Construction and stability of a sixfold repeated artificial gene

The concatenation of an artificial gene consisting of 69 base pairs is described. A simple and versatile procedure was employed which is applicable to any DNA segment bordered by different restriction sites. If applied in the 'continuous duplication mode', it does not necessitate the isolation of any DNA fragment. The addition of adaptor oligonucleotides to one terminus of the segment is required to create complementary ends. The ligation of single-stranded oligonucleotides to single-stranded DNA termini was analyzed and optimized. A simple procedure is described for the separation of excess adaptor molecules. The in vivo stability of constructs containing up to six directly repeated genes was investigated. Up to four copies of the gene appeared stable during transformation of strain HB101. With six copies exact excision of ony copy was occasionally observed after transformations of rec+, recA, and recBC host cells. During long-term growth the hexameric construct appeared stable in all three strains.