Codon Optimization

• Keeping GC ratio around 50%;
• Remove cis-acting DNA sequences such as internal TATA-boxes, chi-sites, and ribosomal entry sites AGGAGG, AGGAGGT, TAAGGAGG; AT-rich or GC-rich sequence stretches; repeat sequences; and RNA
• secondary structures
• ATAAGA to ATCCGT; AGAATA to CGCATT; ATAAGG to ATTCGT; AGAAGG to CGTCGC
• Add two stop codons TAATAA
• Append another strong terminator to the end of your DNA sequence

Transcriptional Efficacy:

GC content CpG dinucleotides content Cryptic splicing sites Negative CpG islands SD sequence TATA boxes Terminal signal

Translation Efficiency:

Codon usage bias GC content mRNA secondary structure Premature PolyA sites RNA instability motif (ARE) Stable free energy of mRNA Internal chi sites and ribosomal binding sites

Protein Refolding:

Codon usage bias Interaction of codon and anti-codon Codon-context RNA secondary structures