Yeast recombination cloning is a straightforward and powerful method for recombining a plasmid backbone with a specific DNA fragment. However, the utility of yeast recombination cloning is limited by the requirement for the backbone to contain an CEN/ARS element, which allows for the recombined plasmids to propagate. Although yeast CEN/ARS plasmids are often suitable for further studies, we demonstrate here that they can vary considerably in copy number from cell to cell and from colony to colony. Variation in plasmid copy number can pose an unacceptable and often unacknowledged source of phenotypic variation. If expression levels are critical to experimentation, then constructs generated with yeast recombination cloning must be subcloned into integrating plasmids, a step that often abrogates the utility of recombination cloning. Accordingly, we have designed a vector that can be used for yeast recombination cloning but can be converted into the integrating version of the resulting vector without an additional subcloning. We call these "ICE" vectors, for "Integrating after CEN Excision." The ICE series was created by introducing a "rare-cutter" NotI-flanked CEN/ARS element into the multiple cloning sites of the pRS series yeast integration plasmids. Upon recovery from yeast, the CEN/ARS is excised by NotI digest and subsequently religated without need for purification or transfer to new conditions. Excision by this approach takes ~3 hr, allowing this refinement in the same time frame as standard recombination cloning.

中文翻译：

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CEN 切除 (ICE) 质粒后的整合：将酵母重组克隆的简便性与基因组整合的稳定性相结合。

酵母重组克隆是重组质粒骨架与特定 DNA 片段的一种简单而强大的方法。然而，酵母重组克隆的效用受到主干要求包含 CEN/ARS 元件的限制，这允许重组质粒繁殖。尽管酵母 CEN/ARS 质粒通常适用于进一步研究，但我们在此证明它们在不同细胞和不同菌落的拷贝数方面可能存在很大差异。质粒拷贝数的变化可能会造成不可接受且通常未被承认的表型变异来源。如果表达水平对实验至关重要，则必须将酵母重组克隆产生的构建体亚克隆到整合质粒中，这一步骤通常会取消重组克隆的效用。因此，我们设计了一种可用于酵母重组克隆的载体，但无需额外的亚克隆即可转化为所得载体的整合版本。我们称这些为“ICE”向量，表示“CEN 切除后的整合”。ICE 系列是通过在 pRS 系列酵母整合质粒的多个克隆位点中引入“稀有切割器”NotI 侧翼 CEN/ARS 元件而创建的。从酵母中回收后，CEN/ARS 被 NotI 消化切除，随后无需纯化或转移到新条件即可重新连接。这种方法的切除需要大约 3 小时，允许在与标准重组克隆相同的时间范围内进行这种改进。