We have constructed a plasmid encoding a mouse dihydrofolate reductase (dhfr) minigene which produces dhfr transcripts with all of the 5' and 3' ends observed from the chromosomal mouse dhfr gene. The minigene contains 5' flanking regions, all dhfr coding sequences, one intervening sequence, 11.5 kb of 3' flanking regions beyond the termination codon, an E. coli plasmid origin of replication and antibiotic resistance, and an SV40 minimal origin of replication; the total size is 17.2 kb. When transfected into cells constitutively producing a temperature sensitive SV40 T antigen, the plasmid minigene replicates at the permissive temperature, but fails to replicate at the nonpermissive temperature. Therefore, transcription can be observed in the presence or absence of minigene replication. In addition, a stable divergently transcribed RNA is produced from the dhfr minigene promoter region, with the same 5' ends that are seen in the chromosomal divergently transcribed gene. We show that deletion of the sole remaining intron of the dhfr minigene significantly lowers the amount of dhfr transcript produced but does not affect the amount of divergent transcript. The promoter region for these transcripts contains four 48 bp repeats; reducing the number of these repeats lowers the amount of both dhfr and divergent transcripts produced from the minigene.