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Standard PCR and subcloning techniques were used to make three PAX6 cDNA constructs in the pDBLeu expression vector (ProQuest Two-Hybrid System, Invitrogen), which generates a protein fused to the yeast GAL4 DNA binding domain. PAX6PST contains the whole PST domain (amino acids 278–422 of the full-length PAX6 protein). Primers were ST001 (forward) 5'-AAA AGT TCG ACT GCC AGC AAC ACA CCT AGT C-3' and ST005 (R) 5'-TTT TGC GGC TTT TTA CTG TAA TCT TGG CCA GTA TTG-3'. PAX6CTP contains the newly defined C-terminal peptide alone (391–422). Primers were ST004 (F) 5'-AAA AGT CGA CTA CCA CTT CAA CAG GAC TCA TT-3' and ST005 (R). PAX6PST-CT contains the PST domain minus the C-terminal peptide (278–390). This was made by cutting PAX6PST with NdeI and NotI to drop out the C-terminal peptide, and inserting a synthetic linker between the two restriction sites. All fragments generated by PCR or with linkers were sequenced to check that no errors had been introduced."}

{"project":"craft-ca-core-ex-dev","denotations":[{"id":"T6780","span":{"begin":1005,"end":1022},"obj":"SO_EXT:0000061"},{"id":"T6779","span":{"begin":960,"end":969},"obj":"SO_EXT:sequence_insertion_process"},{"id":"T6778","span":{"begin":947,"end":954},"obj":"CHEBI_SO_EXT:peptide_or_peptide_region"},{"id":"T6777","span":{"begin":936,"end":946},"obj":"CHEBI_SO_EXT:C_terminus_or_C_terminal_region"},{"id":"T6776","span":{"begin":885,"end":892},"obj":"MOP:0000780"},{"id":"T6775","span":{"begin":849,"end":856},"obj":"CHEBI_SO_EXT:peptide_or_peptide_region"},{"id":"T6774","span":{"begin":838,"end":848},"obj":"CHEBI_SO_EXT:C_terminus_or_C_terminal_region"},{"id":"T6773","span":{"begin":821,"end":827},"obj":"SO_EXT:0000417"},{"id":"T6772","span":{"begin":789,"end":790},"obj":"SO:0001031"},{"id":"T6771","span":{"begin":726,"end":727},"obj":"SO:0001030"},{"id":"T6770","span":{"begin":706,"end":713},"obj":"SO_EXT:0000112"},{"id":"T6769","span":{"begin":681,"end":688},"obj":"CHEBI_SO_EXT:peptide_or_peptide_region"},{"id":"T6768","span":{"begin":670,"end":680},"obj":"CHEBI_SO_EXT:C_terminus_or_C_terminal_region"},{"id":"T6767","span":{"begin":577,"end":578},"obj":"SO:0001031"},{"id":"T6766","span":{"begin":509,"end":516},"obj":"SO:0001030"},{"id":"T6765","span":{"begin":489,"end":496},"obj":"SO_EXT:0000112"},{"id":"T6764","span":{"begin":479,"end":486},"obj":"CHEBI_PR_EXT:protein"},{"id":"T6763","span":{"begin":474,"end":478},"obj":"PR_EXT:000012318"},{"id":"T6762","span":{"begin":435,"end":446},"obj":"CHEBI_SO_EXT:amino_acid"},{"id":"T6761","span":{"begin":427,"end":433},"obj":"SO_EXT:0000417"},{"id":"T6760","span":{"begin":388,"end":394},"obj":"SO_EXT:0000417"},{"id":"T6759","span":{"begin":380,"end":387},"obj":"CHEMINF_GO_EXT:chemical_binding_or_bond_formation"},{"id":"T6758","span":{"begin":376,"end":379},"obj":"CHEBI_SO_EXT:DNA"},{"id":"T6757","span":{"begin":371,"end":375},"obj":"PR_EXT:P04386"},{"id":"T6756","span":{"begin":365,"end":370},"obj":"NCBITaxon_EXT:yeast"},{"id":"T6755","span":{"begin":352,"end":357},"obj":"SO_EXT:sequence_fusion_process"},{"id":"T6754","span":{"begin":344,"end":351},"obj":"CHEBI_PR_EXT:protein"},{"id":"T6753","span":{"begin":277,"end":283},"obj":"SO_EXT:0000440"},{"id":"T6752","span":{"begin":266,"end":276},"obj":"GO:0010467"},{"id":"T6751","span":{"begin":241,"end":251},"obj":"SO_EXT:engineered_biological_sequence"},{"id":"T6750","span":{"begin":236,"end":240},"obj":"SO_EXT:cDNA"},{"id":"T6749","span":{"begin":231,"end":235},"obj":"PR_EXT:000012318"},{"id":"T6748","span":{"begin":136,"end":143},"obj":"SO_EXT:sequence_variant_or_variation"},{"id":"T6747","span":{"begin":128,"end":135},"obj":"SO_EXT:0001023"},{"id":"T6746","span":{"begin":123,"end":127},"obj":"PR_EXT:000012318"},{"id":"T6745","span":{"begin":117,"end":122},"obj":"NCBITaxon:9606"},{"id":"T6744","span":{"begin":88,"end":97},"obj":"SO_EXT:biological_sequence"},{"id":"T6743","span":{"begin":70,"end":77},"obj":"CHEBI_PR_EXT:protein"},{"id":"T6742","span":{"begin":61,"end":65},"obj":"SO_EXT:cDNA"},{"id":"T6741","span":{"begin":56,"end":60},"obj":"PR_EXT:000012318"},{"id":"T6740","span":{"begin":50,"end":55},"obj":"NCBITaxon:9606"},{"id":"T6739","span":{"begin":13,"end":23},"obj":"CHEBI_SO_EXT:amino_acid"},{"id":"T6738","span":{"begin":4,"end":8},"obj":"SO_EXT:cDNA"}],"text":"All cDNA and amino acid numbering is based on the human PAX6 cDNA and protein reference sequences available from the Human PAX6 Allelic Variant Database web site [27]. Standard PCR and subcloning techniques were used to make three PAX6 cDNA constructs in the pDBLeu expression vector (ProQuest Two-Hybrid System, Invitrogen), which generates a protein fused to the yeast GAL4 DNA binding domain. PAX6PST contains the whole PST domain (amino acids 278–422 of the full-length PAX6 protein). Primers were ST001 (forward) 5'-AAA AGT TCG ACT GCC AGC AAC ACA CCT AGT C-3' and ST005 (R) 5'-TTT TGC GGC TTT TTA CTG TAA TCT TGG CCA GTA TTG-3'. PAX6CTP contains the newly defined C-terminal peptide alone (391–422). Primers were ST004 (F) 5'-AAA AGT CGA CTA CCA CTT CAA CAG GAC TCA TT-3' and ST005 (R). PAX6PST-CT contains the PST domain minus the C-terminal peptide (278–390). This was made by cutting PAX6PST with NdeI and NotI to drop out the C-terminal peptide, and inserting a synthetic linker between the two restriction sites. All fragments generated by PCR or with linkers were sequenced to check that no errors had been introduced."}

{"project":"craft-ca-core-dev","denotations":[{"id":"T6719","span":{"begin":509,"end":516},"obj":"SO:0001030"},{"id":"T6718","span":{"begin":489,"end":496},"obj":"SO:0000112"},{"id":"T6717","span":{"begin":474,"end":478},"obj":"PR:000012318"},{"id":"T6716","span":{"begin":427,"end":433},"obj":"SO:0000417"},{"id":"T6715","span":{"begin":388,"end":394},"obj":"SO:0000417"},{"id":"T6714","span":{"begin":371,"end":375},"obj":"PR:P04386"},{"id":"T6713","span":{"begin":277,"end":283},"obj":"SO:0000440"},{"id":"T6712","span":{"begin":266,"end":276},"obj":"GO:0010467"},{"id":"T6711","span":{"begin":231,"end":235},"obj":"PR:000012318"},{"id":"T6710","span":{"begin":128,"end":135},"obj":"SO:0001023"},{"id":"T6709","span":{"begin":123,"end":127},"obj":"PR:000012318"},{"id":"T6708","span":{"begin":117,"end":122},"obj":"NCBITaxon:9606"},{"id":"T6707","span":{"begin":56,"end":60},"obj":"PR:000012318"},{"id":"T6706","span":{"begin":50,"end":55},"obj":"NCBITaxon:9606"},{"id":"T6726","span":{"begin":1005,"end":1022},"obj":"SO:0000061"},{"id":"T6725","span":{"begin":885,"end":892},"obj":"MOP:0000780"},{"id":"T6724","span":{"begin":821,"end":827},"obj":"SO:0000417"},{"id":"T6723","span":{"begin":789,"end":790},"obj":"SO:0001031"},{"id":"T6722","span":{"begin":726,"end":727},"obj":"SO:0001030"},{"id":"T6721","span":{"begin":706,"end":713},"obj":"SO:0000112"},{"id":"T6720","span":{"begin":577,"end":578},"obj":"SO:0001031"}],"text":"All cDNA and amino acid numbering is based on the human PAX6 cDNA and protein reference sequences available from the Human PAX6 Allelic Variant Database web site [27]. Standard PCR and subcloning techniques were used to make three PAX6 cDNA constructs in the pDBLeu expression vector (ProQuest Two-Hybrid System, Invitrogen), which generates a protein fused to the yeast GAL4 DNA binding domain. PAX6PST contains the whole PST domain (amino acids 278–422 of the full-length PAX6 protein). Primers were ST001 (forward) 5'-AAA AGT TCG ACT GCC AGC AAC ACA CCT AGT C-3' and ST005 (R) 5'-TTT TGC GGC TTT TTA CTG TAA TCT TGG CCA GTA TTG-3'. PAX6CTP contains the newly defined C-terminal peptide alone (391–422). Primers were ST004 (F) 5'-AAA AGT CGA CTA CCA CTT CAA CAG GAC TCA TT-3' and ST005 (R). PAX6PST-CT contains the PST domain minus the C-terminal peptide (278–390). This was made by cutting PAX6PST with NdeI and NotI to drop out the C-terminal peptide, and inserting a synthetic linker between the two restriction sites. All fragments generated by PCR or with linkers were sequenced to check that no errors had been introduced."}