Figure 2. Schematic diagram for the round of gene stacking of the first two genes.

(A) Through in vivo Cre/loxP-mediated recombination, the donor plasmid with the first gene was integrated into the recipient vector via loxP. Then the backbone of donor plasmid was excised to form a linear targeting construct. Next, the second gene or DNA fragment was retrieved by in vivo Red-mediated recombination and gap repair. (B) Detailed schematic illustration of how Red-mediated recombination might occur. E1/2, rare restriction enzyme set 1 / 2; Ori, pUC replicon; Amp^r, ampicillin resistance gene; Kan, kanamycin selection gene; pVS1, pVS1 replicon; M, marker gene; LB and RB, left border and right border of the T-DNA.