The promoter design is based on the CYC1 core promoter. The relevant structural elements of the CYC1 core promoter elements, which are required for transcription, were conserved in the synthetic promoter design. These elements comprised two TATA boxes at position -221 and -117 (relative to the start of the CYC1 ORF), and the 5'UTR of the CYC1 core promoter (including transcriptional start site, TSS). In the promoter design, three CggR operator sites were inserted adjacent to the two TATA boxes. All functional elements were conserved (blue colored region) during the optimization of the promoter sequence. Nucleotide sequences between the functional elements (grey colored region) were allowed to be optimized by the algorithm. Nucleotides that got optimized are indicated with a black line. A total of 75 sequence versions were generated, where each sequence differed in one mutation from the progenitor sequence. The sequences were optimized for low nucleosome affinity. After optimization, all sequences were checked for synthesis feasibility. The synthesis of the sequences was feasible (green) until the 46th round. After this round, the sequences (not feasible in red) reached a GC content insufficient for proper synthesis. The promoter sequence, which was generated in round 38 (black), showed the best compromise between minimal nucleosome affinity and the possibility to synthesize the sequence. 