Molecular cloning and mutagenesis The DNA sequences encoding identified potassium binding domains, MNT (list all of them; sequence IDs above), and KRaION1 were synthesized by Tsingke Biotech Co. (China) and swapped with Electra1 gene in the pHybrid expression vector [68] (pHybrid-Electra1, WeKwikGene plasmid #0000202) for screening in E.coli bacteria and cytoplasmic expression in HEK293FT cells. The secretion peptides used in Fig 2D were synthesized de novo with mammalian codon optimization based on the amino acid sequences of the N-termini of aspartic proteinase nepenthesin-1 (Asp, 1-24 aa from Sequence ID: BAF98915.1), azurocidin (Azu, 1-19 aa from Sequence ID: NP_001691.1), corticotropin-releasing factor receptor 1 (CRFR, 1-24 aa from Sequence ID: XP_040124593.1), endothelin receptor type B (EtBR, 1-26 aa from Sequence ID: CAD30645.1).|||The custom DNA oligonucleotides were synthesized by Tsingke Biotechnology Co., PrimeStar Max master mix (Takara, Japan) was used for routine polymerase chain reaction (PCR) amplification.
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