You wish to express human thymidylate kinase in E. coli using pET20b, with a His-tag for purification and no signal sequence. The mRNA sequence of the gene is: DTYMK. txt Download DTYMK. txt

The first few amino acids of the protein are: MAARRGALIV (Single letter code).

Please prepare a short report that covers the following:

1. Give the left and right PCR primers that you would use to amplify the gene from a cDNA library. Use online software to determine Tm.

The Tm for your primers should be 60 C
2. Briefly describe the steps involved in inserting your PCR product into the vector.

3. You plan to have a synthetic gene made that you will then insert into pET20b. Give the DNA sequence for the 5' and 3' end of the DNA that includes any restriction sites and the first three and last three codons of the gene. You should use codons that are more suitable for expression in E. coli. The following table provides codon frequencies of highly expressed genes: Codon Table (Links to an external site.)(class II).