Gblocks gene fragment

Constructs for stable transduction in mammalian cells were cloned into the pHR lentiviral backbone with an SFFV promoter driving the gene of interest. The pHR backbone was linearized using MluI and NotI restriction sites. BcLOV4, iLID, BFP, SOS_cat, and iSH coding DNA fragments were generated via PCR and inserted into the pHR backbone via HiFi cloning mix (New England Biolabs). For expression in Drosophila S2 cells, BcLOV-mCherry, BcLOV-iSH, and BcLOV-SOS_cat were amplified and inserted into the pbphi-nanos promoter-αTubulin 3’UTR vector⁴⁴ between the NheI and BamHI restriction sites. The resulting vectors were digested with NotI and XhoI to replace the nanos protomer with the metallothionein promoter (pMt)⁴⁵ , which was synthesized by gBlocks gene fragments (Integrated DNA Technologies). The pMt promoter permits inducible expression in the presence of heavy metals, e.g. copper. For zebrafish mRNA expression experiments, BcLOV-mCherry, BcLOV-SOS_cat and ERK-KTR-BFP (adapted from Regot et al^{32 (link)}) were amplified with primers containing att sites for Gateway cloning. PCR amplicons were transferred into pDONR221 plasmids and sequence verified. Gateway cloning was used to transfer each insert into pCSDest plasmids ⁴⁶ .