(a) Stall lifetime distribution of G40P stalls on the 1GC substrate. A single exponential fit yields a characteristic lifetime of 79 ± 5 ms. (b) Fraction of all traces with protein activity with complete unwinding on different substrates (AT, 1GC, 2GC, 3GC) and at 21°C and 33°C, respectively (n.s. denotes not significant, *p<0.05, ***p<0.001). (c) Sketch of the intrinsic calibration constructs with x AT base pairs followed by 40-x GC base pairs. Due to its exquisite sequence sensitivity G40P stalls at the GC base pairs. (d) Stall FRET level histograms for four different substrates: 4AT, 7AT, 10AT and 13AT. The Histograms were evaluated with Gaussian fits. The leakage of donor fluorescence to the acceptor channel was determined to give an apparent EFRET = 0.11 ± 0.02. (e) through (h) Sample traces of calibration substrates 4AT, 7AT, 10AT and 13AT, respectively, with the analyzed stall level indicated by arrows.