Qiaprep spin plasmid mini prep kits

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).

All DNA manipulations were carried out in E. coli DH5α as described43 . Plasmid cloning was performed using Gibson Assembly Cloning Kit (New England Biolabs, UK) following the manufacturer’s instructions. Briefly, DNA fragments sharing homologous regions to adjacent DNA fragments were generated via polymerase chain reaction (PCR) or restriction enzymes digestions. The desired DNA fragments were purified with QIAquick Gel Extraction Kit (Qiagen, Valencia, CA) and assembled with the linearized backbone in a single step. The sequences of all PCR primers and templates used are listed in Supplementary Table S1 in the supplemental material. Primers were synthesized by Sigma Aldrich (Singapore). The complete list of fragments for plasmid construction by DNA assembly in this study is summarized in Supplementary Table S1. QIAprep Spin Plasmid Mini-prep Kits (Qiagen, Valencia, CA) were employed to prepare plasmid DNA from E. coli. All restriction enzymes were obtained from New England Biolabs.