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.