The extended CG model is used to sample ɑ(T,ε',ε'') for a chain of N = 10 at ⍴ = 0.01 (Figure ). Complete addressability is now achieved within a polyhedral domain in the (T,ε',ε'') space (Figure ), formed primarily from three planes. The high-T plane, Tc ≃ 0.054, is associated with the ceiling temperature described previously. There are now two low-T limit planes, associated with measuring addressability at low or high ε'/ε. These correspond to the T * /ε'' * and T * /ε' * planes respectively, as the competition between ε' and ε'' determines the extent of addressability. For example, at ε''/ε = 0.05, we observed that the critical T * is largely dominated by the T * /ε' * plane: since ε' < ε'' (|ε'| > |ε''|), all incorrect bonds tend to be ε'-rather than ε''-bonds (Figure ). Consequently, T * /ε' * is due to a kinetic trap from incorrect ε' bonds. As ε''/ε is increased to 0.4, ε''-bonds begin to dominate the incorrect structures whenever ε''/ε > ε'/ε, thereby shrinking the ɑ = 1 domain (Figure ). Practically, however, ε'/ε tends to be much larger than ε''/ε and ε''/ε is near 0, according to our DNA hybridization energy analysis, irrespective of T and sequence length (Figure , S8c). Thus, the T * /ε'' * plane can be ignored, and addressability for DNA-enabled self-assembly will be determined by Tc and T * /ε' * , akin to Figure . The DNA-informed CG models enabled us to start from considering all possible 4 l sequences of a specific length l, and establish rules that will narrow down this complete ensemble to create smaller ensembles prime for programmable self-assembly (Figure , S9). Every sequence within these optimized ensembles has its ε'/ε less than a selected cutoff, (ε'/ε)c = 0.6, with this value indicated by the T * /ε' * boundary of the ɑ = 1 region in our addressability plots (Figure ). We also chose annealing temperatures of T/ε between 0.040 and 0.053 (Figure ) as a higher annealing T slightly below Tc would help suppress the nucleation of incorrect partial structures that could frustrate and prolong successful assembly. Assuming the average energy for a WC nearest neighbor pair 49 at the appropriate temperatures, this imposes an annealing temperature at 20°C for 9 nt and 8 nt sequences (corresponding to T/ε ~ 0.041 and T/ε ~ 0.047, respectively), 10°C for 7 nt sequences (T/ε ~ 0.047), and 4°C for 6 nt sequences (T/ε ~ 0.052), all of which fit within the desired T/ε range. Some rules in the sequence design procedure are based on the identity of each DNA sequence. Palindromic sequences must be eliminated, as they could allow a distinct block to bind to another block in an incorrect reversed orientation. Self-complementary sequences must also be excluded, as a distinct block decorated with a self-complementary sequence could erroneously bind to another block of the same type when multiple copies of blocks are present. Other rules are based on the differences between one sequence and all the other sequences in the ensemble. For example, if a sequence is highly similar to another sequence but differs only by a couple of nucleotides, there may be a high chance of misbinding between the former sequence and the latter sequence's reverse complement. These problematic sequences can be eliminated by using the nearest neighbor model 49 to estimate the critical number of nucleotide mismatches, below which ε'/ε > (ε'/ε)c (specifically the critical number of single internal (ms*), double internal (md*), and terminal (mt*) mismatches, see Figure and Supporting Information, Section S5 for the derivation of Equations S2-S4). We also eliminated sequences which could form dangling ends shorter than or equal to a critical value, dl ≤ dl*, as it would lead to duplexes within the ensemble with ε'/ε > (ε'/ε)c (see Equation S5, and Supporting Information, Section S5).