The article describes a system for shotgun DNA sequencing using the single-stranded DNA phage M13mp7. The system introduces a multipurpose cloning site into the gene for β-galactosidase (β-D-galactosidegalactohydrolase) on the M13mp2 phage, allowing for the cloning of DNA fragments generated by various restriction endonucleases. The cloning site adds 14 additional codons without affecting the intracistronic complementation of the lac gene product. The nucleotide sequences of the cloned DNAs can be determined rapidly by DNA synthesis using a synthetic oligonucleotide primer complementary to 15 bases before the new array of restriction sites. The system's versatility is demonstrated through the insertion of multiple restriction enzyme sites, including SalI, PstI, BamHI, AccI, and HinCII, which can generate cohesive or blunt-ended termini. The article also details the construction of M13mp7, the selection of mutations to remove additional cleavage sites, and the use of a synthetic primer for DNA sequencing. This system provides a rapid and efficient method for sequencing long stretches of nucleotide sequences from higher organisms.The article describes a system for shotgun DNA sequencing using the single-stranded DNA phage M13mp7. The system introduces a multipurpose cloning site into the gene for β-galactosidase (β-D-galactosidegalactohydrolase) on the M13mp2 phage, allowing for the cloning of DNA fragments generated by various restriction endonucleases. The cloning site adds 14 additional codons without affecting the intracistronic complementation of the lac gene product. The nucleotide sequences of the cloned DNAs can be determined rapidly by DNA synthesis using a synthetic oligonucleotide primer complementary to 15 bases before the new array of restriction sites. The system's versatility is demonstrated through the insertion of multiple restriction enzyme sites, including SalI, PstI, BamHI, AccI, and HinCII, which can generate cohesive or blunt-ended termini. The article also details the construction of M13mp7, the selection of mutations to remove additional cleavage sites, and the use of a synthetic primer for DNA sequencing. This system provides a rapid and efficient method for sequencing long stretches of nucleotide sequences from higher organisms.