Reece-Hoyes John S, Walhout Albertha J M
Cold Spring Harb Protoc. 2018 Jan 2;2018(1):pdb.prot094946. doi: 10.1101/pdb.prot094946.
This protocol describes using the Gateway recombinatorial cloning system to simultaneously transfer a promoter and an open reading frame (ORF) from two different Entry clones into the same Destination vector using LR enzymes. A multisite cloning reaction transfers the inserts from multiple Entry clones into a single Destination vector. This type of recombination is much less efficient than transferring a single DNA fragment; however, the variety of Destination clones that can be generated in this manner is vast. In this example protocol, we describe using pDEST-MB14 to make a Destination clone that features a promoter fragment fused upstream to an ORF that is cloned in-frame with a carboxy-terminal green fluorescent protein (GFP) moiety encoded by the plasmid backbone. This method can be used as a guide for other multisite cloning reactions.
本方案描述了使用Gateway重组克隆系统，利用LR酶将来自两个不同入门克隆的启动子和开放阅读框（ORF）同时转移到同一个目的载体中。多位点克隆反应将多个入门克隆的插入片段转移到单个目的载体中。这种重组类型的效率远低于转移单个DNA片段；然而，以这种方式可以产生的目的克隆种类繁多。在本示例方案中，我们描述了使用pDEST-MB14构建一个目的克隆，该克隆的特征是一个启动子片段与一个ORF上游融合，该ORF与质粒骨架编码的羧基末端绿色荧光蛋白（GFP）部分框内克隆。此方法可作为其他多位点克隆反应的指南。
