Patent ID: 7267984
Filing Date: 2007-09-11
Classification: C12N

Abstract:
1. A method for the assembly of large DNA fragments, comprising: a) manipulating a replicon to comprise in order a final excision site, a first fragment, a first excision site and a first recombinase site; b) manipulating a first vector to comprise in order the first recombinase site from step a), undesired vector sequences, the first excision site from step a), a second fragment, and a second excision site; c) inserting the first vector into the replicon using a first recombinase so that the two first site specific excision sites are oriented in an appropriate orientation for excision with undesired vector sequences therebetween; d) treating the replicon with a first excisionase to completely remove the undesired vector sequences including both first excisionase sites and bring the second fragment directly adjacent to the first fragment; e) manipulating a second vector to comprise in order the first recombinase site from step a), undesired vector sequences, the second excision site from step b), a third fragment, and the first excision site from step a); f) inserting the second vector into the replicon using the first recombinase so that the two second site specific excision sites are oriented in an appropriate orientation for excision with undesired vector sequences therebetween; g) treating the replicon with a second excisionase to completely remove the undesired vector sequences including both second excisionase sites and bring the third fragment directly adjacent to the second fragment; h) repeating steps b-g using at least the first and second excisionases to make an assembled DNA, wherein the final vector also comprises the final excision site 5′ to all other sequences and in an appropriate orientation for excision; and i) excising and circularizing the assembled DNA with a final excisionase.