The synthetic gene coding for N-terminal Profinity eXact tagged mature NP was designed on the basis of codon preference in E. coli [26 (link),27 (link)]. A codon optimized Profinity eXact tag was added to the 5’ end of the gene coding for the mature NP (Accession number MN908947) (Fig. S1 ). This synthetic gene was digested with NcoI and BamHI restriction enzymes (TaKaRa Bio, Kusatsu, Japan) and then subcloned into NcoI/BamHI digested pET-3d expression vector (Merck, Rahway, NJ, USA) to produce the pET-3d-eXact-NP that encodes the eXact tag-SARS-CoV-2 NP fusion protein (Fig. S1A ).
Ncoi and bamhi restriction enzymes
Manufactured by Takara Bio
Sourced in Japan, China
NcoI and BamHI are type II restriction enzymes that recognize and cleave specific DNA sequences. NcoI recognizes and cleaves the palindromic DNA sequence 5'-CCATGG-3', while BamHI recognizes and cleaves the palindromic DNA sequence 5'-GGATCC-3'. These enzymes are commonly used in molecular biology and genetic engineering applications for DNA manipulation and analysis.
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Lab products found in correlation
2 protocols using ncoi and bamhi restriction enzymes
1
Codon-optimized SARS-CoV-2 NP Expression
2
Cloning and Expression of SnRK2.1
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The cDNA encoding for SnRK2.1 was provided by Professor Chen from College of Life Sciences, Northwest A&F University, China. The cDNA was amplified by PCR using LA Taq DNA polymerase (Takara, Dalian, China) and primer pairs as follows: 5′-CCGCCATGG ACAAGTATGACGTTG-3′ and 5′-CGGGATCC TTAAGCTTTGTCAGACTCT-3′. Restriction sites (NcoI and BamHI) are underlined and the stop codon TAA introduced is italicized. The PCR product was digested with NcoI and BamHI restriction enzymes (Takara, Dalian, China) and ligated into the identically digested cell-free expression vector pIVEX2.4c (Roche Applied Science, Mannheim, Germany), carrying a His6-tag sequence at the N-terminal, by T4 DNA ligase (Takara, Dalian, China). The construction of the expression vector pIVEX2.4c-SnRK2.1, which encoded a His6-tag at the N-terminal of target protein, was confirmed by restriction enzyme digestion and the correctness of sequence was confirmed by DNA sequencing.
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