Vectors and Cloning: Controls and Synthetic Core Promoters Fused to the PAOX1-R

Ten different controls were created using the genomic wild type P_AOX1 sequence as template: deletion of the entire upstream regulatory region (CRM) upstream of the core promoter, deletion of the core promoter, replacement of the natural AOX1 core promoter with the core promoter of the HHF2 gene^46,47 and seven completely random sequences. For the first control (deletion of CRM) primers C-WO-CRM1 and eGFP-pAOX1–3prime were used. For the remaining controls, pAOX1_Syn_dBamHI_SwaI-forward was used as forward primer, while as reverse primers were C-WO-Core1, C–W–HHF2+10 and R1 to R7, respectively. The primers sequences are provided in Supplementary Table S1.

The synthetic core promoters were ordered as long primers (Ultramer DNA Plate Oligo by Integrated DNA Technologies (Leuven, Belgium) in 96-well microtiter plates), attached by PCR to the P_AOX1-R and cloned into the P. pastoris/E. coli shuttle vector pPpT4_SB-truncatedAOX1-eGFP, reported by Vogl et al.(25) The plasmid genbank file and respective map are available in the Supporting Information and Supplementary Figure S1. The synthetic promoters were amplified using forward primer pAOX1_Syn_dBamHI_SwaI-forward and the reverse primers listed in Supplementary Tables S2–S5.

The final PCR product was gel purified and cloned by assembly cloning into the SwaI and NheI digested vector backbone. All constructs were verified by Sanger sequencing.