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Put an end to codon usage bias with these helpful tips

Most amino acids are determined by more than one codon and these synonymous codons are not equally used, creating codon usage bias. Specific sequences are favored over others when coding for certain amino acids in certain organisms, making it difficult to work with genes in the lab when expression must take place outside the native organism. This makes it important to avoid codon usage bias, as this article explains.

A codon is a sequence of three nucleotides that together correspond to or “code” for a specific amino acid.  With there being 64 different codons but only 20 translated amino acids, it means that many amino acids are encoded for by more than one codon.  This redundancy means that the genetic code is degenerate and within different organisms there is often bias towards one codon encoding the same amino acid over the other options.  The codon table below shows the different codon sequence that corresponds to each of the different amino acids.

Table 1. Universal genetic codon table.

1st position 2nd position 3rd position
U Phe Ser Tyr Cys U
Phe Ser Tyr Cys C
Leu Ser stop stop A
Leu Ser stop Trp G
C Leu Pro His Arg U
Leu Pro His Arg C
Leu Pro Gln Arg A
Leu Pro Gln Arg G
A Ile Thr Asn Ser U
Ile Thr Asn Ser C
Ile Thr Lys Arg A
Met Thr Lys Arg G
G Val Ala Asp Gly U
Val Ala Asp Gly C
Val Ala Glu Gly A
Val Ala Glu Gly G
Amino Acids
Ala:AlanineGln: GlutamineLeu: LeucineSer: Serine
Arg: ArginineGlu: Glutamic acidLys: LysineThr: Threonine
Asn: AsparagineGly: GlycineMet: MethionineTrp: Tryptophane
Asp: Apartic acidHis: HistidinePhe: PhenylalanineTyr: Tyrosine
Cys: CysteineIle: IsoleucinePro: ProlineVal: Valine


What is codon usage bias?

Synonymous codons are not randomly or equally used, and particular sequences are favored over others to encode for certain amino acids. Codon usage bias (CUB) is the non-random or favored use of identical codons [1]. CUB is seen as a code within the genetic code itself and has been designated the second genetic code. It can vary within and among species, as well as between genes within an organism evolving through mutation, natural selection, and genetic drift [1]. It can also vary in a family or group within a kingdom as well. This means that certain codons are optimal and translated more efficiently in comparison to their synonymous partners [2].

Factors that contribute to codon usage bias

The factors that impact CUB include GC content, population size, gene expression levels, protein length, context and position of the codon, tRNA abundance, tRNA interactions, and the mRNA structure [1]. GC content is one of the biggest factors in codon bias and has been shown to be a major determinant of usage variation of specific codons as monocots have a higher GC content (59-61%) than dicots (35-42%) [1]. Larger populations, highly expressed genes, and shorter proteins all tend to have a stronger codon usage bias when compared to their counterparts [1]. There is also a relationship between codon positioning, abundance and interaction of tRNA, and mRNA structure. These factors can help provide insight into ways in which optimal translation can occur whether it be in nature or in the lab. It is in the laboratory where codon usage bias becomes an issue.

Why is codon usage bias a problem?

For an organism in its natural environment, codon usage bias is beneficial and an important part of evolution. If, however, you wish to work with a sequence or gene of interest from one organism and express it in another which is quite common in research, you want to be sure that the gene will in fact be expressed and will function as it is supposed to. This will require optimization of the sequence. 

Tips to avoid codon usage bias

Optimization of your sequence is the most important way to avoid codon usage bias. This can be done using the IDT Codon Optimization Tool. This tool converts your DNA or protein sequence from one host organism for expression to another by reassigning codon usage to make sure that you are using the optimal sequence. It simplifies designing synthetic genes and single-stranded or double-stranded DNA fragments for expression in a variety of organisms. There is also a video available with a step-by-step tutorial for using the Codon Optimization Tool. Plus, you can read how IDT’s Codon Optimization Tool makes designing synthetic genes easy and can help you optimize your sequence.

There are a few key points to keep in mind when designing a sequence if choosing to manually optimize your sequence—these factors are all taken into consideration by IDT’s Codon Optimization Tool:

  • Avoid rare codons
  • Choose codons that have a bias similar to the natural bias of the organism selected for expression. This information can be found in a variety of online codon usage charts
  • Avoid areas of extremely high GC content
  • Avoid areas repetitive sequences
  • Avoid sequences that are likely to create challenging secondary structures

Get started

Instead of spending days designing and constructing your synthetic gene, you can simply 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 video tutorial for using the Codon Optimization Tool.

Contact us

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 us. We can accept non-standard optimizations that fall outside of the rules used by the tool (design fee may apply).



  1. Parvathy ST, Udayasuriyan V, Bhadana V. Codon usage biasMol Biol Rep. 2022;49(1):539-565.
  2. Hia F, Takeuchi O. The effects of codon bias and optimality on mRNA and protein regulationCell Mol Life Sci. 2021;78(5):1909-1928.


Published Nov 28, 2023