The IDT Codon Optimization Tool simplifies designing synthetic genes, Megamer Single-Stranded Gene Fragments, and gBlocks Gene Fragments for expression in a variety of organisms. The main design consideration is to optimize a DNA or protein sequence from one host organism for expression in another by re‑assigning codon usage. The tool also makes it easy to adjust a sequence that has difficult secondary structure, a repetitive motif, or high/low GC content for compatibility with IDT manufacturing requirements for single-stranded and double-stranded gene fragments.
Easy steps for use
Use of the Codon Optimization Tool is straightforward (see Figure 1 for a screen shot of the tool interface):
- Select the Sequence Type by clicking either DNA Bases or Amino Acids.
- Select the desired Product Type of either gBlocks Gene Fragments, Megamer ssDNA Fragment, or Gene to set the optimization rules that the tool will use.
- Select your Organism for expression from the pulldown list (If working with cell lines, select animal or plant origin) .
- Paste in your sequence (Bulk entry is also available in a variety of input formats).
- (optional) To designate a specific part of the sequence for optimization, click on Highlight Codons. You can then edit the sequence by changing the reading frame shift and by indicating start/stop codons.
- Obtain results in one of the following ways:
- Use default optimization rules
- Click on Optimize (lower right orange button).
Note: multiple clicks on the Optimize button will generate slightly different sequences.
- (Optional; available only for amino acid sequences) Click on Manual Optimization (Figure 2) to change codon usage for any amino acid or stop codon in your sequence (Figure 3).
- To manually optimize your original sequence, click on Map Codons Only and then Manual Optimization (Figure 2) to change codon usage for any amino acid or stop codon in your sequence (Figure 3).
- Order sequence using the blue Order item button.
Figure 1. Single sequence entry for the Codon Optimization Tool.
Natural codon bias and optimized sequence ranking
The Codon Optimization Tool randomly chooses codons with a bias similar to the natural bias observed in the selected organism’s genome (Figure 2); however, rare codons present less than 10% of the time are not included (for example, Figure 3, codon 1, AGA). With no one-to-one codon to amino acid assignment by the algorithm, multiple optimization attempts of the same sequence will generate different results, possibly having different restriction enzyme sites, as synonymous codons are assigned based on frequencies found in the selected codon table.
Figure 2. Codon-optimized sequence result. You can also elect to manually optimize the sequence, which allows you to select the exact codons you would like to use. This feature is useful for handling problematic regions of sequence; e.g., optimization of high GC codons that encode Alanine or Glycine. Using Manual Optimization, you could assign codons that are 66% GC, rather than 100% GC, which is useful in high GC genomes.
With the Manual Optimization view (Figure 3), you can make desired changes to individual codons by simply clicking on their locations. Hovering your cursor over a particular codon will generate a pop out window showing codon frequency. Changes made using Manual Optimization are automatically reflected in the results.
Figure 3. Codon view for Manual Optimization. The Manual Optimization screen splits each codon into an individual box. Codons chosen by the tool are shown in blue. Those selected for the final sequence are highlighted as a gray box. Codons used less than 10% of the time in the chosen organism are shown in gray, struck-through text.
A caveat: Does codon optimization equal higher expression?
While codon optimization is important for optimal protein expression, it does not guarantee higher expression levels. Additional factors, such as mRNA stability, protein maturation, translation speed, chaperone expression, promoter design, etc. also influence expression. See the DECODED article, Benefits of codon optimization, for more discussion on this topic.
Instead of spending days designing and constructing your synthetic gene, use the Codon Optimization Tool to order your gene in minutes through IDT, and spend the saved time advancing your research, while we make your genes, Megamer Gene Fragments, or gBlocks Gene Fragments.
Access IDT’s free, online Codon Optimization Tool and get started.
We also provide a step-by-step tutorial for using the Codon Optimization Tool. View it here.