• Journal Article

The Complete Nucleotide Sequences of the pSacBII P1 Cloning Vector and Three Cosmid Cloning Vectors: pTCF, svPHEP, and LAWRIST16

Citation

Pan, H., Wang, S., Chissoe, S., Bodenteich, A., Iyer, K., Clifton, S., ... Roe, B. (1994). The Complete Nucleotide Sequences of the pSacBII P1 Cloning Vector and Three Cosmid Cloning Vectors: pTCF, svPHEP, and LAWRIST16. Genetic Analysis, Techniques and Applications, 11(5-6), 181 - 186.

Abstract

The complete nucleotide sequence of the 16,009-bp SacBII 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-stranded DNA from recombinant P1 and cosmid clones suitable for generating shotgun libraries, also has been developed. The isolated recombinant DNAs were physically sheared to generate 1- to 2-kb fragments that then were blunt-ended and subcloned into double-stranded pUC-based sequencing vectors. The double-stranded sequencing templates were isolated by an alkaline lysis method and subjected to Taq polymerase catalyzed fluorescent end-labeled primer cycle sequencing. After shotgun sequence assembly, contig gaps were closed and ambiguities were resolved via Sequenase catalyzed fluorescent dye-terminator sequencing.