Application of these rules to the complete ensemble of 4 l sequences not only informs the design of this optimal ensemble of different sequences with minimal misbinding, but also provides an estimate for the largest number of unique sequences that can be combined during one-pot selfassembly (Table , see the full procedure in Supporting Information, Section S5). Interestingly, application of the terminal mismatch rule results in the same number of sequences for all the hundred sequence ensembles generated for each l, yet each of them contain a unique set of sequences. This may be because application of the rule to a specific sequence corresponds to eliminating the other 15 (out of 16) different ways nucleotides can be arranged at the terminal positions. We also noticed that due to the discrete nature of the rules in Equations S2-5, there is a non-monotonic reduction of sequences across different lengths. For example, the average number of permitted sequences after application of the double internal mismatch rule for 6 nt sequences is higher than that for 7 nt (46 vs. 39, respectively) as md* jumps from 1 to 2, respectively. This results in the final number of sequences within a collection with ε'/ε ≤ (ε'/ε)c for an ensemble of 7 nt sequences to be comparable with that of 6 nt sequences.