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I want to chemically synthesize a GFP gene for expressing in rice.I am using the IDT Codon Optimization program and found that not all the codons are 100% optimized. Some of them are the 2nd or 3rd in frequency of usage. Also, the stop codon in the gene optimized is the least used in rice. I don't think all the codons can be the ones that are the most used in rice as there is need of translational pauses to get optimal protein expression. But how do I decide which codons are to be used in what positions to get maximum efficiency. Do I just use the automatic codon optimized gene given by IDT or further optimize it manually?
So should the stop codon used be the one which is the most frequently used in rice for proper mRNA termination?
Please help me as I am new to this and want tips and suggestions for getting optimized codons in this gene.
Generally the codon optimization is the most frequent tRNA found in your species of interest. However, your codon must not result in something that disrupts standard molecular biology practices. For instance, if your codon places a restriction site in a unique cloning vector, then the algorithm will just look for the next best codon which does not cause unwanted addition or elimination of a cloning site. Places like IDT will also weigh the cost of the codon vs. the ease of manufacturing in their algorithm (see here.)
What you describe as translational pausing that is 'needed' by the organism to optimize proper gene expression is a consequence and not a necessity of infrequent codons (to my full understanding). See here. That means you don't want to add this translational pausing in optimized gene expression. It is a simply an evolutionary byproduct.
To answer your question more bluntly, I can tell you with some experience to trust the codon optimization algorithms from biotech companies like IDT. I find they work very well. Rice is a common enough organism :)
The optimization done by service providers like IDT should be fine because they weigh in multiple factors like avoiding mRNA secondary structures and specified restriction endonucleases. Even I was considering manual editing but then some research on the internet revealed that codon usage is not the only factor involved :)