Patent ID: 8202727

Claim:
A method for sequentially cloning a plurality of heterologous nucleic acid sequences, comprising: (a) inserting a portable segment that (i) lacks an origin of replication, but (ii) comprises a first marker sequence that confers a first identifiable characteristic on host cells that contain the first marker sequence, into a first recipient construct that (i) lacks the first marker sequence, but (ii) comprises a first heterologous nucleic acid sequence to thereby form a first recombinant construct that comprises a first cassette that (i) is portable into a second recipient construct, (ii) lacks an origin of replication, and (iii) comprises the first marker sequence and the first heterologous nucleic acid sequence; (b) introducing the first recombinant construct into a first set of host cells; (c) screening for the first set of host cells with the first identifiable characteristic to thereby identify a first set of recombinant cells that contain the first recombinant construct; (d) obtaining the first cassette from the first recombinant construct corresponding to the first set of recombinant cells; (e) inserting the first cassette into the second recipient construct that (i) lacks the first marker sequence, but (ii) comprises a second heterologous nucleic acid sequence to form a second recombinant construct comprising a second cassette that (i) is optionally portable into a third recipient construct, (ii) lacks an origin of replication, and (iii) comprises the first cassette and the second heterologous nucleic acid sequence; (f) introducing the second recombinant construct into a second set of host cells, and (g) screening for the second set of hosts cells with the first identifiable characteristic to thereby identify a second set of recombinant cells that contain the second recombinant construct.