Schematic diagram of three-fragment Gateway MultiSite recombination cloning.

Fragments 1, 2, and 3 are amplified by PCR using oligonucleotides that incorporate flanking attB1 and attB4 sites in fragment 1, flanking attB4r and attB3r sites in fragment 2, and flanking attB3 and attB2 sites in fragment 3. Fragment 1 is combined with pDONR 221 P1-P4, fragment 2 with pDONR 221 P4r-P3r, and fragment 3 with pDONR 221 P2-P2 in separate BP reactions. The products of the BP reactions are pENTR attL1-Frag1-attL4, pENTR attR4-Frag2-attR3, and pENTR attL3-Frag3-attL2. In the LR reaction these three entry clones are combined with a destination vector to produce an expression clone containing fragment 1, fragment 2, and fragment 3 in a position and orientation-specific manner. Note that the pENTR R4-R3 and pENTR L3-L2 entry clones are also used in four-fragment Gateway MultiSite recombination cloning. Schematic modified from the Invitrogen MultiSite Gateway Pro user manual.