Cloning and Mutagenesis of SOXE Transcription Factors

Human SOXE and SOX17 expression plasmids were generated by cloning full-length coding sequences in frame with an N-terminal 3FLAG epitope (35 (link)) in the pcDNA3.1(+) vector (Thermo Fisher Scientific, Waltham, MA, USA). These sequences were amplified by PCR using PfuUltra High-Fidelity DNA Polymerase (Agilent Technologies, Santa Clara, CA, USA) and human cDNA using forward and reverse primers containing BamHI and EcoRI sites, respectively (Supplementary Table S2). Plasmids encoding GAL4^DBD/SOXE fusion proteins were generated by cloning SOXE cDNA segments into the pBIND plasmid (Promega, Madison, WI, USA). These segments were generated by PCR using custom-made primers (Supplementary Table S3). Missense mutations were introduced in SOX sequences by QuikChange Site-Directed Mutagenesis (Stratagene, San Diego, CA, USA) using tailored primers (Supplementary Table S4). The integrity of all plasmid inserts was verified by Sanger sequencing.