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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