Co-selection is required for efficient multiplex recombination.

Multiplex insertion was performed using a pBeloBAC11 plasmid (19.5 kb total size) containing the region of the P2rx1 gene shown in S1 Fig. The Zeocin and Gentamicin lagging strand protected cassettes were co-transformed with a Kanamycin resistance plasmid and selected with Zeocin and Kanamycin or Kanamycin only. BAC DNA was prepared using the QIAquick Spin Miniprep kit (Qiagen) from each selection scheme and 100 ng of DNA was transformed into HS996 cells and plated with combined Zeocin and Gentamicin selection. Values represent averages; error bars indicate standard error of mean (n = 3).