SHUTTLE VECTOR YCUP4 AND MICROORGANISM STRAIN HOLDING YCUP4 IN BACTERIA

摘要

PURPOSE:To accomplish cloning of gene fragment in high efficiency by imparting pUC-based plasmid with specific URA3 gene. CONSTITUTION:pUC13 is treated with a restriction enzyme SspI followed by alkali phosphatase (a). And the essential domain is recovered as a DNA fragment (b) so as not to cut centromere sequence and autoreplication sequence from YCp50. Thence, the component (a) is mixed with the component (b) and Eschericha coli is transformed with a reaction liquid obtained by incubating T4 ligase, and the resulting transformant is spread on an antipyrine-contg. L-culture medium to obtain a colony. Thence, plasmid is prepared from this colony and digested by a restriction enzyme followed by agarose gel electrophoresis, thus obtaining a plasmid (PCEN1). This PCEN1 is then treated with a restriction enzyme ECoO109 and alkali phosphatase followed by mixing the resultant product with plain-terminated URA3 gene fragment obtained by digesting YEp24 with a restriction enzyme Hiud II and Sma I and then making an incubation in the presence of T4 ligase followed by transformation with Escherichia coli, thus obtaining the objective shuttle vector YCUp as shown in the figure.