Plasmid Cloning Protocol Using Gibson Assembly

Plasmid cloning was performed using the Gibson Assembly Cloning kit from New England Biolabs following the manufacturer's instructions or using the DNA assembler method46 (link). The list of primers used can be found in Supplementary Table 5. For DNA manipulations, yeast plasmids were isolated using a Zymoprep II yeast plasmid isolation kit (Zymo Research) and transferred into E. coli for amplification. QIAprep Spin Plasmid Mini-prep kits (Qiagen, Valencia, CA) were employed to prepare plasmid DNA from E. coli. All enzymes used for recombinant DNA cloning were from New England Biolabs unless otherwise noted. PCR, digestion and ligation products were purified by QIAquick PCR Purification and Gel Extraction kits (Qiagen). Genomic DNA isolation was performed using the YeaStar Genomic DNA kit (Zymo Research).