EcI5, a group IIB intron with high retrohoming frequency: DNA target site recognition and use in gene targeting

EcI5 intron donor and recipient plasmids used in intron mobility assays. (A) pACDF-EcI5 uses a T7lac promoter (P_T7lac) to express full-length EcI5 with flanking 5′- and 3′-exon sequences (E1 and E2, respectively). (B) pACD2-EcI5 expresses the EcI5-ΔORF intron and flanking exons, with a phage T7 promoter (P_T7; arrowhead in intron) inserted in DIV, and the IEP expressed from a position downstream of the 3′ exon. SD is the Shine–Dalgarno sequence for the intron ORF. (C) pBRR-EcI5 contains an EcI5 target site (ligated E1 and E2 sequences) cloned upstream of a promoterless tet^R gene. Donor plasmids are derivatives of pACYC184 and carry a cam^R marker, while the recipient plasmid is a derivative of pBR322 and carries an amp^R marker. T1 and T2 are E. coli rrnB transcription terminators. T1 terminates both E. coli and phage T7 RNA polymerase, while T2 terminates E. coli but not phage T7 RNA polymerase. Tφ is a phage T7 transcription terminator, which terminates T7 RNA polymerase.