Extended Data Fig. 6: Single-copy branching circuit construction in mammalian cells.

Schematic of the workflow used to generate CHO-K1 or HEK293FT cell lines carrying single-copy, recombinase-driven differentiation circuits. A landing pad containing a PhiC31 attB site, puromycin resistance (PuroR), and left and right homology arms (LHA and RHA) is first inserted into a defined safe-harbour locus using CRISPR–Cas9. After ~2 weeks of puromycin selection, single-cell clones are isolated by limiting dilution, expanded, and screened by digital PCR to identify clones with a single landing-pad copy. Verified clones then undergo PhiC31-mediated integration of the synthetic branching circuit, which carries an attP site, bleomycin resistance (BleoR), and a TagBFP reporter. Zeocin selection enriches recombined cells, and the brightest TagBFP-positive population is isolated by flow cytometry. To eliminate off-target integrations and sorting bias, a second round of clonal isolation and digital PCR is performed to confirm single-copy insertion. Finally, lentiviral delivery of inducible recombinases (e.g., Bxb1 or DHFR–Bxb1), followed by blasticidin (BSD) selection and enrichment of iRFP-positive cells, yields stable lines expressing a single-copy differentiation circuit with tightly regulated recombinase activity.