The TALENs used to facilitate the insertion of Zwitch were designed using TAL Effector Nucleotide Targeter 2.0 software (Doyle et al., 2012) and constructed using the Golden Gate assembly method (Cermak et al., 2011; Sakuma et al., 2013). The TALENs were cloned into pCS2TAL3DDD and pCS2TAL3RRR vectors (provided by Dr. David Grunwald, University of Utah) (Dahlem et al., 2012). TALEN mRNAs were synthesized from linearized vectors using the mMESSAGE mMACHINE SP6 Transcription Kit (Thermo Fisher Scientific, Waltham, MA), and they were co-injected with pZwitch-shha-int1 into one-cell-stage embryos. The modified alleles were characterized using genomic PCR. The sequences of the PCR primers used to characterize the alleles are listed in Supplementary file 1. The offspring of a single founder were propagated and used in all subsequent experiments. The established conditional gene trap line Tg(shha:Zwitch)vcc8Gt is referred to as shhact. A DIG DNA labeling and detection kit (Roche) was used for Southern blot analysis of genomic DNA isolated from adult shhact/+ fish and WT clutch-mates. The detection probe was generated using PCR with WT genomic DNA and the following primers: