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我們使用 pBAtC 向量 (加入編號 KU213971)和 Aar I介導的sgRNA克隆系統(Kim等人,2014年)為Agrobac- 在佩妮的特米里調解的轉換。幻影玫瑰。的 ACO1-sg1 的目標 sgRNA 序列作為插入 退火寡核苷酸進入AarI消化的pBAtC向量 在 AtU6 啟動器和 sgRNA 支架之間,然後 迴圈與T4 ligase孵化(新英格蘭生物實驗室, 伊普斯威奇)在室溫3小時。退火寡利哥努- 核苷酸產物是使用含有混合物的 以下目標寡核苷酸的100 lmol/L 條件:95 °C 5分鐘,線性梯度從95°C到25°C 70 分鐘,在 10°C 下保持。ACO1(第 1 項)目標 sgRNA (gx20) 序列在 阿拉伯的阿利亞納-U6推廣者,和人類合作優化 鏈球菌類球基因Cas9(SpCas9)表達被控制 由35S促進者。

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