Figure 1: Marker recycling strategy. (A, C ) Transformants are generated by integration of the CpPosNeg plasmid into a neutral site within the WT plastome via homologous recombination of left and right homology arms (LHA and RHA) and selection on Spc conferred by aadA. Initial transformants are then re-streaked on plates supplemented with Spc to eliminate all WT copies and generate the intermediate R1 plastome. (B, D ) The R1 strains are then re-streaked on plates supplemented with 5-FC. Negative selection conferred by codA selects for plastome copies where thecodA-aadA marker has been lost via intramolecular recombination of the rbcL direct repeat. Re-streaking on 5-FC results in the final marker-free R2 strain.