Sequencing:Article Title: A Novel A105Y Mutant of CYP17A1 Exhibits Almost Perfect Regioselectivity in the Production of 17α-Hydroxyprogesterone.
Article Snippet: Azenta pPICZA-CPRSc pPICZA carrying CPR gene from S. cerevisiae Azenta pPICZA-CPRHomo pPICZA carrying CPR gene from Homo Azenta pPICZA-CPRAn pPICZA carrying CPR gene from Aspergillus niger Azenta Table S2 Primers used in this study Primer Sequence (5’ to 3’) Site-directed mutagenesis primers A105Y-F GATATTTATTCTAATAATAGAAAAGGTATTGCC A105Y--R ATTAGAATAAATATCCAGGGTGGCCATC A105L-F GATATTCTGTCTAATAATAGAAAAGGTATTGCC A105L--R ATTAGACAGAATATCCAGGGTGGCCATC A105M-F GATTGTAATAGACTCCTGCTGCTG A105M--R ATTAGACATAATATCCAGGGTGGCCATCTG A100I-F ACAGATGATTACCCTGGATATTGCCTC A100I-R CCAGGGTAATCATCTGTGGTCTACCAGAAAAGTCC Q98L-F AGACCACTGATGGCCACCCTGGATATTG Q98L-R GGCCATCATCTGTCTACCAGAAAAGTCCTTACCC Q98S-F AGACCACTGATGGCCACCCTGGATATTG Q98S-R GGCCATCATCAGACTACCAGAAAAGTCCTTACCC Primers for constructing the plasmid pPIC3.5K-HCYP17A1 pPIC3.5K-HCYP17A1-F GATCATAATAGACTCCTGCTGCTG pPIC3.5K-HCYP17A1-R CTATTATGATCAGAAATGGTTGGGGTTC Primers were for constructing the plasmid pPICZx-CPR pPICZA-CPRrat-F CGGATCGGTACCTCGAGATGGGTGATTCTCATG pPICZA-CPRrat-R TCGACTTAAGACCAAACATCCAGAGAATAACGAC pPICZA-CPRSg-F ATCGGTACCTCGAGATGCAGTCTGAATCTTC pPICZA-CPRSg-R GTCGACTTACCAAACATCACGCAGATAACGAC pPICZA-CPRSc-F TCGAGATGCCGTTTGGAATAGACAACACCG pPICZA-CPRSc-R GGTCGACTTACCAGACATCTTCTTGGTATCTACCTG pPICZA-CPRH-F CCTCGAGATGGGTGATTCTCATGTTGATACCTC pPICZA-CPRH-R GGTCGACTTAAGACCAAACATCCAGAGAATAACGACC pPICZA-CPRAn-F CGAGATGGCTCAGCTGGATACCCTGG pPICZA-CPRAn-R GGTCGACTTAAGACCAAACATCTTCTTGATAAGAACCA GAAGAAC Figure S1.
Mutagenesis:Article Title: A Novel A105Y Mutant of CYP17A1 Exhibits Almost Perfect Regioselectivity in the Production of 17α-Hydroxyprogesterone.
Article Snippet: Azenta pPICZA-CPRSc pPICZA carrying CPR gene from S. cerevisiae Azenta pPICZA-CPRHomo pPICZA carrying CPR gene from Homo Azenta pPICZA-CPRAn pPICZA carrying CPR gene from Aspergillus niger Azenta Table S2 Primers used in this study Primer Sequence (5’ to 3’) Site-directed mutagenesis primers A105Y-F GATATTTATTCTAATAATAGAAAAGGTATTGCC A105Y--R ATTAGAATAAATATCCAGGGTGGCCATC A105L-F GATATTCTGTCTAATAATAGAAAAGGTATTGCC A105L--R ATTAGACAGAATATCCAGGGTGGCCATC A105M-F GATTGTAATAGACTCCTGCTGCTG A105M--R ATTAGACATAATATCCAGGGTGGCCATCTG A100I-F ACAGATGATTACCCTGGATATTGCCTC A100I-R CCAGGGTAATCATCTGTGGTCTACCAGAAAAGTCC Q98L-F AGACCACTGATGGCCACCCTGGATATTG Q98L-R GGCCATCATCTGTCTACCAGAAAAGTCCTTACCC Q98S-F AGACCACTGATGGCCACCCTGGATATTG Q98S-R GGCCATCATCAGACTACCAGAAAAGTCCTTACCC Primers for constructing the plasmid pPIC3.5K-HCYP17A1 pPIC3.5K-HCYP17A1-F GATCATAATAGACTCCTGCTGCTG pPIC3.5K-HCYP17A1-R CTATTATGATCAGAAATGGTTGGGGTTC Primers were for constructing the plasmid pPICZx-CPR pPICZA-CPRrat-F CGGATCGGTACCTCGAGATGGGTGATTCTCATG pPICZA-CPRrat-R TCGACTTAAGACCAAACATCCAGAGAATAACGAC pPICZA-CPRSg-F ATCGGTACCTCGAGATGCAGTCTGAATCTTC pPICZA-CPRSg-R GTCGACTTACCAAACATCACGCAGATAACGAC pPICZA-CPRSc-F TCGAGATGCCGTTTGGAATAGACAACACCG pPICZA-CPRSc-R GGTCGACTTACCAGACATCTTCTTGGTATCTACCTG pPICZA-CPRH-F CCTCGAGATGGGTGATTCTCATGTTGATACCTC pPICZA-CPRH-R GGTCGACTTAAGACCAAACATCCAGAGAATAACGACC pPICZA-CPRAn-F CGAGATGGCTCAGCTGGATACCCTGG pPICZA-CPRAn-R GGTCGACTTAAGACCAAACATCTTCTTGATAAGAACCA GAAGAAC Figure S1.
Plasmid Preparation:Article Title: A Novel A105Y Mutant of CYP17A1 Exhibits Almost Perfect Regioselectivity in the Production of 17α-Hydroxyprogesterone.
Article Snippet: Azenta pPICZA-CPRSc pPICZA carrying CPR gene from S. cerevisiae Azenta pPICZA-CPRHomo pPICZA carrying CPR gene from Homo Azenta pPICZA-CPRAn pPICZA carrying CPR gene from Aspergillus niger Azenta Table S2 Primers used in this study Primer Sequence (5’ to 3’) Site-directed mutagenesis primers A105Y-F GATATTTATTCTAATAATAGAAAAGGTATTGCC A105Y--R ATTAGAATAAATATCCAGGGTGGCCATC A105L-F GATATTCTGTCTAATAATAGAAAAGGTATTGCC A105L--R ATTAGACAGAATATCCAGGGTGGCCATC A105M-F GATTGTAATAGACTCCTGCTGCTG A105M--R ATTAGACATAATATCCAGGGTGGCCATCTG A100I-F ACAGATGATTACCCTGGATATTGCCTC A100I-R CCAGGGTAATCATCTGTGGTCTACCAGAAAAGTCC Q98L-F AGACCACTGATGGCCACCCTGGATATTG Q98L-R GGCCATCATCTGTCTACCAGAAAAGTCCTTACCC Q98S-F AGACCACTGATGGCCACCCTGGATATTG Q98S-R GGCCATCATCAGACTACCAGAAAAGTCCTTACCC Primers for constructing the plasmid pPIC3.5K-HCYP17A1 pPIC3.5K-HCYP17A1-F GATCATAATAGACTCCTGCTGCTG pPIC3.5K-HCYP17A1-R CTATTATGATCAGAAATGGTTGGGGTTC Primers were for constructing the plasmid pPICZx-CPR pPICZA-CPRrat-F CGGATCGGTACCTCGAGATGGGTGATTCTCATG pPICZA-CPRrat-R TCGACTTAAGACCAAACATCCAGAGAATAACGAC pPICZA-CPRSg-F ATCGGTACCTCGAGATGCAGTCTGAATCTTC pPICZA-CPRSg-R GTCGACTTACCAAACATCACGCAGATAACGAC pPICZA-CPRSc-F TCGAGATGCCGTTTGGAATAGACAACACCG pPICZA-CPRSc-R GGTCGACTTACCAGACATCTTCTTGGTATCTACCTG pPICZA-CPRH-F CCTCGAGATGGGTGATTCTCATGTTGATACCTC pPICZA-CPRH-R GGTCGACTTAAGACCAAACATCCAGAGAATAACGACC pPICZA-CPRAn-F CGAGATGGCTCAGCTGGATACCCTGG pPICZA-CPRAn-R GGTCGACTTAAGACCAAACATCTTCTTGATAAGAACCA GAAGAAC Figure S1.
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