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Lab #4 "Putting it all together" Disaster. The protein you and your colleagues thought would inhibit SARS CoV-2 entry doesn't work. Lab experiments show it doesn't bind SARS CoV-2 spike protein. You want to test another small protein, which requires that you clone its encoding DNA into a plasmid, express it, and purify it. The sequence of this gene is: 5'- ATG CAT CAC CAT CAC CAT CAC ACA TGG TAC GTA AAA ATC ATC GAC CGG CGT GCA GCA GCA TGC GAA TCT TGG GTA CCT TTT CAC CAG GAA AAC TGA-3' (15 points) 1a. Circle the start codon 1b. Underline the His6 sequence 1c. Box the stop codon You wish to clone this protein into an expression vector, and only have BamH1 and Xho1 restriction enzymes in lab. The vector you use has BamH1 and Xho1 restriction sites in the multiple cloning site. BamH1 restriction site 51 GGATCC 3' 3'. CCTAGG. 5' Xho1 restriction site 5' CTCGAG...3 3' GAGCTC...5" (15 points) 2. Design primers that will generate an amplicon that can digested with BamH1 and Xho1 and cloned into doubly digested (BamH1 / Xho1) expression vector. Hint: a primer is typically ~20 nucleotides in length.

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