Codon Optimization
How to codon optimize a gene
- Paste a protein sequence, or a coding DNA sequence whose length is a multiple of three.
- Choose the host. The sequence is recoded with that host's codon usage.
- Add any restriction sites you need to keep out, then copy or download the FASTA.
Frequently asked questions
What does codon optimization actually do?
It swaps each codon for another that codes for the same amino acid but is used more often by the host. The protein is unchanged; what changes is how readily the host's ribosomes and tRNA pool translate the message.
Which codon choice should I use?
"The most used codon" gives the highest adaptation index and is what most gene synthesis orders use. "In proportion to use" matches the host's natural mix and avoids the long stretches of identical codons that can empty a tRNA pool. "Most used, but vary repeats" is a middle course that only breaks up runs.
What is the codon adaptation index?
The geometric mean of how often each codon in the gene is used, relative to the best codon for that amino acid. 1.0 means every codon is the host's favourite. Natural highly expressed genes sit around 0.7 to 0.9.
Where do the usage tables come from?
They are the relative codon usage of each organism from the published codon usage tables, with one fraction per codon. You can paste your own table instead and choose "The table below", which is worth doing if your lab has figures for a particular strain or for highly expressed genes only.
Will optimization definitely improve expression?
No. It usually helps when a gene from one organism is expressed in another, but expression also depends on mRNA folding near the start, the promoter and ribosome binding site, GC content, protein toxicity and solubility. Rare codons are only one of the things that can be wrong.
Why avoid runs of the same base?
Long homopolymer runs make a sequence harder and more expensive to synthesise, and can cause slippage during PCR and sequencing. Six or more of one base is the usual limit.
Does it keep my restriction sites out?
It tries. Each codon that would create one of the listed sites is swapped for the next best codon for that amino acid. Some sites cannot be avoided without changing the protein, and those are named in the status line.
Is my sequence uploaded?
No. Everything runs in your browser, so your sequences never leave your device.