ORFeome Cloning and Systems Biology: Standardized Mass Production of the Parts From the Parts-List

The Gateway recombinational cloning system. (A) ORFs flanked by recombinational sites B1 and B2 are directionally cloned into a P1::ccdB::P2 Donor vector using the BP reaction. The resulting Entry clones are selected using the kanamycin-resistance marker (Km^r). (B) ORFs flanked by L1 and L2 sites are directionally transferred from Entry clones into R1::ccdB::R2 Destination vectors using the LR reaction. The resulting Destination clones are selected using the ampicillin resistance marker (Amp^r). (C) Promoters flanked by recombinational sites B4 and B1r are directionally cloned into a P4::ccdB::P1r Donor vector using the BP reaction. The resulting Entry clones are selected using the kanamycin-resistance marker (Km^r). (D) ORFs flanked by L1 and L2 sites and promoters flanked by L4 and R1 are directionally transferred right adjacent to each other from their respective Entry clones into R4::ccdB::R2 Destination vectors using the LR reaction. The resulting Destination clones are selected using the ampicillin-resistance marker (Amp^r).