Kuhn I, Stephenson F H, Boyer H W, Greene P J
Gene. 1986;42(3):253-63. doi: 10.1016/0378-1119(86)90229-5.
The construction and use of a series of positive-selection vectors are described. These plasmids encode EcoRI endonuclease, the synthesis of which is under the control of the lacUV5 promoter. The pKG2 plasmid encodes a wild-type EcoRI endonuclease. In the absence of EcoRI methylase, the endonuclease is lethal. Cloning into any of the unique restriction sites within the endonuclease-coding gene allows survival of the transformed EcoRI-methylase-less host. The pKGW and pKGS plasmids encode an altered EcoRI endonuclease which, when repressed in a lacIQ host, allows survival in the absence of the methylase. Induction with IPTG, however, results in cell death as a result of high-level EcoRI synthesis. Cloning into any of the unique restriction sites within the EcoRI gene of pKGW or pKGS allows survival of derepressed transformed cells. These vectors strongly select for cloning events which inactivate the endonuclease gene.
本文描述了一系列正选择载体的构建和使用。这些质粒编码EcoRI核酸内切酶，其合成受lacUV5启动子控制。pKG2质粒编码野生型EcoRI核酸内切酶。在没有EcoRI甲基化酶的情况下，该核酸内切酶具有致死性。克隆到核酸内切酶编码基因内的任何一个独特限制位点可使无EcoRI甲基化酶的转化宿主存活。pKGW和pKGS质粒编码一种改变的EcoRI核酸内切酶，当在lacIQ宿主中受到抑制时，在没有甲基化酶的情况下可存活。然而，用IPTG诱导会因高水平的EcoRI合成导致细胞死亡。克隆到pKGW或pKGS的EcoRI基因内的任何一个独特限制位点可使去阻遏的转化细胞存活。这些载体强烈选择使核酸内切酶基因失活的克隆事件。
