Unravelling the core promoter sequence-function relationship is fundamental for engineering transcription initiation and thereby a feasible "tuning knob" for fine-tuning expression in synthetic biology and metabolic engineering applications. Here a systematic replacement studies of the core promoter and 5' untranslated region (5'UTR) of the exceptionally strong and tightly methanol regulated Komagataella phaffii (syn. Pichia pastoris) alcohol oxidase 1 (AOX1) promoter at unprecedented resolution is performed. Adjacent triplets of the 200 bp long core promoter are mutated at a time by changing the wild-type sequence into cytosine or adenine triplets, resulting in 130 variants that are cloned upstream of an eGFP reporter gene providing a library for expression fine-tuning. Mutations in the TATA box motif, regions downstream of the transcription start site or next to the start codon in the 5'UTR had a significant effect on the eGFP fluorescence. Surprisingly, mutations in most other regions are tolerated, indicating that yeast core promoters can show a high tolerance toward small mutations, supporting regulatory models of degenerate motifs, or redundant design. The authors exploited these neutral core promoter positions, not affecting expression, to introduce extrinsic sequence elements such as cloning sites (allowing targeted core promoter/5'UTR modifications) and bacterial promoters (applicable in multi host vectors).