Figure 6

Use of a retrotranscription-activated marker ( RAM ) to select for targetron integrations. A targetron with a RAM cassette is expressed from a donor plasmid. The RAM cassette consists of a selectable marker gene, such as an antibiotic-resistance gene (kan^R), inserted within the intron DIV in the antisense orientation, but interrupted by an efficiently self-splicing group I intron (the phage T4 td intron) in the sense orientation, thereby preventing expression of the kan^R marker gene from the donor plasmid. Transcription of the targetron carrying the RAM from the donor plasmid yields a primary transcript from which the group I intron is spliced to reconstitute the kan^R marker and the group II intron is spliced to yield RNPs that retrohome into a DNA target site. After retrohoming, the reconstituted kan^R marker is expressed from its own promoter, enabling selection for targetron integrations.