HinLVA flips adjacent hixC -flanked segments independently. (A) Four pancake stack arrangements can be distinguished by quantitative multiplex PCR. Black arrows indicate primers for multiplex PCR. Bars indicate sizes of PCR amplicons. Whole stack inversions that generate the other four possible arrangements cannot be distinguished in this assay. (B) Quantitative PCR of an equimolar mix of all eight manually assembled BPP plasmids (top row), BPP plasmid (-2, 1) alone (middle row), or a single 10-hour colony of cells carrying BPP plasmid (-2, 1) in the presence of HinLVA (bottom row). The first lane in each gel contains a DNA molecular weight marker (M). (C) Histogram showing the frequency of detectable bands at 18, 20, 22, 24 and 26 PCR cycles (n = 46 for each band at each cycle). At 10 – 11 hours following cotransformation of (-2, 1) and HinLVA, the starting pancake arrangement predominates (400 bp band). A single flip of either pancake alone (producing a 500 bp or a 600 bp amplicon) is next most frequent occurrence, while two successive flips (700 bp) is least common.