For codon optimization, what steps are taken to maintain wild-type protein expression levels?

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.  

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