基因序列最佳化系統使用指南 (Codon Optimizer User Guide)

根據各物種真實密碼子使用頻率，將蛋白質或核酸序列轉換為最適合目標宿主表達的 DNA 序列，並可自動迴避指定限制酶切位。

Converts protein or nucleic acid sequences into optimized DNA sequences based on species-specific codon usage frequencies, with automatic avoidance of selected restriction enzyme recognition sites.

Step 1　序列輸入 (Sequence Input)

• 中文說明：選擇序列類型（Protein／DNA／RNA），將序列貼入輸入框，系統會自動驗證格式與字元是否正確。支援 FASTA 格式，標題行會自動去除。DNA／RNA 序列長度須為 3 的倍數。

• English Instructions: Select the sequence type (Protein / DNA / RNA) and paste your sequence into the input box. The system will automatically validate the format. FASTA format is supported with header lines removed automatically. DNA/RNA length must be a multiple of 3.

Step 2　目標表達物種 (Host Organism)

• 中文說明：從下拉選單中選擇目標表達宿主，涵蓋細菌、藻類、酵母、植物、昆蟲、兩棲類及哺乳類等共 36 個物種，系統將依據該物種的密碼子偏好性進行最佳化。若需使用特定物種的密碼子頻率資料，可勾選「使用自定義密碼子頻率」，前往 www.kazusa.or.jp/codon 搜尋目標物種，將密碼子頻率表整段複製後貼入輸入框即可。

• English Instructions: Select your target expression host from the dropdown menu, covering 36 species across bacteria, algae, yeast, plants, insects, amphibians, and mammals. The system will optimize the sequence based on that species' codon preference. To use codon usage data for a specific species not listed, check "Use Custom Codon Usage", visit www.kazusa.or.jp/codon to search for your target species, and paste the entire codon frequency table into the input box.

Step 3　限制酶切位迴避 (Avoid Cleavage Sites of Restriction Enzymes)　— 選填 / Optional

• 中文說明：若載體構建需保留特定酶切位，可在此勾選需要迴避的限制酶。支援搜尋欄快速篩選。若因密碼子選擇空間不足而無法完全消除某切位，結果欄將顯示警告提示。

• English Instructions: If your cloning strategy requires certain restriction sites to remain intact elsewhere, check the enzymes whose sites should be avoided in the sequence. Use the search bar to filter quickly. If a site cannot be eliminated due to limited codon choices, a warning will appear in the result panel.

Step 4　執行運算與取得結果 (Run Optimization)

• 中文說明：點擊「▶ 開始最佳化運算」按鈕，系統將自動完成密碼子最佳化、5' 端降頻處理、GC 含量調整（目標 30–70%）、mRNA 髮夾結構消除、類 Shine-Dalgarno 序列移除及限制酶切位消除。完成後結果欄顯示最佳化 DNA 序列及統計資訊，點擊「⧉ 複製序列」即可複製。

• English Instructions: Click "▶ Run Optimization". The system will perform codon optimization, 5' end de-optimization, GC content adjustment (target 30–70%), mRNA hairpin elimination, Shine-Dalgarno-like motif removal, and restriction site avoidance. Once complete, the result panel displays the optimized DNA sequence with statistics including length and GC content. Click "⧉ Copy" to copy the sequence.