| 摘要 |
The present invention provides novel vectors and methods for assembling complex DNA molecules starting with a plurality of input gene sequences. The input gene sequences (which overlap with each other by a defined number of bases) are cloned into a vector at a unique restriction site that is flanked on each side by class IIS restriction endonuclease sites. When the clones are digested with the class IIS restriction enzyme, the inserts are released from the vector with a defined number of bases removed from either the 5' or 3' termini, corresponding to the overlap sequences. The overlap sequences, which are unique, non-palindromic sequence strings, permit the fragments to self-assemble. When the fragments are ligated, a seamless, unambiguous linear array fragments is created. The invention can be used for assembling synthetic genes, constructs, vectors and chromosomes.
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