Q16 - Designing Cloning Primers with Restriction Sites for Vector A

Part of the sequence of vector A, which is for protein expression using Escherichia coli as a host, is shown. It was planned to express a plant-derived gene X using vector A. Vector A is a plasmid vector that expresses a protein fused to the N-terminus His-tag, which enables efficient purification of the expressed protein. As shown in Figure 1, translation of the protein occurs from the start codon immediately before the His tag with six consecutive His residues. The DNA sequences of the 5’ and 3’ regions of gene X are shown in Figure 2. We planned to clone gene X using restriction enzyme sites, EcoRI, SmaI, or SalI in vector A. When the gene X is amplified by PCR, a fragment with a restriction enzyme site at the end can be amplified using the primer with a restriction enzyme site. Since the restriction enzyme site is not recognized if it is located at the end of the DNA fragment, three “Cs” were also attached in addition to the restriction enzyme site. Choose true if the primer is a correct one to use, if not, choose false.

DNA sequence of the cloning region of vector A (double strands).

DNA sequence of the gene X showing 5’ region and 3’ region: 1566 base pair +)63#  4'26’/%‘0(5‘)’/‘!*

Using the information and data, determine which of the statements are true or which are false.

Question reproduced from IBO 2020, Theoretical Exam 1, licensed under CC BY-NC-SA 4.0 - attributed to the International Biology Olympiad. Open the full exam PDF · Community solutions (unofficial)