{"status":"ok","message-type":"work","message-version":"1.0.0","message":{"indexed":{"date-parts":[[2026,9,18]],"date-time":"2026-09-18T18:47:48Z","timestamp":1789757268019,"version":"4.0.1"},"reference-count":36,"publisher":"Oxford University Press (OUP)","issue":"14","license":[{"start":{"date-parts":[[2026,7,20]],"date-time":"2026-07-20T00:00:00Z","timestamp":1784505600000},"content-version":"vor","delay-in-days":3,"URL":"https:\/\/creativecommons.org\/licenses\/by-nc\/4.0\/"}],"funder":[{"DOI":"10.13039\/100000002","name":"National Institutes of Health","doi-asserted-by":"publisher","award":["GM142886"],"award-info":[{"award-number":["GM142886"]}],"id":[{"id":"10.13039\/100000002","id-type":"DOI","asserted-by":"publisher"}]},{"DOI":"10.13039\/100000002","name":"National Institutes of Health","doi-asserted-by":"publisher","award":["AI178151"],"award-info":[{"award-number":["AI178151"]}],"id":[{"id":"10.13039\/100000002","id-type":"DOI","asserted-by":"publisher"}]},{"DOI":"10.13039\/100000002","name":"National Institutes of Health","doi-asserted-by":"publisher","award":["AI161275"],"award-info":[{"award-number":["AI161275"]}],"id":[{"id":"10.13039\/100000002","id-type":"DOI","asserted-by":"publisher"}]}],"content-domain":{"domain":[],"crossmark-restriction":false},"short-container-title":[],"published-print":{"date-parts":[[2026,7,17]]},"abstract":"<jats:title>Abstract<\/jats:title>\n                  <jats:p>Typical mammalian overexpression systems test protein sequence variants with little control over expression levels and steady-state protein abundances, hindering interpretations of how protein sequence and expression converge to yield phenotypic outcomes. We explored the translation initiation sequence, commonly referred to as the Kozak sequence, as a means to systematically modulate protein steady-state abundance and cellular function. We performed sort-seq on a randomized library of the 6 nt preceding the start codon, amounting to 4042 sequences, with a single sequence integrated at a shared genomic integration site in each HEK 293T cell. Calibrating the scores revealed a \u223c100-fold range of protein steady-state abundances possible through manipulation of the Kozak sequence. We identified human germline variants with predicted expression-reducing Kozak substitutions in disease-associated genes. Modulating the cell surface abundance of the host cell receptor ACE2 controlled the rate at which those cells became infected by SARS-like coronavirus spike pseudotyped particles. We demonstrated the potential of the approach by simultaneously testing Kozak libraries with a small panel of coding variants for ACE2 and STIM1. This approach lays the methodological groundwork for linking the causal relationships between protein sequence, abundance, and functional outcome.<\/jats:p>","DOI":"10.1093\/nar\/gkag728","type":"journal-article","created":{"date-parts":[[2026,7,11]],"date-time":"2026-07-11T11:52:15Z","timestamp":1783770735000},"source":"Crossref","is-referenced-by-count":1,"title":["Kozak sequence libraries for systematically characterizing transgenes across expression levels"],"prefix":"10.1093","volume":"54","author":[{"ORCID":"https:\/\/orcid.org\/0000-0001-5912-7039","authenticated-orcid":false,"given":"Nidhi","family":"Shukla","sequence":"first","affiliation":[{"name":"Department of Pathology, Case Western Reserve University School of Medicine , Cleveland, OH 44106 ,","place":["United States"]}],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0001-8969-0631","authenticated-orcid":false,"given":"Nisha D","family":"Kamath","sequence":"additional","affiliation":[{"name":"Department of Pathology, Case Western Reserve University School of Medicine , Cleveland, OH 44106 ,","place":["United States"]}],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0003-2683-6108","authenticated-orcid":false,"given":"John C","family":"Snell","sequence":"additional","affiliation":[{"name":"Department of Pathology, Case Western Reserve University School of Medicine , Cleveland, OH 44106 ,","place":["United States"]}],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0003-1695-5349","authenticated-orcid":false,"given":"Anna M","family":"Bruchez","sequence":"additional","affiliation":[{"name":"Department of Pathology, Case Western Reserve University School of Medicine , Cleveland, OH 44106 ,","place":["United States"]}],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0001-9149-551X","authenticated-orcid":false,"given":"Kenneth A","family":"Matreyek","sequence":"additional","affiliation":[{"name":"Department of Pathology, Case Western Reserve University School of Medicine , Cleveland, OH 44106 ,","place":["United States"]}],"role":[{"vocabulary":"crossref","role":"author"}]}],"member":"286","published-online":{"date-parts":[[2026,7,20]]},"reference":[{"key":"2026072014430189200_B1","doi-asserted-by":"crossref","first-page":"5","DOI":"10.1186\/1472-6750-13-5","article-title":"Graded or threshold response of the tet-controlled gene expression: all depends on the concentration of the transactivator","volume":"13","author":"Heinz","year":"2013","journal-title":"BMC Biotechnol"},{"key":"2026072014430189200_B2","doi-asserted-by":"publisher","first-page":"431","DOI":"10.1038\/s41589-018-0021-8","article-title":"The dTAG system for immediate and target-specific protein degradation","volume":"14","author":"Nabet","year":"2018","journal-title":"Nat Chem Biol"},{"key":"2026072014430189200_B3","doi-asserted-by":"publisher","DOI":"10.1038\/s41587-025-02854-y","article-title":"Programmable promoter editing for precise control of transgene expression","author":"Kabaria","year":"2025","journal-title":"Nat Biotechnol"},{"key":"2026072014430189200_B4","doi-asserted-by":"publisher","first-page":"194","DOI":"10.1101\/2024.03.12.583048","article-title":"miRNA modules for precise, tunable control of gene expression","volume":"86","author":"Du","year":"2026","journal-title":"Mol Cell"},{"key":"2026072014430189200_B5","doi-asserted-by":"publisher","first-page":"5233","DOI":"10.1093\/nar\/9.20.5233","article-title":"Possible role of flanking nucleotides in recognition of the AUG initiator codon by eukaryotic ribosomes","volume":"9","author":"Kozak","year":"1981","journal-title":"Nucl Acids Res"},{"key":"2026072014430189200_B6","doi-asserted-by":"publisher","first-page":"229","DOI":"10.1083\/jcb.108.2.229","article-title":"The scanning model for translation: an update","volume":"108","author":"Kozak","year":"1989","journal-title":"J Cell Biol"},{"key":"2026072014430189200_B7","doi-asserted-by":"publisher","first-page":"947","DOI":"10.1016\/0022-2836(87)90418-9","article-title":"At least six nucleotides preceding the AUG initiator codon enhance translation in mammalian cells","volume":"196","author":"Kozak","year":"1987","journal-title":"J Mol Biol"},{"key":"2026072014430189200_B8","doi-asserted-by":"crossref","first-page":"748","DOI":"10.15252\/msb.20145136","article-title":"Quantitative analysis of mammalian translation initiation sites by FACS-seq","volume":"10","author":"Noderer","year":"2014","journal-title":"Mol Syst Biol"},{"key":"2026072014430189200_B9","first-page":"e127","article-title":"Modular 5\u2032-UTR hexamers for context-independent tuning of protein expression in eukaryotes","volume":"46","author":"Petersen","year":"2018","journal-title":"Nucleic Acids Res"},{"key":"2026072014430189200_B10","doi-asserted-by":"publisher","first-page":"4018","DOI":"10.1038\/s41598-018-22330-9","article-title":"Changes in global translation elongation or initiation rates shape the proteome via the Kozak sequence","volume":"8","author":"Acevedo","year":"2018","journal-title":"Sci Rep"},{"key":"2026072014430189200_B11","doi-asserted-by":"publisher","first-page":"10756","DOI":"10.1093\/nar\/gkac799","article-title":"Translational enhancement by base editing of the Kozak sequence rescues haploinsufficiency","volume":"50","author":"Ambrosini","year":"2022","journal-title":"Nucleic Acids Res"},{"key":"2026072014430189200_B12","doi-asserted-by":"publisher","first-page":"e1009715","DOI":"10.1371\/journal.ppat.1009715","article-title":"Mutants of human ACE2 differentially promote SARS-CoV and SARS-CoV-2 spike mediated infection","volume":"17","author":"Shukla","year":"2021","journal-title":"PLoS Pathog"},{"key":"2026072014430189200_B13","doi-asserted-by":"publisher","first-page":"3352","DOI":"10.1021\/acssynbio.3c00355","article-title":"Mammalian genomic manipulation with orthogonal Bxb1 DNA recombinase sites for the functional characterization of protein variants","volume":"12","author":"Roelle","year":"2023","journal-title":"ACS Synth Biol"},{"key":"2026072014430189200_B14","doi-asserted-by":"publisher","first-page":"e1012044","DOI":"10.1371\/journal.ppat.1012044","article-title":"Pseudotyped virus infection of multiplexed ACE2 libraries reveals SARS-CoV-2 variant shifts in receptor usage","volume":"20","author":"Shukla","year":"2024","journal-title":"PLoS Pathog"},{"key":"2026072014430189200_B15","doi-asserted-by":"publisher","first-page":"614","DOI":"10.1093\/bioinformatics\/btt593","article-title":"PEAR: a fast and accurate Illumina Paired-End reAd mergeR","volume":"30","author":"Zhang","year":"2014","journal-title":"Bioinformatics"},{"key":"2026072014430189200_B16","doi-asserted-by":"publisher","first-page":"150","DOI":"10.1186\/s13059-017-1272-5","article-title":"A statistical framework for analyzing deep mutational scanning data","volume":"18","author":"Rubin","year":"2017","journal-title":"Genome Biol"},{"key":"2026072014430189200_B17","doi-asserted-by":"publisher","first-page":"874","DOI":"10.1038\/s41588-018-0122-z","article-title":"Multiplex assessment of protein variant abundance by massively parallel sequencing","volume":"50","author":"Matreyek","year":"2018","journal-title":"Nat Genet"},{"key":"2026072014430189200_B18","doi-asserted-by":"publisher","first-page":"165","DOI":"10.1186\/s13073-021-00984-x","article-title":"Integrating thousands of PTEN variant activity and abundance measurements reveals variant subgroups and new dominant negatives in cancers","volume":"13","author":"Matreyek","year":"2021","journal-title":"Genome Med"},{"key":"2026072014430189200_B19","doi-asserted-by":"publisher","first-page":"e102","DOI":"10.1093\/nar\/gkx183","article-title":"A platform for functional assessment of large variant libraries in mammalian cells","volume":"45","author":"Matreyek","year":"2017","journal-title":"Nucleic Acids Res"},{"key":"2026072014430189200_B20","first-page":"e1","article-title":"An improved platform for functional assessment of large protein libraries in mammalian cells","volume":"48","author":"Matreyek","year":"2020","journal-title":"Nucleic Acids Res"},{"key":"2026072014430189200_B21","doi-asserted-by":"publisher","first-page":"283","DOI":"10.1016\/0092-8674(86)90762-2","article-title":"Point mutations define a sequence flanking the AUG initiator codon that modulates translation by eukaryotic ribosomes","volume":"44","author":"Kozak","year":"1986","journal-title":"Cell"},{"key":"2026072014430189200_B22","doi-asserted-by":"publisher","first-page":"803","DOI":"10.1038\/s41587-019-0164-5","article-title":"Human 5\u2032 UTR design and variant effect prediction from a massively parallel translation assay","volume":"37","author":"Sample","year":"2019","journal-title":"Nat Biotechnol"},{"key":"2026072014430189200_B23","doi-asserted-by":"publisher","first-page":"D1062","DOI":"10.1093\/nar\/gkx1153","article-title":"ClinVar: improving access to variant interpretations and supporting evidence","volume":"46","author":"Landrum","year":"2018","journal-title":"Nucleic Acids Res"},{"key":"2026072014430189200_B24","doi-asserted-by":"publisher","first-page":"100866","DOI":"10.1016\/j.gim.2023.100866","article-title":"ACMG SF v3.2 list for reporting of secondary findings in clinical exome and genome sequencing: a policy statement of the American College of Medical Genetics and Genomics (ACMG)","volume":"25","author":"Miller","year":"2023","journal-title":"Genet Med"},{"key":"2026072014430189200_B25","first-page":"100298","article-title":"In vitro data suggest a role for PMS2 Kozak sequence mutations in Lynch syndrome risk","volume":"5","author":"Matoy","year":"2024","journal-title":"HGG Adv"},{"key":"2026072014430189200_B26","doi-asserted-by":"publisher","first-page":"315","DOI":"10.1016\/j.ajhg.2017.07.014","article-title":"Variant interpretation: functional assays to the rescue","volume":"101","author":"Starita","year":"2017","journal-title":"Am Hum Genet"},{"key":"2026072014430189200_B27","doi-asserted-by":"crossref","first-page":"2061","DOI":"10.3390\/v14092061","article-title":"Receptor-binding domain (RBD) antibodies contribute more to SARS-CoV-2 neutralization when target cells express high levels of ACE2","volume":"14","author":"Farrell","year":"2022","journal-title":"Viruses"},{"key":"2026072014430189200_B28","doi-asserted-by":"publisher","first-page":"e3001738","DOI":"10.1371\/journal.pbio.3001738","article-title":"Expanded ACE2 dependencies of diverse SARS-like coronavirus receptor binding domains","volume":"20","author":"Roelle","year":"2022","journal-title":"PLoS Biol"},{"key":"2026072014430189200_B29","doi-asserted-by":"publisher","first-page":"iyaf146","DOI":"10.1093\/genetics\/iyaf146","article-title":"Comprehensive mutational characterization of the calcium-sensing STIM1 EF-hand reveals residues essential for structure and function","volume":"231","author":"Kamath","year":"2025","journal-title":"Genetics"},{"key":"2026072014430189200_B30","doi-asserted-by":"crossref","first-page":"224","DOI":"10.1046\/j.1365-2141.2003.04754.x","article-title":"Beta+45 G \u2192 C: a novel silent beta-thalassaemia mutation, the first in the Kozak sequence","volume":"124","author":"De\u00a0Angioletti","year":"2004","journal-title":"Br J Haematol"},{"key":"2026072014430189200_B31","doi-asserted-by":"publisher","first-page":"2732","DOI":"10.1002\/ajmg.a.36703","article-title":"A mutation in the Kozak sequence of GATA4 hampers translation in a family with atrial septal defects","volume":"164","author":"Mohan","year":"2014","journal-title":"Am J Med Genet A"},{"key":"2026072014430189200_B32","doi-asserted-by":"publisher","first-page":"10075","DOI":"10.1093\/nar\/gkad687","article-title":"Precise genome editing of the Kozak sequence enables bidirectional and quantitative modulation of protein translation to anticipated levels without affecting transcription","volume":"51","author":"Xie","year":"2023","journal-title":"Nucleic Acids Res"},{"key":"2026072014430189200_B33","doi-asserted-by":"publisher","first-page":"689","DOI":"10.1038\/msb.2013.47","article-title":"Protein synthesis rate is the predominant regulator of protein expression during differentiation","volume":"9","author":"Kristensen","year":"2013","journal-title":"Mol Syst Biol"},{"key":"2026072014430189200_B34","doi-asserted-by":"publisher","first-page":"e0149221","DOI":"10.1128\/JVI.01492-21","article-title":"Susceptibilities of human ACE2 genetic variants in coronavirus infection","volume":"96","author":"Ren","year":"2022","journal-title":"J Virol"},{"key":"2026072014430189200_B35","doi-asserted-by":"publisher","first-page":"2715","DOI":"10.1093\/molbev\/msab060","article-title":"Identifying primate ACE2 variants that confer resistance to SARS-CoV-2","volume":"38","author":"Bhattacharjee","year":"2021","journal-title":"Mol Biol Evol"},{"key":"2026072014430189200_B36","doi-asserted-by":"publisher","first-page":"11016","DOI":"10.1128\/JVI.01242-09","article-title":"A quantitative affinity-profiling system that reveals distinct CD4\/CCR5 usage patterns among human immunodeficiency virus type 1 and simian immunodeficiency virus strains","volume":"83","author":"Johnston","year":"2009","journal-title":"J Virol"}],"container-title":["Nucleic Acids Research"],"original-title":[],"language":"en","link":[{"URL":"https:\/\/academic.oup.com\/nar\/article-pdf\/54\/14\/gkag728\/69570820\/gkag728.pdf","content-type":"application\/pdf","content-version":"vor","intended-application":"syndication"},{"URL":"https:\/\/academic.oup.com\/nar\/article-pdf\/54\/14\/gkag728\/69570820\/gkag728.pdf","content-type":"unspecified","content-version":"vor","intended-application":"similarity-checking"}],"deposited":{"date-parts":[[2026,7,20]],"date-time":"2026-07-20T18:43:11Z","timestamp":1784572991000},"score":1,"resource":{"primary":{"URL":"https:\/\/academic.oup.com\/nar\/article\/doi\/10.1093\/nar\/gkag728\/8738336"}},"subtitle":[],"short-title":[],"issued":{"date-parts":[[2026,7,17]]},"references-count":36,"journal-issue":{"issue":"14","published-print":{"date-parts":[[2026,7,17]]}},"URL":"https:\/\/doi.org\/10.1093\/nar\/gkag728","relation":{},"ISSN":["0305-1048","1362-4962"],"issn-type":[{"value":"0305-1048","type":"print"},{"value":"1362-4962","type":"electronic"}],"subject":[],"published-other":{"date-parts":[[2026,8,12]]},"published":{"date-parts":[[2026,7,17]]},"article-number":"gkag728"}}