THE COMPLETE NUCLEOTIDE-SEQUENCES OF THE SACBII-KAN DOMAIN OF THE P1-PAD10-SACBII CLONING VECTOR AND 3 COSMID CLONING VECTORS - PTCF, SVPHEP, AND LAWRIST16

The complete nucleotide sequence of the 16,009-bp SacBIl Kan domain of the P1 pAD10-SacBII cloning vector and the sequences of three cosmid cloning vectors, pTCF (7941 bp), sVPHEP (9201 bp), and LAWRIST16 (5194 bp) have been determined. A modified diatomaceous earth (Prep-A-Gene) -based procedure, which rapidly yields highly supercoiled double-stran ded DNA from recombinant P1 and cosmid clones suitable for generating shotgun libraries, also has been developed. The isolated recombinant D NAs were physically sheared to generate 1- to 2-kb fragments that then were blunt-ended and subcloned into double-stranded pUC-based sequenc ing vectors. The double-stranded sequencing templates were isolated by an alkaline lysis method and subjected to Tag polymerase catalyzed fl uorescent end-labeled primer cycle sequencing. After shotgun sequence assembly, contig gaps were closed and ambiguities were resolved via Se quenase catalyzed fluorescent dye-terminator sequencing.