Fig. 4. Large duplications and rearrangements and restoration to desired structure.
(A) Karyotypic analysis of synX(A-R), yYW0077 and corrected synX strain, yYW0115 by pulsed-field gel electrophoresis. (B) Two large duplications and rearrangements occurred in the synX(A-R) strain. Sequencing coverage of synX(A-R) strain revealed two large duplications of synthetic fragments. The NotI* site and loxPsym site mediated junction of duplicated segments. (C) Massive duplications and rearrangements occurred during integrative transformation. Intermediate assembly strain semi-synX(A-C) (yYW0007) and synX(A-R) have identical copy numbers in the duplicated region. (D) Meiotic crossover to generate a synX strain lacking amplified segments. (E) Verification of the absence of duplication regions in synX strain yYW0115 by means of junction primers.