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Exon 7 encodes 24 amino acids in the active-site region, including Asn286, one of the catalytic triad residues. The short (upstream) arm of the targeting construct was provided by a 2.7-kb HindIII-BamHI fragment, containing exons 5 and 6, which was inserted into the pPNT vector upstream of the PGK-Neo cassette [48]. During cloning, the BamHI site at the 3' end of the short arm was abolished. The loss of this BamHI site in the mutant allele, coupled with the introduction of a new BamHI site at the 3' end of the PGK-Neo cassette, provided a basis for distinguishing the wild-type and mutant alleles by Southern blotting. The long (downstream) arm of homology was provided by a 7-kb HindIII-KpnI fragment, extending from intron 7 to intron 12, inserted between the PGK-Neo and thymidine kinase (tk) cassettes."}

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{"project":"craft-ca-core-ex-dev","denotations":[{"id":"T16708","span":{"begin":956,"end":965},"obj":"SO_EXT:0005853"},{"id":"T16707","span":{"begin":952,"end":954},"obj":"GO_PR_EXT:thymidine_kinase"},{"id":"T16706","span":{"begin":934,"end":950},"obj":"GO_PR_EXT:thymidine_kinase"},{"id":"T16705","span":{"begin":934,"end":943},"obj":"CHEBI_EXT:thymine_nucleobase_or_nucleoside_or_nucleotide_molecular_entity_or_group"},{"id":"T16704","span":{"begin":926,"end":929},"obj":"CHEBI_GO_EXT:neomycin_or_neomycin_phosphotransferase"},{"id":"T16703","span":{"begin":922,"end":925},"obj":"GO_EXT:phosphoglycerate_kinase"},{"id":"T16702","span":{"begin":901,"end":909},"obj":"SO_EXT:sequence_insertion_process"},{"id":"T16701","span":{"begin":890,"end":896},"obj":"SO_EXT:0000188"},{"id":"T16700","span":{"begin":878,"end":884},"obj":"SO_EXT:0000188"},{"id":"T16699","span":{"begin":840,"end":847},"obj":"PR_EXT:P43870"},{"id":"T16698","span":{"begin":838,"end":839},"obj":"CHEBI_SO_EXT:base"},{"id":"T16697","span":{"begin":789,"end":799},"obj":"SO_EXT:sequence_downstreamness"},{"id":"T16696","span":{"begin":749,"end":756},"obj":"SO_EXT:0001023"},{"id":"T16679","span":{"begin":403,"end":411},"obj":"SO_EXT:sequence_insertion_process"},{"id":"T16677","span":{"begin":351,"end":356},"obj":"PR_EXT:P23940"},{"id":"T16676","span":{"begin":343,"end":350},"obj":"PR_EXT:P43870"},{"id":"T16675","span":{"begin":341,"end":342},"obj":"CHEBI_SO_EXT:base"},{"id":"T16674","span":{"begin":308,"end":317},"obj":"SO_EXT:engineered_biological_sequence"},{"id":"T16673","span":{"begin":277,"end":285},"obj":"SO_EXT:sequence_upstreamness"},{"id":"T16672","span":{"begin":256,"end":264},"obj":"CHEBI_EXT:residue"},{"id":"T16671","span":{"begin":240,"end":249},"obj":"GO_MOP_EXT:catalysis"},{"id":"T16670","span":{"begin":172,"end":183},"obj":"CHEBI_SO_EXT:amino_acid"},{"id":"T16669","span":{"begin":161,"end":168},"obj":"SO_EXT:sequence_coding_function"},{"id":"T16668","span":{"begin":154,"end":158},"obj":"SO_EXT:0000147"},{"id":"T16667","span":{"begin":133,"end":141},"obj":"SO_EXT:0005853"},{"id":"T16666","span":{"begin":129,"end":132},"obj":"CHEBI_GO_EXT:neomycin_or_neomycin_phosphotransferase"},{"id":"T16665","span":{"begin":125,"end":128},"obj":"GO_EXT:phosphoglycerate_kinase"},{"id":"T16664","span":{"begin":110,"end":114},"obj":"SO_EXT:0000704"},{"id":"T16663","span":{"begin":104,"end":109},"obj":"PR_EXT:000005015"},{"id":"T16662","span":{"begin":90,"end":94},"obj":"SO_EXT:0000147"},{"id":"T16661","span":{"begin":61,"end":68},"obj":"PR_EXT:P43870"},{"id":"T16660","span":{"begin":55,"end":60},"obj":"PR_EXT:P23940"},{"id":"T16659","span":{"begin":52,"end":54},"obj":"SO_EXT:0000028"},{"id":"T16658","span":{"begin":38,"end":45},"obj":"SO_EXT:sequence_substitution_process"},{"id":"T16657","span":{"begin":12,"end":21},"obj":"SO_EXT:engineered_biological_sequence"},{"id":"T16695","span":{"begin":742,"end":748},"obj":"SO_EXT:sequence_altered_entity_or_alteration_process"},{"id":"T16694","span":{"begin":728,"end":737},"obj":"SO_EXT:wild_type_entity_or_quality"},{"id":"T16693","span":{"begin":678,"end":686},"obj":"SO_EXT:0005853"},{"id":"T16692","span":{"begin":674,"end":677},"obj":"CHEBI_GO_EXT:neomycin_or_neomycin_phosphotransferase"},{"id":"T16691","span":{"begin":670,"end":673},"obj":"GO_EXT:phosphoglycerate_kinase"},{"id":"T16690","span":{"begin":638,"end":643},"obj":"PR_EXT:P23940"},{"id":"T16689","span":{"begin":591,"end":597},"obj":"SO_EXT:0001023"},{"id":"T16688","span":{"begin":584,"end":590},"obj":"SO_EXT:sequence_altered_entity_or_alteration_process"},{"id":"T16687","span":{"begin":566,"end":571},"obj":"PR_EXT:P23940"},{"id":"T16686","span":{"begin":492,"end":497},"obj":"PR_EXT:P23940"},{"id":"T16685","span":{"begin":479,"end":486},"obj":"SO_EXT:sequence_cloning_process"},{"id":"T16684","span":{"begin":457,"end":465},"obj":"SO_EXT:0005853"},{"id":"T16683","span":{"begin":453,"end":456},"obj":"CHEBI_GO_EXT:neomycin_or_neomycin_phosphotransferase"},{"id":"T16682","span":{"begin":449,"end":452},"obj":"GO_EXT:phosphoglycerate_kinase"},{"id":"T16681","span":{"begin":433,"end":441},"obj":"SO_EXT:sequence_upstreamness"},{"id":"T16680","span":{"begin":426,"end":432},"obj":"SO_EXT:0000440"},{"id":"T16678","span":{"begin":378,"end":383},"obj":"SO_EXT:0000147"}],"text":"A targeting construct was designed to replace a 785 bp BamHI-HindIII fragment, containing exon 7 of the Capn2 gene, with the PGK-Neo cassette (Figure 1). Exon 7 encodes 24 amino acids in the active-site region, including Asn286, one of the catalytic triad residues. The short (upstream) arm of the targeting construct was provided by a 2.7-kb HindIII-BamHI fragment, containing exons 5 and 6, which was inserted into the pPNT vector upstream of the PGK-Neo cassette [48]. During cloning, the BamHI site at the 3' end of the short arm was abolished. The loss of this BamHI site in the mutant allele, coupled with the introduction of a new BamHI site at the 3' end of the PGK-Neo cassette, provided a basis for distinguishing the wild-type and mutant alleles by Southern blotting. The long (downstream) arm of homology was provided by a 7-kb HindIII-KpnI fragment, extending from intron 7 to intron 12, inserted between the PGK-Neo and thymidine kinase (tk) cassettes."}