Patent ID: 9631204
Date: 2017-04-25
CPC Classifications: C12N

Claim:
1. A method for making recombinant vectors suitable for homologous recombination with each other in yeast comprising: i) providing a first recombinant vector (CDS vector) comprising a protein coding sequence (CDS sequence) wherein the CDS is flanked on its 5′ and 3′ ends by first Type IIS restriction enzyme recognition sites, the CDS vector further comprising a first antibiotic resistance gene; ii) providing a second recombinant vector (PRO vector) comprising a promoter sequence (PRO sequence) wherein the PRO sequence is flanked on its 5′ and 3′ ends by the first Type IIS restriction enzyme recognition sites, the PRO vector further comprising the first antibiotic resistance gene; iii) providing a third recombinant vector (TER vector) comprising a transcription termination sequence (TER sequence) wherein the TER sequence is flanked on its 5′ and 3′ ends by the first restriction Type IIS enzyme recognition sites, the TER vector further comprising the first antibiotic resistance gene; iv) providing a fourth recombinant vector (LVA vector) comprising a first left adapter polynucleotide sequence (LVA sequence) wherein the LVA sequence is flanked on its 5′ and 3′ ends by the first Type IIS restriction enzyme recognition sites, the LVA vector further comprising the first antibiotic resistance gene; v) providing a fifth recombinant vector (RVA vector) comprising a first right adapter polynucleotide sequence (RVA sequence) wherein the RVA sequence is flanked on its 5′ and 3′ ends by the first Type IIS restriction enzyme recognition sites, the RVA vector further comprising the first antibiotic resistance gene; wherein the LVA sequence comprises the sequence: and/or wherein the RVA sequence comprises the sequence: vi) providing a sixth recombinant vector (acceptor vector) comprising a segment, the segment comprising a polynucleotide sequence encoding a detectable marker (detectable marker sequence), wherein the detectable marker sequence is flanked by the first Type IIS restriction enzyme recognition sites, and wherein the segment is flanked by a second Type IIS restriction enzyme recognition sites, wherein the acceptor vector comprises a second antibiotic resistance gene but does not comprise the first antibiotic resistance gene; vii) incubating the CDS vector, the PRO vector, the TER vector, the LVA vector, the RVA vector, and the acceptor vector in a single reaction container with a first Type IIS restriction endonuclease that recognizes the first Type IIS restriction endonuclease recognition site and a DNA ligase enzyme such that ligated vectors are produced, wherein the ligated vectors comprise sequentially the LVA sequence, the PRO sequence, the CDS sequence, the TER sequence, and the RVA sequence (LVA-TU-RVA vectors), wherein the PRO, CDS and TER sequences comprise a transcription unit (TU), and wherein the LVA-TU-RVA vectors comprise the second antibiotic resistance gene, but do not comprise the first antibiotic resistance gene, wherein the LVA-TU-RVA vectors do not comprise the detectable marker sequence, and wherein the ligated vectors do not comprise the first Type IIS restriction site, but do comprise the second Type IIS restriction site; viii) introducing the LVA-TU-RVA vectors from vii) into bacteria and culturing the bacteria with a culture medium comprising an antibiotic to which bacteria comprising the LVA-TU-RVA vectors are resistant via expression of the second antibiotic resistance gene such that clonal colonies of the bacteria comprising the VEGAS vectors are formed, wherein the clonal colonies do not express the detectable marker; and viii) isolating the LVA-TU-RVA vectors from the colonies that do not express the detectable marker to obtain isolated LVA-TU-RVA vectors.