The mfap4 promoter was PCR amplified using the primers 5′-CATGTTCTCGAGGCGTTTCTTGGTACAGCTGG-3′ and 5′-CATGTTGGATCCCACGATctaaagtcatgaagaaaga-3′. The product was subsequently cloned into p5E MCS using XhoI and BamHI sites. The native start codon was mutated using the primer 5′-CTGAGCTGTTGAGGAGAGAGTGAGAAG[ATT]GCAGTAAGTTCTGTGGCTGTTTTATTCC-3′ by inverse PCR with the backbone primers 5′-GTAAGTTCTGTGGCTGTTTTATTC-3′ and 5′-CTTCTCACTCTCTCCTCAACAG-3′. The final p5E mfap4 was then assembled by Gibson assembly using this single stranded oligo and the backbone.
To generate pME Turquoise2, we used the primers 5′-GGGGACAAGTTTGTACAAAAAAGCAGGCTggaccatggtgagcaagggcgaggag-3′ and 5′-GGGGACCACTTTGTACAAGAAAGCTGGGTttacttgtacagctcgtccat-3′ to amplify off pmTurquoise2 H2A (Addgene plasmid #3620235 (link)). The PCR product was subsequently cloned into pDONR221 by BP cloning (Invitrogen) to generate pME Turquoise2.
The mfap4:turquoise transgene construct was subsequently constructed by recombining p5E mfap4, pME Turquoise and p3E polyA into pDestTol2pA2 to generate pDestTol2; mfap4:turquoise.
To generate pME Turquoise2, we used the primers 5′-GGGGACAAGTTTGTACAAAAAAGCAGGCTggaccatggtgagcaagggcgaggag-3′ and 5′-GGGGACCACTTTGTACAAGAAAGCTGGGTttacttgtacagctcgtccat-3′ to amplify off pmTurquoise2 H2A (Addgene plasmid #3620235 (link)). The PCR product was subsequently cloned into pDONR221 by BP cloning (Invitrogen) to generate pME Turquoise2.
The mfap4:turquoise transgene construct was subsequently constructed by recombining p5E mfap4, pME Turquoise and p3E polyA into pDestTol2pA2 to generate pDestTol2; mfap4:turquoise.
