Construction of Novel Transcription Vectors for Production of Biologically Active Transcripts from Viral cDNAs

Abstract

A novel transcription vector, pUT118 which starts transcription to be directed to the natural 5' end of viral cDNA insert was constructed. After cleavage at a Xba I site just downstream of the cDNA insert, in vitro transcription can be performed by T7 RNA polymerase to produce full-length copies of viral RNAs. Biologically active CMV satellite RNA (Y strain) was successfully synthesized by inserting complete cDNA copy into a unique Nsi I site in pUT118 and transcribing the clone. To overcome the low transcription efficiency due to the modified T7 promoter in pUT118, pUT118GG was also created from pUT118 to add two extra G residues to the 5' end of the transcripts, allowing high efficiency of transcription initiation of the cDNA insert.