Figure 1.

Generation of episomal vectors for conditional expression of microRNAs. Flow chart showing the steps necessary to generate pEMI-vectors harboring microRNA cassettes. AmpR, ampicillin resistance; CmR, chloramphenicol resistance; EBNA-1, EBV nuclear antigen required for Ori P function; EBV, Epstein–Bar virus; H2R, homology 2 region; Hyg B, hygromycin B; LTR, long-terminal repeats; MAGIC, mating-assisted genetically integrated cloning; miR-30, precursor microRNA; mRFP, monomeric red fluorescent protein; OriP, EBV origin of replication; pEMI, plasmid for episomal microRNA expression; pheS, phenylalanine synthase relaxed-specificity allele Gly294; P_PGK, PGK-promoter; pSHUMI, plasmid for shuttling of microRNAs; pSM2c, pSHAG-MAGIC 2c retroviral vector for microRNA expression; P_tet-bi-1, bidirectional tet-responsive promoter; rtTA2^S-M2, reverse tetracycline controlled transactivator; tTS^KRAB, tetracycline repressed silencer.