A system for shotgun DNA sequencing was developed by introducing a multipurpose cloning site into the gene for β-galactosidase on the single-stranded DNA phage M13mp2 using synthetic DNA. This site adds 14 additional codons and does not affect the function of the lac gene product. Two restriction endonuclease cleavage sites in the viral gene II were removed by single base-pair mutations. The new phage M13mp7 allows for the direct cloning of DNA fragments generated by various restriction enzymes. The nucleotide sequences of the cloned DNAs can be determined rapidly using DNA synthesis with chain terminators and a synthetic oligonucleotide primer complementary to 15 bases preceding the new array of restriction sites. The system utilizes the E. coli lac system and the filamentous single-stranded DNA phage M13 to prepare cloned templates in a single-stranded form required for DNA sequencing with chain terminators. The phage M13mp7 is a versatile vehicle for preparing fragment banks from any DNA. It is an excellent vector for a shotgun approach to rapidly obtain the nucleotide sequence of any DNA of interest. The DNA of interest is converted into sets of fragments, each set generated by one of the restriction endonucleases listed. When cloned, these sets yield banks of recombinant M13mp7 phage in which the large DNA is represented piece-meal with the two complementary DNA strands present in separate clones. The entire primary structure of the DNA can be reconstructed by compiling the data obtained from sequencing several recombinant M13mp7 banks and aligning the nucleotide sequences by overlaps and complementarities. The large amount of data processing necessary for reconstructing long sequences can be efficiently assisted by computer.A system for shotgun DNA sequencing was developed by introducing a multipurpose cloning site into the gene for β-galactosidase on the single-stranded DNA phage M13mp2 using synthetic DNA. This site adds 14 additional codons and does not affect the function of the lac gene product. Two restriction endonuclease cleavage sites in the viral gene II were removed by single base-pair mutations. The new phage M13mp7 allows for the direct cloning of DNA fragments generated by various restriction enzymes. The nucleotide sequences of the cloned DNAs can be determined rapidly using DNA synthesis with chain terminators and a synthetic oligonucleotide primer complementary to 15 bases preceding the new array of restriction sites. The system utilizes the E. coli lac system and the filamentous single-stranded DNA phage M13 to prepare cloned templates in a single-stranded form required for DNA sequencing with chain terminators. The phage M13mp7 is a versatile vehicle for preparing fragment banks from any DNA. It is an excellent vector for a shotgun approach to rapidly obtain the nucleotide sequence of any DNA of interest. The DNA of interest is converted into sets of fragments, each set generated by one of the restriction endonucleases listed. When cloned, these sets yield banks of recombinant M13mp7 phage in which the large DNA is represented piece-meal with the two complementary DNA strands present in separate clones. The entire primary structure of the DNA can be reconstructed by compiling the data obtained from sequencing several recombinant M13mp7 banks and aligning the nucleotide sequences by overlaps and complementarities. The large amount of data processing necessary for reconstructing long sequences can be efficiently assisted by computer.