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. 2018 May 28;14(5):e7985. doi: 10.15252/msb.20177985

Figure EV2. Recipient toolkit construction.

Figure EV2

  1. Two constructs were built to generate recipients. The first fragment, preR1, contained loxP/lox2272 sites flanking a klURA3 marker and two 20‐bp unique barcodes flanking these loci. In this study, only the upstream barcode was used for further steps, and for simplification, the downstream barcode was omitted from Fig 1.
  2. The second construct, preR2, contained the can1Δ::P STE2 ‐spHis5‐T STE2 mating type marker.
  3. The two fragments were assembled in vivo using a derivative of the delitto perfetto construct.
  4. Resulting proRecipients were arrayed and Sanger‐sequenced to confirm integrity of preR1 loci. ProRecipients with confirmed preR1 loci were mated with SGA query strains to generate strains carrying both a uniquely barcoded recipient construct and a gene deletion of interest. Then, they were sporulated and the haploid MAT a progeny were selected using the mating type maker indicated in panel (C). Details on selective media are shown in Fig EV3.