Cloning method

A system for producing a nucleic acid construct of interest incorporated at a target locus, said system comprising:

• I) a system for preparing two or more standardized expression cassettes, said system comprising:
• a) two or more sets of element sequences,
• each set of element sequences together comprising at least one functional expression cassette,
• each element sequence being flanked on both sides by a type Ils restriction endonuclease cleavage site followed by the recognition site thereof,
• the type IIs restriction endonuclease recognition sites and cleavage sites being selected so that the sets of element sequences may be assembled into a functional expression cassette;
• b) at least two backbone entry vectors,
• each backbone entry vector comprising in this order (i) a restriction enzyme cleavage site with its recognition site and a first connector sequence (LF);
• (ii) a vector backbone comprising a selectable marker gene;
• and (iii) a second connector sequence (RF) and a restriction enzyme recognition site with its cleavage sequence, and;
• (iv) optionally, an insert between the recognition sites of (i) and (iii),
• the connector sequences, RF and LF, on any backbone entry vector being selected so that they can assemble with a LF or RF connector sequence respectively on the same or a different backbone entry vector;
• and
• II) at least two double-stranded integration sequences, one of which is capable of recombination with a first expression cassette comprising at least one of the two or more sets of element sequences of I) a) and a sequence flanking the target locus, and the second of which is capable of recombination with a second expression cassette comprising at least one of the two or more sets of element sequences of I) a) and a sequence flanking the other side of the target locus.