Molecular cloning of LRP16 gene promoter and constructing LRP16 promoter-pGL3-Basic vector

Objective:To clone LRP16 gene promoter and construct LRP16 gene promoter_pGL3_Basic vector.Methods:①A 2.7?kb DNA sequence of LRP16 5′_end was obtained from NCBI by BLAST software.②After the 2.7?kb target sequence from a healthy blood donor DNA sample was amplified by PCR method,the product of PCR was inserted into pGEM_T Easy vector by T4 DNA ligase and the vector was transferred into JM109 E.coli . The positive clone was identified by nest PCR, double digestion with Kpn Ⅰ and Bam HⅠ and DNA sequencing.③The identified target LRP16 promoter DNA sequence was regained from LRP16 promoter_pGEM_T Easy vector by Sac Ⅰ and Hin dⅢ cutting, then was inserted into pGL3_Basic vector to construct the target LRP16 promoter_pGL3_Basic vector.Results and Conclusion:A 2.7?kb LRP16 promoter was successfully cloned and the LRP16 promoter_pGL3_Basic vector was well constructed.A known gene promoter sequence can be freely obtained from NCBI database, which is very useful for the gene promoter cloning and promoter vector construction.