**Title:** Introducing a Nuclear Localization Sequence into Yeast Cytoplasmic Proteins**Objective:** To understand how to add a nuclear localization sequence (NLS) of Lys-Lys-Lys-Arg-Lys to a cytoplasmic protein in yeast.**Content:**You aim to add a nuclear localization sequence (NLS) of Lys-Lys-Lys-Arg-Lys to a protein typically found in the cytoplasm of a yeast cell. To achieve this, you should introduce the nucleotide sequence encoding the NLS into the gene that encodes the cytoplasmic protein of interest.**a. Nucleotide Insert Size:**- **Question:** What is the size of the nucleotide insert that will encode the NLS? Briefly explain.**b. Gene Insertion Diagram:**Below is a diagram illustrating the gene encoding the cytoplasmic protein of interest within the yeast genome. The goal is to insert the NLS at the carboxyl (C) terminus of the protein. Determine at which location (A-E) the NLS should be inserted and provide a brief explanation.**Diagram Explanation:**- **5′ to 3′ Direction:** Shows the DNA strand coding process.- **Promoter Region:** Located between the arrows labeled "promoter," featuring sequences like TATAA and ATATT.- **Locations A-E:** Indicate potential insertion points for the NLS: - **A:** After the promoter sequence. - **B:** After the transcription start site (+1). - **C:** After the start codon (ATG). - **D:** Mid-sequence between the start codon and stop codon. - **E:** After the stop codon (TAA).- **Correction:** Typically, the NLS should be placed near the C-terminus for its role in nuclear import, which generally means inserting it at location E, after the stop codon of the cytoplasmic protein's coding sequence. This ensures proper expression and function.

To insert a nuclear localization signal (NLS) into a yeast gene, it is important to consider the location of the insertion. The NLS should be inserted after the stop codon (TAA) to ensure that it is translated and incorporated into the protein. This will allow the protein to be transported into the nucleus, where it can function.a. The size of the nucleotide insert that will encode the NLS is 15 nucleotides. This is because each amino acid is encoded by 3 nucleotides, and the NLS contains 5 amino acids.b. To insert the NLS at the carboxyl (C) terminus of the protein, the nucleotide sequence encoding the NLS should be inserted after the stop codon (TAA). This is because the stop codon signals the end of translation, and any nucleotides inserted after the stop codon will not be translated into protein.Therefore, the best location to insert the NLS is at position E in the diagram.Explanation:The diagram shows a gene encoding a cytoplasmic protein of interest in the yeast genome. The gene has a promoter region, a coding region, and a terminator region. The promoter region contains the sequences necessary to initiate transcription of the gene. The coding region contains the codons that specify the amino acid sequence of the protein. The terminator region contains the sequences necessary to terminate transcription of the gene.The stop codon (TAA) is located at the end of the coding region. It signals the end of translation, and any nucleotides inserted after the stop codon will not be translated into protein.Therefore, to insert the NLS at the C terminus of the protein, the nucleotide sequence encoding the NLS should be inserted after the stop codon. This will ensure that the NLS is translated and incorporated into the protein.The best location to insert the NLS is at position E in the diagram, after the stop codon (TAA).