette (165-2086; Bio-Rad, Hercules, CA) by using a Gene PulserXcell electroporation system (Bio-Rad), following the manufacturer’s protocol.

PCR amplification with Phusion High Fidelity Polymerase (Thermo Fisher Scientific) was performed according to the manufacturer's instructions using PAGE-purified oligonucleotide primers (Sigma-Aldrich, St. Louis, MO, USA). Diagnostic PCR was done using DreamTaq polymerase (Thermo Fisher Scientific) and desalted primers (Sigma-Aldrich). All primer sequences are shown in Table 4. DNA fragments obtained by PCR were separated by gel electrophoresis. Gel purification was carried out using the Zymoclean Gel DNA Recovery Kit (Zymo Research, Irvine, CA, USA). PCR purification was performed using either the GenElute PCR Clean-Up Kit (Sigma-Aldrich) or GeneJET PCR purification kit (Thermo Fisher Scientific). Gibson assembly was done using the NEBuilder HiFi DNA Assembly Master Mix (New England Biolabs, Ipswich, MA, USA) according to the manufacturer's recommendations. Restriction digest with PdmI and SmaI was performed using FastDigest enzymes (Thermo Fisher Scientific), according to the manufacturer's instructions. Escherichia coli strain XL1-blue was used for plasmid transformation, amplification and storage. Plasmids were isolated from E. coli with the GenElute Plasmid Miniprep Kit (Sigma-Aldrich).