The in vitro transcripts of phage SP6 RNA polymerase usually contain extraneous 5`` and 3`` plasmid sequences in addition to the cloned gene sequence. Template plasmids were constructed to eliminate these extraneous sequences. The 5 S RNA gene of Xenopus borealis somatic cell was inserted at the transcription initiation site (BamHI site) of PCKSP6 so that the transcription starts from the 5`` end of the gene. The restriction enzyme DraI site was inserted at the 3`` end of 5 S RNA structrual gene by oligonucleotide-directed site-specific mutagenesis. Now, when the recombinant DNA is digested with DraI, the resulting in vitro run-off transcripts of the linearized template using phage SP6 RNA polymerase contain the authentic 5`` and 3``ends. Thus, the large quantity of authentic 5 S RNA and any mutant RNAs, which could easily be radioactively labeled, can be produced for structure-function relationship studies, RNA folding studies, and other biophysical studies that have been limited by sample quantities.