Constructs were synthesized using the PCR scheme developed by Zhang and Crothers (32 (link)), as described previously (3 (link)). In short, PCR reactions (50 μl) contained 1× PrimeSTAR buffer, 0.2 mM dNTP mix, 2 μM of each of the primers, 30 nM of the top and bottom templates and 0.04 units of PrimeSTAR DNA polymerase (Takara, Japan). Cyclization kinetics measurements were carried out as described before (3 (link),32 (link)). Ligase concentration was varied as a function of phasing length (0.3 U/μl for the in-phase 156L14 and 156L16 constructs and 1.2 U/μl for all other phasing constructs) and total length (from 0.2 U/μl for the 157 constructs, to 0.8 U/μl for the 154–155 and 158–160 constructs and 2.2 U/μl for the 150–153 constructs). Quantitative data on the conformational properties of the tested DNA molecules was derived by the simulation program developed by Zhang and Crothers (33 (link)), as previously described (3 (link)). The outcome of the simulations are the bend angle, the twist angle, the roll and tilt fluctuations (defined as the thermal fluctuations of the roll and tilt angle between adjacent base pairs, 32 (link),33 (link)), and the twist fluctuations (defined as the thermal fluctuations of the twist angle between adjacent bases, 32 (link),33 (link)), per DNA sequence (p53 half-site).