DNA vector including multiple cloning site - for cloning, specific mutagenesis, sequencing and optimised expression of inserted genes, and construction of gene banks

摘要

New vector is a circular, double-stranded DNA molecule contg. at least the elements, (1) origin of replication (ori1); (2) promoter; (3) ribosome binding site (RBS); (4) multiple cloning site (MCS) and (5) origin of replication (ori2) of a single-stranded DNA phage. The elements 2, 3 and 4 exist, in the order quoted, in the transcription direction. Also new are kits contg. this vector. The promoter is pref. strong and esp. inducible; specifically the lambda-PL, tac or rac promoters. the MCS has, at its 3'-end, a stop codon for all 3 reading frames and also a start codon at the 5'-end. It restriction sites which allows cloning in all 3 frames. Ori2 is derived from the phages fl, M13 or SP6, and the vector may include a termination sequence, esp. the T1 and T2 sequences of the rrnB protein. USE/ADVANTAGE - The vectors are useful for cloning, targeted alteration, sequencing and/or expression of genes inserted into MCS, and for construction of (partial) gene banks. It combines the properties of a strong expression vector with the advantages of a single-stranded DNA phage, i.e. it is simple to use for site-directed mutagenesis; allows the RBS/start codon to be optimised, and almost all genetic engineering operations can be carried out in the same vector without recloning.