Integrase-catalyzed Plasmid DNA Insertion

This assay was performed using the standard protocol described previously^{24 (link),27 (link)}. Briefly, 25 nM PFV intasome, 50 ng of 3 kb supercoiled (SC) plasmid DNA (pGEM-T Easy, Promega), 10 mM Bis-tris propane, pH 7.5, 110 mM NaCl, 5 mM MgSO₄, 4 μM ZnCl₂, and 10 mM DTT in 15 μl total volume were incubated at 37 °C for 5 min. The reactions were terminated by adding 0.1% SDS, 2.5 mM EDTA, 1 mg/ml proteinase K and incubated at 55 °C for an hour. The products were mixed with 5% glycerol before resolving on a 1% agarose gel in 1X TAE at 105 V for an hour. Gels were stained with 0.1 μg/mL ethidium bromide and scanned on a Sapphire Biomolecular Imager (Azure Biosystems). Unreacted SC plasmid and linear concerted integration products were quantified with AzureSpot software (Azure Biosystems) as described previously^{24 (link),27 (link)} and presented in Supplementary Fig. 1.