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
                  


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>G5A, codon optimized, with flanking EcoRV restriction sites and partial Kozak sequence
GATATCACCATGAGCAAGGGAGAGGAGCTGTTCACCGGAGTGGTGCCTATCCTGGTGGAGCTGGACGGCGATGTGAATGGCCACAAGTTTTCTGTGAGCGGAGAGGGAGAGGGCGACGCAACCTACGGCAAGCTGACACTGAAGTTCATCTGCACCACAGGCAAGCTGCCCGTGCCTTGGCCAACCCTGGTGACCACACTGACATACGGCGTGCAGTGTTTTTCTCGCTATCCTGACCACATGAAGCAGCACGATTTCTTTAAGAGCGCCATGCCAGAGGGCTACGTGCAGGAGAGGACAATCTTCTTTAAGGACGATGGCAACTATAAGACCCGCGCCGAGGTGAAGTTCGAGGGCGACACACTGGTGAACCGGATCGAGCTGAAGGGCATCGACTTTAAGGAGGATGGCAATATCCTGGGCCACAAGCTGGAGTACAACTATAATTCCCACAACGTGTACATCATGGCCGATAAGCAGAAGAACGGCATCAAGGCCAACTTCAAGATCCGCCACAATATCGAGGACGGCTCTGTGCAGCTGGCCGATCACTACCAGCAGAACACCCCAATCGGCGACGGACCCGTGCTGCTGCCTGATAATCACTATCTGTCTACACAGAGCGCCCTGTCCAAGGACCCCAACGAGAAGCGGGATCACATGGTGCTGCTGGAGTTCGTGACCGCAGCAGGCATCACACACGGCATGGATGAGCTGTATAAGTCCGGCGGCTCCGGCTCTGGCGGCCAGTCTGGAGGAAGCGGATCCGGAGGACAGAGCGGAGGCTCTGGCAGCGGCGGCCAGAGCGGAGGAAGCGGATCCGGCGGCCAGTCTGGCGGCAGCGGCAGCGGAGGCCAGTCTGGCCTGCGGAGCGTGAAGCTGACCAGCGACTTTGATAACCCTCGGTGGATCGGCAGACACAAGCACATGTTCAATTTTCTGGACGTGAACCACAATGGCAAGATCAGCCTGGACGAGATGGTGTACAAGGCCTCCGACATCGTGATCAACAATCTGGGCGCCACCCCAGAGCAGGCCAAGAGACACAAGGATGCAGTGGAGGCCTTCTTTGGAGGAGCAGGAATGAAGTACGGAGTGGAGACAGACTGGCCCGCCTATATCGAGGGCTGGAAGAAGCTGGCCACCGATGAGCTGGAGAAGTACGCCAAGAATGAGCCTACACTGATCAGGATCTGGGGCGACGCCCTGTTCGACATCGTGGACAAGGATCAGAACGGCGCCATCACCCTGGACGAGTGGAAGGCCTATACAAAGGCCGCCGGCATCATCCAGAGCTCCGAGGATTGCGAGGAGACCTTCCGGGTGTGCGACATCGATGAGTCCGGCCAGCTGGACGTGGATGAGATGACCAGGCAGCACCTGGGCTTCTGGTACACAATGGACCCAGCCTGCGAGAAGCTGTATGGCGGCGCCGTGCCCTGAGATATC
