Cloning of the chimeric Gqi9DF protein: Cloning strategy: NEBuilder HiFi Assembly kit Vector for insert: pcDNA3.1(+), digested with EcoRV Template: Gqi9 from AddGene (Plasmid #125711) cloning strategy: primers have extensions for Gibson cloning into a EcoRV digested pcDNA3.1(+) vector Forward primer pcDNA3.1(+) overlap: GTCCAGTGTGGTGGAATTCTGCAGAT Forward primer Kozak extension: CGCCACC Forward gene specific primer: ATGGGGTGCACCCTGAGC Reverse primer pcDNA3.1(+) overlap: AGCGGCCGCCACTGTGCTGGAT Reverse gene specific primer with mutation sites: TCAGAAAAGGCCACAGTCCTTCAG Full forward primer: GTCCAGTGTGGTGGAATTCTGCAGATCGCCACCATGGGGTGCACCCTGAGC Full reverse primer: AGCGGCCGCCACTGTGCTGGATTCAGAAAAGGCCACAGTCCTTCAG -> Reverse primer changes stop codon from TAA to TGA, and mutates the C-terminus of the Gqi9 protein from YCGLC to DCGLF, similar to the Gqi5 protein C-terminus from Conklin et al 1993 >Gqi9 like ATGGGGTGCACCCTGAGCGAGGAGGCCAAGGAAGCCCGGAGGATCAACGACGAGATCGAGCGGCAGCTGCGCAGGGACAAGCGCGACGCCCGCCGGGAGCTCAAGCTGCTGCTGCTGGGGACAGGGGAGAGTGGCAAGTCGACCTTCATCAAGCAGATGAGGATCATCCACGGGTCGGACTACTCTGACGAAGACAAGCGCGGCTTCACCAAGCTGGTGTATCAGAACATCTTCACGGCCATGCAGGCCATGATCAGAGCGATGGACACACTCAAGATCCCATACAAGTATGAACACAATAAGGCTCATGCACAATTGGTTCGAGAGGTTGATGTGGAGAAGGTGTCTGCTTTTGACGTCCCCGACTACGCGGCAATAAAGAGCTTGTGGAATGATCCTGGAATCCAGGAGTGCTACGACAGACGACGGGAATATCAGTTATCTGACTCTACCAAATACTATCTGAATGACTTGGACCGTGTAGCCGACCCTTCCTATCTGCCTACACAACAAGACGTGCTTAGAGTTCGAGTCCCCACTACAGGGATCATCGAATACCCCTTTGACTTACAAAGTGTCATTTTCAGAATGGTCGATGTAGGGGGCCAAAGGTCAGAGAGAAGAAAATGGATACACTGCTTTGAAAATGTCACCTCCATCATGTTTCTAGTAGCGCTTAGCGAATATGATCAAGTTCTTGTGGAGTCAGACAATGAGAACCGCATGGAGGAGAGCAAAGCACTCTTTAGAACAATTATCACCTACCCCTGGTTCCAGAACTCCTCTGTGATTCTGTTCTTAAACAAGAAAGATCTTCTAGAGGAGAAAATCATGTATTCCCACCTAGTCGACTACTTCCCAGAATATGATGGACCCCAGAGAGATGCCCAGGCAGCTCGAGAATTCATCCTGAAAATGTTCGTGGACCTGAACCCCGACAGTGACAAAATCATCTACTCCCACTTCACGTGCGCCACAGATACCGAGAACATCCGCTTCGTCTTTGCAGCCGTCAAGGACACCATCCTGCAGCTGAACCTGAAGGACTGTGGCCTTTTCTGA ------------------------------------------------------------------------------------------------------- ------------------------------------------------------------------------------------------------------- >G5A, codon optimized, with flanking EcoRV restriction sites and partial Kozak sequence GATATCACCATGAGCAAGGGAGAGGAGCTGTTCACCGGAGTGGTGCCTATCCTGGTGGAGCTGGACGGCGATGTGAATGGCCACAAGTTTTCTGTGAGCGGAGAGGGAGAGGGCGACGCAACCTACGGCAAGCTGACACTGAAGTTCATCTGCACCACAGGCAAGCTGCCCGTGCCTTGGCCAACCCTGGTGACCACACTGACATACGGCGTGCAGTGTTTTTCTCGCTATCCTGACCACATGAAGCAGCACGATTTCTTTAAGAGCGCCATGCCAGAGGGCTACGTGCAGGAGAGGACAATCTTCTTTAAGGACGATGGCAACTATAAGACCCGCGCCGAGGTGAAGTTCGAGGGCGACACACTGGTGAACCGGATCGAGCTGAAGGGCATCGACTTTAAGGAGGATGGCAATATCCTGGGCCACAAGCTGGAGTACAACTATAATTCCCACAACGTGTACATCATGGCCGATAAGCAGAAGAACGGCATCAAGGCCAACTTCAAGATCCGCCACAATATCGAGGACGGCTCTGTGCAGCTGGCCGATCACTACCAGCAGAACACCCCAATCGGCGACGGACCCGTGCTGCTGCCTGATAATCACTATCTGTCTACACAGAGCGCCCTGTCCAAGGACCCCAACGAGAAGCGGGATCACATGGTGCTGCTGGAGTTCGTGACCGCAGCAGGCATCACACACGGCATGGATGAGCTGTATAAGTCCGGCGGCTCCGGCTCTGGCGGCCAGTCTGGAGGAAGCGGATCCGGAGGACAGAGCGGAGGCTCTGGCAGCGGCGGCCAGAGCGGAGGAAGCGGATCCGGCGGCCAGTCTGGCGGCAGCGGCAGCGGAGGCCAGTCTGGCCTGCGGAGCGTGAAGCTGACCAGCGACTTTGATAACCCTCGGTGGATCGGCAGACACAAGCACATGTTCAATTTTCTGGACGTGAACCACAATGGCAAGATCAGCCTGGACGAGATGGTGTACAAGGCCTCCGACATCGTGATCAACAATCTGGGCGCCACCCCAGAGCAGGCCAAGAGACACAAGGATGCAGTGGAGGCCTTCTTTGGAGGAGCAGGAATGAAGTACGGAGTGGAGACAGACTGGCCCGCCTATATCGAGGGCTGGAAGAAGCTGGCCACCGATGAGCTGGAGAAGTACGCCAAGAATGAGCCTACACTGATCAGGATCTGGGGCGACGCCCTGTTCGACATCGTGGACAAGGATCAGAACGGCGCCATCACCCTGGACGAGTGGAAGGCCTATACAAAGGCCGCCGGCATCATCCAGAGCTCCGAGGATTGCGAGGAGACCTTCCGGGTGTGCGACATCGATGAGTCCGGCCAGCTGGACGTGGATGAGATGACCAGGCAGCACCTGGGCTTCTGGTACACAATGGACCCAGCCTGCGAGAAGCTGTATGGCGGCGCCGTGCCCTGAGATATC