Use of pRosa26-DEST for Cre-regulated expression of cDNA constructs. Entry vectors carrying Gateway-compatible cDNA constructs without any regulatory sequences can be shuttled into pRosa26-DEST using a LR reaction in which the ccdB bacterial negative counter-selection gene is lost. The construct is recombined via gene targeting into the endogenous Rosa26 locus. The first exon of the Rosa26 locus (outside the targeting construct) will splice to the splice acceptor site in the construct but transcription is interrupted by the PGK-neo-pA and three copies of the SV40 polyA signal, which function as a STOP cassette. Cre-mediated removal of this cassette links Rosa26 exon 1 to the cDNA and thus the cDNA is expressed. As the endogenous Rosa26 transcripts are not translated into protein, the first start codon in the cDNA will be used for initiation of translation. Fragments shown in green can be expressed, fragments in red cannot; the ccdB counter-selection gene in blue is expressed in bacteria only.