Novel kanamycin/neomycin phosphotransferase cassette increases transformation efficiency in E-coli

Stable transformation depends on the efficient delivery of DNA into cells a nd the robust expression of genes that encode proteins which provide resist ance to selective (cytotoxic) compounds. We have examined the possibility t hat altering the 5' untranslated region (UTR) of a selectable marker may in crease transformation efficiency. A 15-nucleotide synthetic UTR (the so-cal led universal translational enhancer [UTE]) was placed upstream of a kanamy cin/neomycin phosphotransferase (kanaR) gene to create a novel expression c assette, UTE-kanaR. In comparison to a wild-type version of kanaR, UTE-kana R produced up to 30-fold more transformants in E. coli. The superior perfor mance of UTE-kanaR was independent of the promoter strength, indicating tha t the gene may find general use in routine transformation experiments.