Synthetic Biology
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Codon optimization tool makes synthetic gene design easy

Free, online Codon Optimization Tool

The IDT Codon Optimization Tool simplifies designing synthetic genes 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 gene or gBlocks Gene Fragments manufacturing requirements.

Easy steps for use

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 or Gene to set the optimization rules that the tool will use.
  3. Select your Organism (if working with cell lines, select animal or plant origin) for expression from the pulldown list.
  4. Paste in your sequence (Bulk entry available in a variety of input formats).
  5. (optional) To designate a specific part of the sequence for optimization, click on Custom Reading Frame.
  6. Obtain results in one of the following ways:
    1. Use default optimization rules
      • Click on Optimize (lower right orange button).
        Note: multiple clicks on the Optimize button will generate slightly different sequences.
      • (optional) Click on Manual Optimization (Figure 2) to change codon usage for any amino acid or stop codon in your sequence (Figure 3).
    2. 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 one of the blue buttons.
codon optimization tool screenshot

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.

codon optimization tool screenshot

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 Manual Optimization, the codon usage result is displayed in an interactive Manual Optimization view (Figure 3). From here, 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.

codon optimization tool screenshot

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 < 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.

Get started

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 gene 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.

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 our Genes Support group at: We can accept non-standard optimizations that fall outside of the rules used by the tool (design fee may apply).

Product focus—custom dsDNA fragments

gBlocks® Gene Fragments

These double-stranded, sequence-verified, DNA genomic blocks, 125–3000 bp in length, are designed by you, and are shipped in 2–5 working days for affordable and easy gene construction or modification. They have been used in a wide range of applications including CRISPR-mediated genome editing, antibody research, codon optimization, mutagenesis, CRISPR genome editing, and aptamer expression. They can also be used for generating qPCR standards.

gBlocks Gene Fragments Libraries

gBlocks Gene Fragments are also available as dsDNA fragment pools that contain up to 18 consecutive variable bases (N or K) for recombinant antibody generation or protein engineering.

Learn more about gBlocks Gene Fragments at

Additional resources

The OligoAnalyzer® Program—Get quick instructions on how to use this free IDT web tool that allows you to determine the physical characteristics of your oligonucleotides.

Benefits of codon optimization—Planning to express a gene in a heterologous system? Learn how rebalancing codon usage is important for optimizing protein expression. While there are no known methods to predict protein expression, as numerous factors contribute to ultimate protein yield, codon optimization plays a critical role.

Review other DECODED Online newsletter articles on synthetic biology applications.

You can also browse our DECODED Online newsletter for additional application reviews, lab tips, and citation summaries to facilitate your research.

Authors: Hans Packer, PhD, is a scientific writer, and Sam Shen, PhD, is a product applications specialist; both at IDT.

© 2016, 2017 Integrated DNA Technologies. All rights reserved. Trademarks contained herein are the property of Integrated DNA Technologies, Inc. or their respective owners. For specific trademark and licensing information, see

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