Identification of recombination events by PCR screening

PCR of DNA extracted from the clones (adherent or suspension cultures) was performed using GoTaq Flexi DNA polymerase (Promega) to amplify genomic recombination junctions using the primers listed in the figure descriptions and 500 ng of genomic DNA from each recombinant clone or parental cells as a template in 50 μL reactions. The thermal cycling parameters for the PCR was as follows: initial denaturation at 95°C for 2 min, 35 cycles of denaturation at 95°C for 1 min, annealing at 55°C for 1 min and extension at 72°C for 1 min per kb, and a final step of 72°C for 5 min. The PCR products were analysed by electrophoresis in 0.8% agarose (Seakem Agarose, Lonza, USA) gels in 0.5X TBE (Tris-Boric acid-EDTA buffer) containing 0.5 μg/ml ethidium bromide. PCR-generated products were compared with DNA standard markers and digitally documented under UV illumination (Quantum Vilber Lourmat, Germany). PCR-amplified products were analysed by sequencing. Primers are listed in Supplementary Table S1.