To maintain wild-type protein expression:

• We avoid using rare codons (those with a codon frequency of <8%).
• We ensure sequences do not have strong hairpins (ΔG < -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.
• We avoid introducing promoter sequences internal to expression sequences 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).

To improve the likelihood of successful manufacture:

• We won’t introduce any repeat longer than 20 base pairs.
• We avoid homopolymer runs of 10 or more bases.
• We avoid fitted sequences that create global GC% of less than 25% or more than 65% and local GC windows (50 bp) of less than 35% or more than 65%.

Please note that codon optimization is useful for improving chances of a successful synthesis, not protein expression.