Patent Document ID: 10041067
Application ID: 15140105
Patent Flag: 1

Claim One:
1. A method for in vitro assembly of genetic modules, comprising: (a) providing recombinant transcription units Tu1, Tu2 and TuN wherein N>=3, each recombinant transcription unit being present in a separate stage 1 vector and flanked by a first pair of restriction sites of a first type IIs enzyme, wherein the first pair of restriction sites for each recombinant transcription unit are pre-designed such that upon digestion by the first type IIs enzyme, compatible cohesive ends are generated to allow ligation of the recombinant transcription units into a linear DNA molecule having a predetermined order 5′-Tu1-TuN-Tu2-3′; (b) providing a stage 2 vector having a second pair of restriction sites of the first type IIs enzyme, wherein the second pair of restriction sites are pre-designed such that upon digestion by the first type IIs enzyme, a first and second cohesive end are generated to allow direct ligation of the first cohesive end with Tu1 at its 5′ end and direct ligation of the second cohesive end with Tu2 at its 3′ end; and (c) assembling the recombinant transcription units and the stage 2 vector into a plasmid in a one-pot reaction comprising the first type IIs enzyme and a ligase.