{"status":"ok","message-type":"work","message-version":"1.0.0","message":{"indexed":{"date-parts":[[2026,4,21]],"date-time":"2026-04-21T10:55:25Z","timestamp":1776768925401,"version":"3.51.2"},"reference-count":20,"publisher":"Wiley","issue":"5","license":[{"start":{"date-parts":[[2014,3,20]],"date-time":"2014-03-20T00:00:00Z","timestamp":1395273600000},"content-version":"vor","delay-in-days":0,"URL":"http:\/\/onlinelibrary.wiley.com\/termsAndConditions#vor"}],"content-domain":{"domain":[],"crossmark-restriction":false},"short-container-title":["Yeast"],"published-print":{"date-parts":[[2014,5]]},"abstract":"<jats:title>Abstract<\/jats:title><jats:p>Many yeast experiments require strains modified by recombinant DNA methods. Some experiments require precise insertion of a DNA segment into the genome without a selectable marker remaining. For these applications, we developed a new PCR\u2010based method for marker\u2010free DNA transplant. The current PCR\u2010based method requires the labour\u2010intensive construction of a PCR template plasmid with repeats of the DNA segment flanking <jats:italic>URA3<\/jats:italic>. The design of a new vector, IpO, reduces the work in cloning a single copy of the DNA segment between overlapping <jats:italic>URA3<\/jats:italic> fragments present in the vector. Two PCRs are performed that capture the DNA segment and one or the other <jats:italic>URA3<\/jats:italic> fragment. When the PCR products are co\u2010transformed into yeast, recombination between the overlapping <jats:italic>URA3<\/jats:italic> fragments restores <jats:italic>URA3<\/jats:italic> and transposes the cloned DNA segment inside out, creating a repeat\u2010<jats:italic>URA3<\/jats:italic>\u2010repeat cassette. Sequences designed into the PCR primers target integration of the cassette into the genome. Subsequent selection with 5\u2010fluoro\u2010orotic acid yields strains that have 'popped out' <jats:italic>URA3<\/jats:italic> via recombination between the DNA repeats, with the result being the precise insertion of the DNA segment minus the selectable marker. An additional advantage of the IpO method is that it eliminates PCR artifacts that can plague the current method's repeat\u2010containing templates. Copyright \u00a9 2014 John Wiley &amp; Sons, Ltd.<\/jats:p>","DOI":"10.1002\/yea.3006","type":"journal-article","created":{"date-parts":[[2014,3,6]],"date-time":"2014-03-06T14:42:40Z","timestamp":1394116960000},"page":"185-193","source":"Crossref","is-referenced-by-count":2,"title":["IpO: plasmids and methods for simplified, PCR\u2010based DNA transplant in yeast"],"prefix":"10.1002","volume":"31","author":[{"given":"Joe","family":"Horecka","sequence":"first","affiliation":[{"name":"Department of Biochemistry Stanford University  CA USA"}],"role":[{"role":"author","vocabulary":"crossref"}]},{"given":"Angela M.","family":"Chu","sequence":"additional","affiliation":[{"name":"Department of Biochemistry Stanford University  CA USA"}],"role":[{"role":"author","vocabulary":"crossref"}]},{"given":"Ronald W.","family":"Davis","sequence":"additional","affiliation":[{"name":"Department of Biochemistry Stanford University  CA USA"}],"role":[{"role":"author","vocabulary":"crossref"}]}],"member":"311","published-online":{"date-parts":[[2014,3,20]]},"reference":[{"key":"e_1_2_6_2_1","volume-title":"Methods in Yeast Genetics: A Cold Spring Harbor Laboratory Course Manual","author":"Amberg DC","year":"2005"},{"key":"e_1_2_6_3_1","volume-title":"Current Protocols in Molecular Biology","author":"Ausubel FM","year":"1995"},{"key":"e_1_2_6_4_1","doi-asserted-by":"publisher","DOI":"10.1007\/BF00330984"},{"key":"e_1_2_6_5_1","doi-asserted-by":"publisher","DOI":"10.1007\/978-1-59745-321-9_14"},{"key":"e_1_2_6_6_1","doi-asserted-by":"crossref","first-page":"1112","DOI":"10.2144\/00286st01","article-title":"PCR\u2010 and ligation\u2010mediated synthesis of split\u2010marker cassettes with long flanking homology regions for gene disruption in Candida albicans","volume":"28","author":"Hoogt R","year":"2000","journal-title":"Biotechniques"},{"key":"e_1_2_6_7_1","doi-asserted-by":"publisher","DOI":"10.1002\/(SICI)1097-0061(199611)12:14<1439::AID-YEA37>3.0.CO;2-O"},{"key":"e_1_2_6_8_1","doi-asserted-by":"publisher","DOI":"10.1038\/nprot.2007.13"},{"key":"e_1_2_6_9_1","doi-asserted-by":"publisher","DOI":"10.1002\/yea.2992"},{"key":"e_1_2_6_10_1","doi-asserted-by":"publisher","DOI":"10.1371\/journal.pone.0023794"},{"key":"e_1_2_6_11_1","doi-asserted-by":"publisher","DOI":"10.1016\/0092-8674(87)90491-0"},{"key":"e_1_2_6_12_1","doi-asserted-by":"publisher","DOI":"10.1073\/pnas.1016459108"},{"key":"e_1_2_6_13_1","doi-asserted-by":"publisher","DOI":"10.1002\/(SICI)1097-0061(199807)14:10<953::AID-YEA293>3.0.CO;2-U"},{"key":"e_1_2_6_14_1","doi-asserted-by":"publisher","DOI":"10.1007\/BF00291998"},{"key":"e_1_2_6_15_1","doi-asserted-by":"publisher","DOI":"10.1016\/S0076-6879(02)50968-X"},{"key":"e_1_2_6_16_1","doi-asserted-by":"publisher","DOI":"10.1073\/pnas.76.10.4951"},{"key":"e_1_2_6_17_1","doi-asserted-by":"publisher","DOI":"10.1002\/yea.320111306"},{"key":"e_1_2_6_18_1","doi-asserted-by":"publisher","DOI":"10.1093\/genetics\/122.1.19"},{"key":"e_1_2_6_19_1","doi-asserted-by":"publisher","DOI":"10.1016\/0076-6879(91)94008-Z"},{"key":"e_1_2_6_20_1","doi-asserted-by":"publisher","DOI":"10.1002\/yea.320101310"},{"key":"e_1_2_6_21_1","doi-asserted-by":"publisher","DOI":"10.1074\/jbc.272.13.8402"}],"container-title":["Yeast"],"original-title":[],"language":"en","link":[{"URL":"https:\/\/api.wiley.com\/onlinelibrary\/tdm\/v1\/articles\/10.1002%2Fyea.3006","content-type":"unspecified","content-version":"vor","intended-application":"text-mining"},{"URL":"https:\/\/onlinelibrary.wiley.com\/doi\/pdf\/10.1002\/yea.3006","content-type":"unspecified","content-version":"vor","intended-application":"similarity-checking"}],"deposited":{"date-parts":[[2023,10,5]],"date-time":"2023-10-05T05:02:32Z","timestamp":1696482152000},"score":1,"resource":{"primary":{"URL":"https:\/\/onlinelibrary.wiley.com\/doi\/10.1002\/yea.3006"}},"subtitle":[],"short-title":[],"issued":{"date-parts":[[2014,3,20]]},"references-count":20,"journal-issue":{"issue":"5","published-print":{"date-parts":[[2014,5]]}},"alternative-id":["10.1002\/yea.3006"],"URL":"https:\/\/doi.org\/10.1002\/yea.3006","archive":["Portico"],"relation":{},"ISSN":["0749-503X","1097-0061"],"issn-type":[{"value":"0749-503X","type":"print"},{"value":"1097-0061","type":"electronic"}],"subject":[],"published":{"date-parts":[[2014,3,20]]}}}