(A) CRE sequences were designed using 130 bp regions with 30 bp steps that covered the 250 bp upstream of the transcription start site (TSS). (B) Intra-specific CREs include the Oak (YJF153, black) and ChII (SX6, green) alleles, as well as each variant substituted into the Oak and ChII background. Inter-specific CREs include the S. cerevisiae (black) and S. uvarum (green) alleles as well as two chimeric alleles designed with recombination in the center of CREs. (C) Synthesized 200 bp sequences contained priming sequences to amplify the library, restriction sites (RS) for cloning, fill sequences to compensate for any difference length due to insertion deletion polymorphisms (InDels), barcodes (BC), and CREs. (D) Synthesized sequences were cloned into pIM202, then YFP with the TSA1 core promoter was inserted between the CRE and barcode. This eliminated the fill sequence and placed the BC in the 3' UTR of YFP. (E) The reporter gene (YFP) with the barcoded CREs was integrated at the URA3 locus. The A and P1 primers were used for barcode sequencing of extracted RNA and DNA.