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Two inherited disorders cause hereditary cobalamin deficiency: the Imerslund-Grasbeck syndrome and congenital pernicious anemia (intrinsic factor deficiency)

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coli , gene synthesized (Biomatik), and inserted into pET28a vector using Eco R1 and Nde 1 sites in frame with an N-terminal 6x-His tag using a previously described cloning method (Schenck et al., For site directed mutagenesis, plasmid template was diluted 100-fold, mixed with 0.04 U/μL Phusion DNA polymerase (Thermo), 0.2 mM dNTP's, 0.5 μM forward and reverse mutagenesis primers and 1x Phusion reaction buffer (Thermo), and then placed in a thermocycler for 98°C for 30 s followed by 20 cycles of 10 s at 98°C, 20 s at 70°C, 4.5 min at 72°C with a final extension at 72°C for 10 min