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Here, leveraging a high-throughput phenotypic reporter, we systematically dissect how T-DNA vector architecture, plasmid biology, and regulatory element choice shape transformation outcomes in\n                  <jats:italic>Arabidopsis thaliana<\/jats:italic>\n                  . We discover a pronounced trade-off between transformation efficiency and T-DNA copy number, uncovering the virulence enhancing overdrive sequence as a major determinant of this relationship. Guided by these insights, we engineered a new T-DNA vector that balances efficient transformation with predominantly single-copy integration. Additionally, we replaced viral elements, such as the widely used CaMV 35S promoter, with Arabidopsis-derived regulatory elements to minimise undesired enhancer effects, and developed a streamlined workflow for efficient T-DNA insertion mapping in the genome. Together, these advances form the T1 vector series, an Arabidopsis-optimised T-DNA vector system that enables clean, single-copy, and readily mappable transgene integration with predictable expression in the first generation after transformation.\n                <\/jats:p>","DOI":"10.64898\/2026.06.08.730999","type":"posted-content","created":{"date-parts":[[2026,6,10]],"date-time":"2026-06-10T14:31:22Z","timestamp":1781101882000},"source":"Crossref","is-referenced-by-count":0,"title":["Rational design of T-DNA vectors enables predictable, single-copy integration in\n                  <i>Arabidopsis thaliana<\/i>"],"prefix":"10.64898","author":[{"given":"William M.","family":"Shaw","sequence":"first","affiliation":[{"name":"Biological Design Center, Boston University, Boston, MA, USA"},{"name":"Department of Biomedical Engineering, Boston University, Boston, MA, USA"},{"name":"Whitehead Institute for Biomedical Research, Cambridge, MA, USA"},{"name":"Earlham Institute, Norwich Research Park, Norwich, Norfolk, NR4 7UZ, UK"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Anjali","family":"Gajendiran","sequence":"additional","affiliation":[{"name":"Biological Design Center, Boston University, Boston, MA, USA"},{"name":"Department of Biomedical Engineering, Boston University, Boston, MA, USA"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Elizabeth I.","family":"Tchantouridze","sequence":"additional","affiliation":[{"name":"Biological Design Center, Boston University, Boston, MA, USA"},{"name":"Bioinformatics Program, Boston University, Boston, MA, USA"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Lindsey L.","family":"Bechen","sequence":"additional","affiliation":[{"name":"Whitehead Institute for Biomedical Research, Cambridge, MA, USA"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Sean G.","family":"Clarke","sequence":"additional","affiliation":[{"name":"Whitehead Institute for Biomedical Research, Cambridge, MA, USA"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Sarah","family":"Guiziou","sequence":"additional","affiliation":[{"name":"Earlham Institute, Norwich Research Park, Norwich, Norfolk, NR4 7UZ, UK"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Mary","family":"Gehring","sequence":"additional","affiliation":[{"name":"Whitehead Institute for Biomedical Research, Cambridge, MA, USA"},{"name":"Department of Biology, Massachusetts Institute of Technology, Cambridge, MA, USA"},{"name":"Department of Biological Engineering, Massachusetts Institute of Technology, Cambridge, MA, USA"},{"name":"Howard Hughes Medical Institute, Whitehead Institute for Biomedical Research, Cambridge, MA, USA"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0002-8214-0546","authenticated-orcid":false,"given":"Ahmad S.","family":"Khalil","sequence":"additional","affiliation":[{"name":"Biological Design Center, Boston University, Boston, MA, USA"},{"name":"Department of Biomedical Engineering, Boston University, Boston, MA, USA"},{"name":"John A. Paulson School of Engineering and Applied Sciences, Harvard University, Boston, MA, USA"},{"name":"Department of Molecular and Cellular Biology, Harvard University, Cambridge, MA, USA"},{"name":"Broad Institute of MIT and Harvard, Cambridge, MA, USA"}],"role":[{"vocabulary":"crossref","role":"author"}]}],"member":"54368","reference":[{"key":"2026070804550505000_2026.06.08.730999v2.1","doi-asserted-by":"publisher","DOI":"10.1128\/MMBR.67.1.16-37.2003"},{"key":"2026070804550505000_2026.06.08.730999v2.2","doi-asserted-by":"publisher","DOI":"10.1016\/j.copbio.2006.01.009"},{"key":"2026070804550505000_2026.06.08.730999v2.3","doi-asserted-by":"publisher","DOI":"10.1199\/tab.0186"},{"key":"2026070804550505000_2026.06.08.730999v2.4","doi-asserted-by":"publisher","DOI":"10.1016\/0092-8674(89)90892-1"},{"key":"2026070804550505000_2026.06.08.730999v2.5","doi-asserted-by":"publisher","DOI":"10.1104\/pp.107.113001"},{"key":"2026070804550505000_2026.06.08.730999v2.6","doi-asserted-by":"publisher","DOI":"10.1038\/303179a0"},{"key":"2026070804550505000_2026.06.08.730999v2.7","doi-asserted-by":"crossref","first-page":"342","DOI":"10.1111\/tpj.16496","article-title":"Genomic consequences associated with Agrobacterium-mediated transformation of plants","volume":"117","year":"2024","journal-title":"Plant J"},{"key":"2026070804550505000_2026.06.08.730999v2.8","doi-asserted-by":"publisher","DOI":"10.1105\/tpc.104.024547"},{"key":"2026070804550505000_2026.06.08.730999v2.9","doi-asserted-by":"publisher","DOI":"10.1046\/j.1365-313X.1999.t01-1-00602.x"},{"key":"2026070804550505000_2026.06.08.730999v2.10","doi-asserted-by":"crossref","first-page":"478","DOI":"10.1007\/BF00331618","article-title":"T-DNA structure and gene expression in petunia plants transformed by Agrobacterium tumefaciens C58 derivatives","volume":"207","year":"1987","journal-title":"Molecular and General Genetics MGG"},{"key":"2026070804550505000_2026.06.08.730999v2.11","doi-asserted-by":"crossref","first-page":"79","DOI":"10.1007\/s11032-005-4929-9","article-title":"Approaches to Minimize Variation of Transgene Expression in Plants","volume":"16","year":"2005","journal-title":"Molecular Breeding"},{"key":"2026070804550505000_2026.06.08.730999v2.12","doi-asserted-by":"publisher","DOI":"10.1111\/j.1365-313X.2004.02312.x"},{"key":"2026070804550505000_2026.06.08.730999v2.13","doi-asserted-by":"publisher","DOI":"10.1111\/nph.19650"},{"key":"2026070804550505000_2026.06.08.730999v2.14","doi-asserted-by":"publisher","DOI":"10.1038\/318624a0"},{"key":"2026070804550505000_2026.06.08.730999v2.15","doi-asserted-by":"publisher","DOI":"10.1094\/MPMI-11-12-0267-R"},{"key":"2026070804550505000_2026.06.08.730999v2.16","doi-asserted-by":"publisher","DOI":"10.1046\/j.1365-313x.1998.00343.x"},{"key":"2026070804550505000_2026.06.08.730999v2.17","doi-asserted-by":"publisher","DOI":"10.1023\/A:1006440221718"},{"key":"2026070804550505000_2026.06.08.730999v2.18","doi-asserted-by":"publisher","DOI":"10.1073\/pnas.162304099"},{"key":"2026070804550505000_2026.06.08.730999v2.19","doi-asserted-by":"crossref","first-page":"1708","DOI":"10.1038\/s41587-024-02462-2","article-title":"Binary vector copy number engineering improves Agrobacterium-mediated transformation","volume":"43","year":"2025","journal-title":"Nat Biotechnol"},{"key":"2026070804550505000_2026.06.08.730999v2.20","doi-asserted-by":"crossref","first-page":"1640","DOI":"10.1021\/acssynbio.6c00064","article-title":"Quantitative Dissection of Agrobacterium Virulence to Generate a Synthetic Ti Plasmid","volume":"15","year":"2026","journal-title":"ACS Synth Biol"},{"key":"2026070804550505000_2026.06.08.730999v2.21","doi-asserted-by":"publisher","DOI":"10.1038\/342837a0"},{"key":"2026070804550505000_2026.06.08.730999v2.22","doi-asserted-by":"publisher","DOI":"10.1186\/1472-6750-2-20"},{"key":"2026070804550505000_2026.06.08.730999v2.23","doi-asserted-by":"crossref","first-page":"152","DOI":"10.1038\/s41438-020-00390-1","article-title":"A reporter for noninvasively monitoring gene expression and plant transformation","volume":"7","year":"2020","journal-title":"Hortic Res"},{"key":"2026070804550505000_2026.06.08.730999v2.24","doi-asserted-by":"crossref","first-page":"1729","DOI":"10.1093\/plphys\/kiae421","article-title":"Seeing the invisible: Tools to teach and study plant transcriptional responses","volume":"196","year":"2024","journal-title":"Plant Physiology"},{"key":"2026070804550505000_2026.06.08.730999v2.25","doi-asserted-by":"crossref","unstructured":"Rowan, B.A. et al. 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