All cycling programs and oligos can be found in Supplementary Tables S1 and S2 . More details of the cloning procedure can be found in the Supplementary Materials and Methods section. In short, the evolution plasmids pEVO-loxHEX-L, pEVO-loxHEX-R, pEVO-loxHEX-R1 and pEVO-loxHEX-R2 were generated in two-steps from the previously described pEVO-loxP (30 (link),33 (link)) using the Cold Fusion Cloning Kit (SystemBiosciences). The additional ribosome binding site and appropriate restriction sites to express two recombinases from one plasmid were introduced while preparing the pEVO-loxHEX1+2. All PCR reactions were performed with a high-fidelity polymerase following the manufacturer's manual (Herculase II Fusion DNA Polymerase, Agilent).
The expression plasmid pIRES-NLS-EGFP was generated in two steps using the previously described pIRESneo-Cre vector and standard restriction and ligation cloning (30 (link)).
The loxHEX reporter plasmid was generated by a four-step cloning using the plasmids pCAG-loxPSTOPloxP-ZsGreen (Addgene #51269) and pCAGGS-loxP-mCherry-loxP-EGFP (31 (link),34 (link))
The expression plasmid pIRES-NLS-EGFP was generated in two steps using the previously described pIRESneo-Cre vector and standard restriction and ligation cloning (30 (link)).
The loxHEX reporter plasmid was generated by a four-step cloning using the plasmids pCAG-loxPSTOPloxP-ZsGreen (Addgene #51269) and pCAGGS-loxP-mCherry-loxP-EGFP (31 (link),34 (link))
