Construction of Integrative Plasmids

The integrative plasmids (Table S7) were constructed using gene and promoter BioBricks (Table S8). Specific primers (Table S8) were used to amplify the fragments using Phusion U polymerase (ThermoFisher Scientific, Waltham, MA, USA). The native genes were amplified from S. cerevisiae CEN.PK genomic DNA, and the heterologous genes were synthetized by GeneArt (ThermoFisher Scientific, Waltham, MA, USA). The empty integrative vectors were digested with FastDigest SfaAI (ThermoFisher Scientific, Waltham, MA, USA) restriction endonuclease, nicked with Nb.BsmI (New England BioLabs, Ipswich, MA, USA), and finally assembled together with the PCR-amplified genes and promoters. To express the transporter coding gene in the oocytes, it was cloned downstream of the T7 promoter in the USER compatible Xenopus expression vector pNB1u as described previously [2 (link),3 (link)]. The empty vector was digested by PacI and nicked by Nt.BbvCI (New England BioLabs, Ipswich, MA, USA) for USER-cloning of the amplified transporter ORF. The reaction was transformed into chemically competent E. coli cells. Finally, plasmid DNAs from single colonies were sequenced and confirmed.