Why use codon optimization?
Species-specific codon usage is an important factor to consider for improving protein expression. In many cases, optimizing the sequence of codons to systematically utilize abundant tRNAs during translation has significantly improved protein expression.
To begin, select the part of a DNA sequence you wish to optimize and right click:
Alternatively, click “Analyze” on the menu bar, and then "Optimize Codons." You can choose to optimize the forward or reverse strand.
Specify your parameters
You have several parameters to set:
- GC Content
Organism: Specify the target organism in which you wish to express the sequence. The algorithm will create a sequence whose codons match the codon frequencies of the specified organism.
GC Content: Select a range of desired GC content. The algorithm will attempt to create a sequence that matches the specified range.
Uridine: Select or deselect "mRNA uridine depletion." Depleting uridine in transcribed mRNA has been implicated to reduce immunogenicity of RNA.
Identify protected regions
Specify regions of the sequence you wish to remain the same in the optimization. You can choose annotations or select any portion of the sequence.
Avoid creating new restriction sites or preserve existing ones
Avoided cut sites: Select restriction enzymes to avoid creating new recognition sites for those enzymes. This will also remove any existing cut site for that enzyme.
Preserved cut sites: Add a cut site to ensure the recognition site is maintained in the optimized sequence. No additional cut sites of this type will be created.
Preview your optimization
After specifying your parameters, click "Preview Optimization."
Based on your inputs, the codon optimization algorithm generates an optimized sequence. Review the summary of changes to determine whether you're satisfied with the result.
If you are satisfied, click "Save as new sequence." Alternatively, if you wish to change the nucleotides of the DNA sequence you are currently viewing, click "Save." If you are not satisfied with the result, click "Back" and change your input parameters.
Perform back translations
Want to back translate an optimized sequence? Check out this article to learn more.