Are there any sequence limitations/design guidelines for genes which I should follow?
Synthesis issues are driven mainly by repetitive structures and extreme GC content. The following rules can guide your design. However, note that you can increase the likelihood of success by limiting repeats and evening-out GC content.
For example, if you have a gene with 66% overall GC content, you'll have to reduce it to at least 65% to have it synthesized, but if you can lower it even further to ~60% the gene will be more likely to succeed. The same holds true for eliminating repeats, reducing homopolymer stretches, and evening out extreme variations in GC windows. The more you can avoid or reduce such structures in your gene, the more likely your gene is to succeed in synthesis.
- Avoid repeats of ≥ 20bp or Tm ≥ 60C Global GC content must be between 25% and 65%
- Avoid extreme differences in GC content within a gene (i.e. the difference in GC content between the highest and lowest 50 bp stretch should be no greater than 52%)
- Minimize homopolymers Minimize the number/length of small repeats scattered throughout the sequence
- For HIS tags use a combination of CAC and CAT codons i.e. CACCAT
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