How do I change or optimize codons in a coding sequence?
In this example we optimize the codon usage of a bacterial CDS using a custom codon usage table (CUT). The CUT specifies the preferred codons of highly expressed proteins secreted by the fungal expression host Trichoderma reesei.
Open a Coding Sequence (CDS)
Open a DNA sequence and select the CDS feature. To add a CDS feature to a unannotated sequence see Create a Translated Feature.
Choose Alternative Codons
Click on the CDS feature to select it.
Click menu Features → Codons → Choose Alternative Codons.
Set the Genetic Code and Select a Codon Usage Table
Click the "Genetic Code" drop down menu to set the genetic code for your target recombinant host.
Click the "Codon Usage Table" dropdown menu to choose a codon usage table.
Choose from" Standard Tables" (sourced from https://www.kazusa.org.jp/codon/ †) or choose a custom table from "Additional Tables". See Import or Create a Codon Usage Table to learn how to import or create a custom codon usage table.
† SnapGene provides a selection of "default" codon usage tables sourced from https://www.kazusa.or.jp/codon/. We recommend all users consult the literature for advice on the appropriate codon usage table to use to optimize a CDS for a target heterologous host.
Manually Replace Rare Codons
- Set the arbitrary threshold at which a synonymous codon frequency is considered rare based on the selected codon usage table (select from a threshold of 6-16%). Rare codons will be highlighted yellow on the sequence.
- Use the arrows to step between all "rare" codons.
- Click an amino acid name and choose an alternative synonymous codon from the dropdown.
- Modified codons will be highlighted blue.
- A summary of rare codons and modified codons is shown in the bar.
- Optional: Check the option to "Make a note that rare codons were removed". This will add a
/notequalifier to the CDS feature indicating that "x rare codons [based on] a_codon_usage_table were removed".
Once all modifications are made, click OK to accept the codon changes.
The DNA sequence now differs from the original (although the CDS will encode the same protein sequence) and will be unsaved.
Click File → Save As to save the modified sequence with an appropriate name.
Let SnapGene Automatically Replace Rare Codons
Click the "Genetic Code:" drop down to set the genetic code for your target recombinant host.
Click the "Codon Usage Table" dropdown menu to choose a codon usage table.
Choose from" Standard Tables" (sourced from https://www.kazusa.org.jp/codon/ †) or choose a custom table from "Additional Tables". See Import or Create a Codon Usage Table to learn how to import or create a custom codon usage table.
Click Replace Codons and choose whether to optimize to Replace Rare Codons or Replace Non-Preferred Codons. In this example we choose to Replace Rare Codons.
How do the replacement alternatives differ?
Replace Rare Codons - This method replaces only those synonymous codons that are rarely used by the target host (selection is based on the specified "rare" threshold set for the codon usage table).
- Will usually make fewer changes to the CDS sequence
- Makes use of all "good" synonymous codon options
Replace Non-Preferred Codons - This method replaces all synonymous codons to the single highest frequency (Preferred) synonymous codon for each amino acid/stop codon.
- All available synonymous codons will not be used in the revised sequence
Rare codons that have been substituted will be shown highlighted blue. NOTE: if you select the wrong "Replace" option, click menu Edit → Undo Replace Rare Codons to revert back to the original sequence.
Optional: Check the option to "Make a note that rare codons were removed". This will add a /note qualifier to the CDS feature indicating that "x rare codons [based on] a_codon_usage_table were removed".
Click OK to accept the suggested Codon changes.
"Save As" to Save the Modified Sequence
Consider renaming the CDS feature to clearly indicate that the feature is no longer encoded by the native DNA sequence.
Click menu File → Save As and save the "Codon optimized" Sequence as a new file.
