The IDT Codon Optimization Tool simplifies designing synthetic genes, single-stranded, and double-stranded DNA 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 reassigning codon usage. For example, if a human protein from nature is going to be expressed using an E. coli system, the DNA sequence should be changed taking the codon usage of E. coli into account, which would be considered codon optimization for E. coli expression. 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 genes and gene fragments.
Easy steps for using the Codon Optimization Tool
Use of the Codon Optimization Tool is straightforward (see Figure 1 for a screen shot of the tool interface):
1. Select the Sequence Type by clicking either DNA Bases or Amino Acids.
2. Select the desired Product Type of either gBlocks™ Gene Fragments, Megamer™ ssDNA Fragment, eBlocks™ Gene Fragments, or Gene to set the complexity rules that the tool will use.
3. Select your Organism for expression from the pulldown list (If working with cell lines, select the animal or plant origin).
4. Paste in your sequence (Bulk entry is also available in a variety of input formats).
5. (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.
6. 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).
a. 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).
7. Order sequence using the blue Order item button.
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. For example, during codon optimization for E. coli expression the Codon Optimization Tool will try to maintain the natural codon bias and remove any codons that are not used naturally by E. coli at least 10% of the time.
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.
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, Using a codon optimization tool --how it works and advantages, for more discussion on this topic.
Get started using the Codon Optimization Tool
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 or 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.
Contact Gene Support
If you are working with an organism not listed in the Codon Optimization Tool’s Organism list, or do not see the information you need, contact firstname.lastname@example.org optimizations that fall outside of the rules used by the tool.