Codon Optimization

Codons play a critical role in protein synthesis by constituting the genetic code that governs the process of translating information contained in a DNA or RNA sequence into the corresponding protein sequence.

The same amino acid may be encoded by multiple codons and different organisms exhibit preferential usage of certain codons over the others. Although the reasons underlying the existence of such codon usage bias is not known, it does have a significant impact on protein expression. Therefore, it is critical to optimize a gene sequence based on an organism’s codon bias to achieve optimal expression levels in recombinant protein expression studies. This is particularly important in heterologous protein expression systems where the gene of interest (GOI) derived from a particular organism is expressed in a host organism that does not naturally express that gene. In addition to this, codon optimization of sequences might be necessary for a variety of other applications including improving DNA cloning efficiency by optimizing GC content and repetitive sequences, improving mRNA stability, and improving transcription or translation efficiency.

VectorBuilder’s codon optimization tool is designed to help you achieve the optimal codon adaptation index (CAI) for your GOI in any organism of your choice. It includes a comprehensive list of species and is seamlessly incorporated into our online vector design platform enabling you to optimize your GOIs while designing vectors. Additionally, it allows you to avoid cleavage sites of selected restriction enzymes while codon optimizing your target sequence. Our tool can be used for optimizing sequences with extreme GC content and simple repeats for highly efficient gene synthesis and DNA cloning applications.

Start codon optimization

Below are some examples illustrating various functionalities of our codon optimization tool:

1. Optimizing sequences for codon usage in desired target species - Figure 1 below illustrates that when the sequence of native piggyBac transposase from Trichoplusia ni was optimized for expression in human using our tool, it resulted in an optimized sequence with a CAI of 0.93. The CAI of the piggyBac transposase gene prior to optimization was found to be 0.69. The CAI for a species is a quantification of the frequency of usage of favored codons in highly expressed genes. CAI values range between 0 and 1. The higher the CAI value of a gene for a specific target species, the greater the chances are for it to be expressed optimally in that species.

Figure 1. Optimizing a sequence for codon usage in a target species using VectorBuilder’s codon optimization tool.

2. Optimizing sequences with high GC content - Figure 2 illustrates that when the mouse Hoxa4 gene with an overall GC content of 69.3% was optimized using our tool, it resulted in the GC content to drop to 59.5%. For genes requiring synthesis during the cloning process, an optimal GC content of approximately 60% is recommended to increase the chances for the gene synthesis to succeed.

Figure 2. Optimizing a sequence with high GC content using VectorBuilder’s codon optimization tool.

3. Optimizing sequences with repetitive regions - Figure 3 below illustrates dot plots comparing the human immunoglobulin heavy chain sequence against itself, before and after codon optimization with our tool. While the before optimization dot plot shows the presence of highly repetitive regions within the sequence indicated by the multiple diagonal lines, optimization of the sequence resulted in a significant decrease in the repeats, as shown by the after optimization dot plot.

Figure 3. Optimizing a sequence for reducing repetitive regions using VectorBuilder' s codon optimization tool.

Note: Sequences in both GenBank and FASTA formats can be recognized by this tool.