The Twist Codon Optimizer Tool randomly assigns synonymous codons based on the selected organism's codon preferences. Here is a list of considerations during optimization: We assign more common codons more frequently than less common ones We avoid using rare codons (those with a codon frequency of <8%) We ensure sequences do not have strong hairpins (∆G <-8) in the first 48 base pairs of resulting sequences We check both strands to ensure they don't contain the enzyme cut sites you asked us to avoid creating We avoid introduction of promoter sequences internal to expression sequence by avoiding the creation of strong sigma70 binding sites We avoid sequences that create strong ribosome binding sites (GGAGG and TAAGGAG) We avoid sequences that create terminator sequences (TTTTT or AAAAA) Since the most preferred codon is not always assigned, you should not expect to see the same output after each optimization attempt (no 1:1 codon to amino acid assignment). You should expect to see a different optimized sequence if you optimize the same sequence multiple times. Please note that optimal expression cannot be guaranteed for any sequence that goes through Twist optimization as there are many factors that go into quality expression, some of which are not well understood. Additional factors, such as mRNA stability, protein maturation, translational speed, chaperone expression, promoter design, expression vector etc. also influence expression.