‘HoneySweet’ leaf DNA was extracted using a CTAB protocol33,34; starting with ~1 g of fresh leaf and 7.5 ml of extraction buffer. Primer design used MacVector to predict the best sites that flanked the junction sequences as well as other locations in the transgenes (Table S8). Primers were synthesized by IDT (Coralville, IA). PCR used ~50 ng of DNA per 20 µl reaction using Applied Biosystems™ AmpliTaq™ DNA Polymerase with Buffer II (ThermoFisher Scientific) according to manufacturer’s directions. The standard cycle conditions were 5 min at 95 °C, then 30 s at 95 °C, 30 s at 55 °C, and 30 s at 72 °C for 25 cycles followed by 7 min at 72 °C. Variations were added to obtain single fragments of expected sizes, especially for longer fragments, including raising the temperature of annealing from 55 up to 70 °C, changing Mg concentration, adding DMSO, changing cycling program by adding increasing extension times at 72 °C, increasing cycle numbers and using Phusion TAQ polymerase—Phusion (NEB, Ipswitch, MA). The products were run on agarose gels and visualized with Typhoon FLA scanner (GE Healthcare Life Sciences, Marlborough, MA).

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