GaszKO is a replacement deletion, where we inserted a 3xP3-RFP insertion instead of most of the coding region of Gasz. This knock-in results in a premature stop codon and only gives rise to a 28aa protein starting from the endogenous Gasz start codon. The following sequences are up- and down-stream of the deletion/3xP3-RFP cassette.
gaszKO upstream
ATGATGAGCAACCTTTGCAAATACGGACCGCCCCCAGATTCCGACTCTGATAGCAGCTAC
gaszKO downstream
TTCATTGACTTCAACGAAATACTCGCAGAGCGAAAACGCgAAAAGGTGCGCAGCTACTTC
Below is the sequence of the 3xP3-RFP cassette:
GGATCTAATTCAATTAGAGACTAATTCAATTAGAGCTAATTCAATTAGGATCCAAGCTTATCGATTTCGAACCCTCGACCGCCGGAGTATAAATAGAGGCGCTTCGTCTACGGAGCGACAATTCAATTCAAACAAGCAAAGTGAACACGTCGCTAAGCGAAAGCTAAGCAAATAAACAAGCGCAGCTGAACAAGCTAAACAATCGGGCGGCCGCACTAGAGCCGGTCGCCACCATGAGGTCTTCCAAGAATGTTATCAAGGAGTTCATGAGGTTTAAGGTTCGCATGGAAGGAACGGTCAATGGGCACGAGTTTGAAATAGAAGGCGAAGGAGAGGGGAGGCCATACGAAGGCCACAATACCGTAAAGCTTAAGGTAACCAAGGGGGGACCTTTGCCATTTGCTTGGGATATTTTGTCACCACAATTTCAGTATGGAAGCAAGGTATATGTCAAGCACCCTGCCGACATACCAGACTATAAAAAGCTGTCATTTCCTGAAGGATTTAAATGGGAAAGGGTCATGAACTTTGAAGACGGTGGCGTCGTTACTGTAACCCAGGATTCCAGTTTGCAGGATGGCTGTTTCATCTACAAGGTCAAGTTCATTGGCGTGAACTTTCCTTCCGATGGACCTGTTATGCAAAAGAAGACAATGGGCTGGGAAGCCAGCACTGAGCGTTTGTATCCTCGTGATGGCGTGTTGAAAGGAGAGATTCATAAGGCTCTGAAGCTGAAAGACGGTGGTCATTACCTAGTTGAATTCAAAAGTATTTACATGGCAAAGAAGCCTGTGCAGCTACCAGGGTACTACTATGTTGACTCCAAACTGGATATAACAAGCCACAACGAAGACTATACAATCGTTGAGCAGTATGAAAGAACCGAGGGACGCCACCATCTGTTCCTTTAGCGGCCATCGAATTCGAGCTCGCCCACTAAGCGTCGCGCCACTTCAACGCTCGATGGGAGCGTCATTGGTGGGCGGGGTAACCGTCGAAATCAGTGTTTACGCTTCCAATCGCAACAAAAAATTCACTGCAACACTGAAAAGCATACGAAAACGATGAAGATTGTACGAGAAACCATAAAGTATTTTATCCACAAAGACACGTATAGCAGAAAAGCCAAGTTAACTCGGCGATAAGTTGTGTACACAAGAATAAAATCGGCCAGATTCAGTGTTGTCAGAAATAAGAAAACCCCACTATGTTTTTCTTTGCCTTTTCTTTCTCCCAGCGATCATTCATTTCGTGGTGAAAGAACGGGGTCATTGCACGGAGTTTCGACTGCGGGAAAGCAGAGCTGCCGTTCACTTCGTCTATAATTAGCGCTTTCTATTTTCCCCGATTCGGGCCGCTGCTGCGCTTTTCCGCCTGCTGTTTGTGGCAAGTGTAGCAGCAGGCTGTGCACGCAGTGTGGCATGCACTTGGCTTTCCACCGTTGGTATCGATTCTCTGGGACGATGAGTCATTCCTTTCGGGGCCACAGCATAATCGTTGCCAGCTCACCGAAATGGTGACTTCATTTCTTAACTGCCGTCAAGCATGCGATTGTACATACATACATATTTATATATGTACATATTTATGTGACTATGGTAGGTCGATATAATAGCAATCAACGCAAGCAAATGTGTCAGTCCTGCTTACAGGAACGATTCTATTTAGTAATTTTCGTTGTATAAAGTAATTATGTATGTATGTAAGCCCCATAAATCTGAAACAATTAGGCAAAACCATGCGAAGCT
See the annotated sequence at https://benchling.com/s/seq-jhPeULgEL5qi2DaTpeim.