A plasmid that consists of an 812-base-pair segment containing the replication origin of plasmid ColE1 and of a 1240-base-pair segment containing a beta-lactamase gene has been constructed. The plasmid DNA has three principal sites where transcription is initiated in vitro. One is located in the ColE1 segment 555 nucleotides upstream from the origin. Most transcription from this site extends past the origin; some of the transcripts form hybrids spontaneously with the template at their 3' portions. Cleavage of these transcripts by RNase H generates 3' termini at the origin region. When DNA polymerase I is included in the reaction along with RNA polymerase and RNase H, dAMP or dCMP is added directly onto the cleaved RNA molecules, most of which retain the intact 5' terminus. The addition of a deoxyribonucleotide to the cleaved RNA can be regarded as the first step of ColE1 DNA synthesis. Once it has served as a primer, the RNA is eliminated from the product by RNase H.