Multiplex recombination is limited by availability of DNA template.

(A) Insertion assay. A Gentamicin lagging strand protected cassette was inserted at a site of the P2rx1 gene. (B) Multiplex insertion. Two different Gentamicin and Zeocin lagging strand protected cassettes were inserted at two different sites of the P2rx1 gene. Recombination assays were performed with different amounts of each DNA cassette in the electroporation. Values represent averages; error bars indicate standard error of mean (n = 4). (C) Multiplex insertion PTO assay. Three different Zeocin, Gentamicin and Blasticidin resistance cassettes were PTO protected or unmodified and inserted at three different sites of the P2rx1 gene (n = 3). (D) SPI PTO assay. SPI was performed using a p15A dhfrII subcloning plasmid and the Zeocin and Gentamicin cassettes used in (A) (n = 3).