Abstract: Disclosed are novel DNA segments, vectors and plasmids containing multiple promoters for use with various polymerases in order to transcribe cloned DNA into RNA. A preferred vector, termed pTRIPLEscript.TM., is described which contains the SP6, T7, and T3 phage promoters in the same orientation and on the same side of a multiple cloning site. This vector efficiently synthesizes in vitro transcripts from all three promoters under conditions of both limiting and saturating nucleotide concentrations. This vector also promotes transcription without crosstalk, i.e., without nonspecific initiation at inappropriate promoters.