Cloning Tol2-based Betta Transgenesis Plasmids

To clone the Tol2-based transgenesis plasmids, we first amplified the 4,165-bp region upstream of the transcription start site of the betta actb gene using Q5 polymerase (M0491S, New England Biolabs) from an ornamental betta. We then replaced the ubiquitin promoter in the pDestTol2pA2_ubi:EGFP plasmid [Addgene plasmid #27323 (Mosimann et al., 2011 (link))]; with the amplified actb promoter region using bacterial in vivo assembly (García-Nafría et al., 2016 (link)). We then purified plasmids for injection using the ZymoPURE plasmid miniprep kit (D4210, Zymo Research). We fully sequenced all plasmids prior to injection to confirm intended sequences. To create the bicistronic plasmid, we first linearized actb:EGFP and then amplified a 1.3-kb region from pBH-mcs (a gift from Michael Nonet) containing the 268-bp of the zebrafish cmlc2 (myl7) promoter region, mCherry, and the polyadenylation and large T antigen signals. We then carried out all subsequent steps as described above.