Production of bacterial artificial chromosomes (BACs)

The cDNA amplicons were inserted into the BAC vector pBeloBAC11 (New England BioLabs) using the In-Fusion HD Eco-Dry Cloning Kit (Clontech). Briefly, pBeloBAC11 was converted to a linearized form using the long PCR as above, with primers Kos15_NotI_pBelF and Kos15_NotI_pBelR (Additional file 1), which contain 15-nt overhangs identical to regions of the primers used for the preparation of the cDNA amplicons. The linear product was gel purified and quantified as described above. The linearized pBeloBAC11 vector was mixed with amplicons (ca. 1:2 molar ratio) in a volume of 10 μl, which was transferred to the In-Fusion HD EcoDry tube. The mixture was incubated at 37 °C for 15 min and then at 50 °C for 15 min. A 2.5 μl aliquot was used to transform E. coli Stellar competent cells according to the manufacturer’s protocol, and colonies grown on LB plates containing chloramphenicol (CAM) (15 μg/ml).