Plasmid construction for spectral characterization of PpPHY and PpCRY

The Escherichia coli strains Mach1 T1^R (Thermo Fisher Scientific) and DH5α (TaKaRa) were used for DNA cloning. These cells were grown on Lysogeny Broth (LB) agar medium at 37 °C. The transformed cells were selected by 20 µg mL⁻¹ kanamycin or 100 µg mL⁻¹ ampicillin.

The PpPHY region (1–1986 bp in PpDUC1) was cloned into Nde I and BamH I sites of the pET28a vector with N-terminal His-tag (Novagen) using the Gibson Assembly System (New England Biolabs, Japan). The DNA fragment of the PpPHY region was amplified by PCR using KOD One^TM PCR Master Mix (Toyobo Life Science) with a codon-optimized synthetic gene for expression in E. coli (Genscript) and an appropriate nucleotide primer set (Supplementary Table ²). The pET28a vector was amplified by PCR using DNA polymerase with template DNA and an appropriate nucleotide primer set (Supplementary Table ²).

The PpCRY region (3115–5073 bp in PpDUC1) was cloned into the EcoR I and Xba I sites of a pCold GST DNA vector with an N-terminal GST-tag (TaKaRa) using restriction enzymes and ligase. A DNA fragment of the PpCRY region was amplified by PCR using PrimeSTAR Max DNA Polymerase (TaKaRa) with genomic DNA from P. provasolii and an appropriate nucleotide primer set (Supplementary Table ²). All of the plasmid constructs were verified by nucleotide sequencing (FASMAC).