We report results from scanning tunneling microscopy experiments in which supercoiling of the plasmid, pTNT, leads to localized duplex denaturation at a single location. Measurements of the imaged strand-separated region suggest an extent of approximately 60 base pairs (bp's), in excellent agreement with the prediction of a statistical mechanical analysis of the pTNT base sequence that accounts for the topology-mediated global coupling of melting behaviors. Notwithstanding that the sequence of pTNT includes a 30-bp run of poly-A, where thermodynamic considerations alone predict significant instability, based on the statistical result, we propose the location of denaturation is an approximately 60-bp run, spanning positions 2050-2110, that is 82% AT-rich, terminally flanks the beta-lactamase coding region, and is known to contain two sites susceptible to cleavage by the Dral restriction enzyme.