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. 2021 Dec 17;1(12):e316. doi: 10.1002/cpz1.316

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Published 2021. This article is a U.S. Government work and is in the public domain in the USA. Current Protocols published by Wiley Periodicals LLC.

This is an open access article under the terms of the http://creativecommons.org/licenses/by-nc-nd/4.0/ License, which permits use and distribution in any medium, provided the original work is properly cited, the use is non‐commercial and no modifications or adaptations are made.

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Scheme of the vectors used in the IIS‐alphoid^tetO‐HAC system. (A) A139 vector expressing ΦC31 integrase and Cre recombinase and (B) Type I carrier vector A167 to deliver the first genomic DNA fragment (DNA1) and to perform every odd‐numbered round of DNA integration. A167 vector has a promoterless Puro‐mCherry‐FcyFur (PCF) marker and a constitutively active eGFP‐hph‐TK (GHT) marker under the CAGG promoter in its vector backbone. (C) A135‐JH vector expressing ΦBT1 integrase and Cre recombinase and (D) Type II carrier vector A169 to deliver the second genomic DNA fragment (DNA2) and to perform every even‐numbered round of DNA integration. A169 vector has a promoterless GHT marker and a constitutively active PCF marker under the CAGG promoter in its vector backbone. A139 and A135‐JH expression vectors carry zeomycin resistance (Zeo^R). Both Type I and Type II carrier vectors are used to integrate DNA segments of interest into the platform cassette of the IIS‐alphoid^tetO‐HAC. These vectors contain both a BAC cassette containing F′ origin of replication (low‐copy maintenance) and a pBR322 origin of replication (ColE1) to make the vectors multicopy. ColE1 origin is removed once a large DNA segment of interest (DNA1, DNA2, or DNAn) is added to the vector. (E) DNA fragments are inserted into Type I and Type II carrier vectors A167 and A169 via ligation into unique 8‐bp restriction sites, i.e., AscI/PacI/FseI/NotI (marked in blue). (F) DNA fragments are inserted into Type I and Type II carrier vectors A167 and A169 by TAR cloning in yeast S. cerevisiae (Kouprina et al., 2021). In this case, AscI/PacI and FseI/NotI sites are used to insert the hook sequences homologous to the 5′ and 3′ ends of DNA segments of interest. For TAR isolation of DNA segments of interest from total genomic DNA, A167 and A169 vectors contain CEN6 (a yeast centromere sequence) and HIS3 (a yeast selectable marker) for proper propagation and selection of the TAR‐cloned material in yeast. A BAC cassette allows direct transfer of the TAR‐cloned DNA material from yeast to bacterial cells for further BAC DNA isolation. Abbreviations: BAC, bacterial artificial chromosome; TAR, transformation‐associated recombination.