cell-free-expression

Codons to Avoid in E. coli CFPS

Codon	Amino Acid	Issue	tRNA Abundance
AGG	Arg	Very rare, stalling	0.2%
AGA	Arg	Very rare, stalling	0.4%
CUA	Leu	Low abundance	0.4%
AUA	Ile	Rare	0.5%
CGA	Arg	Inefficient decoding	0.6%
CCC	Pro	Can cause pausing	0.5%
GGA	Gly	Moderate	1.1%

Design Rules

1. First 30 codons: Most critical - use only high-frequency codons
2. Rare codon clusters: Avoid 2+ rare codons within 10 nt
3. Rare codon content: Keep overall <5% of coding sequence
4. GC content: Target 40-60% for balanced expression
5. Avoid runs: No >6 consecutive G or C residues (secondary structure)
6. Strategic slow codons: Place rare codons between domains (aids folding!)

When to Use Rare Codons

• Domain boundaries (allow cotranslational folding)
• Before complex structural elements
• When protein is prone to misfolding

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