LOCUS       sequence-401344-        7816 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  yeast codon optimized mRuby3 alpha-tubulin plasmid for integration 
            into the endogenous TUB1 ORF.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 7816)
  AUTHORS   Seitz BC, Mucelli X, Majano M, Wallis Z, Dodge AC, Carmona C, Durant
            M, Maynard S, Huang LS
  TITLE     Meiosis II spindle disassembly requires two distinct pathways.
  JOURNAL   Mol Biol Cell. 2023 Sep 1;34(10):ar98. doi: 10.1091/mbc.E23-03-0096.
            Epub 2023 Jul 12.
  PUBMED    37436806
REFERENCE   2  (bases 1 to 7816)
  AUTHORS   .
  TITLE     Direct Submission
  JOURNAL   Exported Dec 15, 2025 from SnapGene Server 8.0.1
            https://www.snapgene.com
COMMENT     SGRef: number: 1; type: "Journal Article"; journalName: "Mol Biol 
            Cell."; date: "2023-09-1"; volume: "34(10):ar98. doi"; pages: " 
            10.1091/mbc.E23-03-0096. Epub 2023 Jul 12"
COMMENT     Sequence Label: pHIS3p:yomRuby3-Tub1+3'UTR::LEU2
FEATURES             Location/Qualifiers
     source          1..7816
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     72..94
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     primer_bind     complement(132..150)
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     promoter        218..322
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(323..391,392..1183)
                     /codon_start=1
                     /gene="bla"
                     /product="beta-lactamase"
                     /label=AmpR
                     /note="confers resistance to ampicillin, carbenicillin, and
                     related antibiotics"
                     /translation="MSIQHFRVALIPFFAAFCLPVFAHPETLVKVKDAEDQLGARVGYI
                     ELDLNSGKILESFRPEERFPMMSTFKVLLCGAVLSRIDAGQEQLGRRIHYSQNDLVEYS
                     PVTEKHLTDGMTVRELCSAAITMSDNTAANLLLTTIGGPKELTAFLHNMGDHVTRLDRW
                     EPELNEAIPNDERDTTMPVAMATTLRKLLTGELLTLASRQQLIDWMEADKVAGPLLRSA
                     LPAGWFIADKSGAGERGSRGIIAALGPDGKPSRIVVIYTTGSQATMDERNRQIAEIGAS
                     LIKHW"
     primer_bind     complement(541..560)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      1354..1942
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     1843..1862
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     2096..2113
                     /label=L4440
                     /note="L4440 vector, forward primer"
     primer_bind     2200..2219
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        2200..2218
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     CDS             complement(2776..3870)
                     /codon_start=1
                     /gene="S. cerevisiae LEU2"
                     /product="3-isopropylmalate dehydrogenase, required for 
                     leucine biosynthesis"
                     /label=LEU2
                     /note="yeast auxotrophic marker"
                     /translation="MSAPKKIVVLPGDHVGQEITAEAIKVLKAISDVRSNVKFDFENHL
                     IGGAAIDATGVPLPDEALEASKKADAVLLGAVGGPKWGTGSVRPEQGLLKIRKELQLYA
                     NLRPCNFASDSLLDLSPIKPQFAKGTDFVVVRELVGGIYFGKRKEDDGDGVAWDSEQYT
                     VPEVQRITRMAAFMALQHEPPLPIWSLDKANVLASSRLWRKTVEETIKNEFPTLKVQHQ
                     LIDSAAMILVKNPTHLNGIIITSNMFGDIISDEASVIPGSLGLLPSASLASLPDKNTAF
                     GLYEPCHGSAPDLPKNKVNPIATILSAAMMLKLSLNLPEEGKAIEDAVKKVLDAGIRTG
                     DLGGSNSTTEVGDAVAEEVKKILA"
     promoter        complement(3871..4278)
                     /gene="S. cerevisiae LEU2"
                     /label=LEU2 promoter
     promoter        4641..4827
                     /gene="S. cerevisiae HIS3"
                     /label=HIS3 promoter
     RBS             7030..7038
                     /label=Shine-Dalgarno sequence
                     /note="full consensus sequence for ribosome-binding sites 
                     upstream of start codons in E. coli; complementary to a 
                     region in the 3' end of the 16S rRNA (Chen et al., 1994)"
     promoter        complement(7670..7688)
                     /label=SP6 promoter
                     /note="promoter for bacteriophage SP6 RNA polymerase"
     primer_bind     complement(7671..7688)
                     /label=SP6
                     /note="SP6 promoter, forward primer"
ORIGIN
        1 aacacccgct gacgcgccct gacgggcttg tctgctcccg gcatccgctt acagacaagc
       61 tgtgaccgtc tccgggagct gcatgtgtca gaggttttca ccgtcatcac cgaaacgcgc
      121 gagacgaaag ggcctcgtga tacgcctatt tttataggtt aatgtcatga taataatggt
      181 ttcttagacg tcaggtggca cttttcgggg aaatgtgcgc ggaaccccta tttgtttatt
      241 tttctaaata cattcaaata tgtatccgct catgagacaa taaccctgat aaatgcttca
      301 ataatattga aaaaggaaga gtatgagtat tcaacatttc cgtgtcgccc ttattccctt
      361 ttttgcggca ttttgccttc ctgtttttgc tcacccagaa acgctggtga aagtaaaaga
      421 tgctgaagat cagttgggtg cacgagtggg ttacatcgaa ctggatctca acagcggtaa
      481 gatccttgag agttttcgcc ccgaagaacg ttttccaatg atgagcactt ttaaagttct
      541 gctatgtggc gcggtattat cccgtattga cgccgggcaa gagcaactcg gtcgccgcat
      601 acactattct cagaatgact tggttgagta ctcaccagtc acagaaaagc atcttacgga
      661 tggcatgaca gtaagagaat tatgcagtgc tgccataacc atgagtgata acactgcggc
      721 caacttactt ctgacaacga tcggaggacc gaaggagcta accgcttttt tgcacaacat
      781 gggggatcat gtaactcgcc ttgatcgttg ggaaccggag ctgaatgaag ccataccaaa
      841 cgacgagcgt gacaccacga tgcctgtagc aatggcaaca acgttgcgca aactattaac
      901 tggcgaacta cttactctag cttcccggca acaattaata gactggatgg aggcggataa
      961 agttgcagga ccacttctgc gctcggccct tccggctggc tggtttattg ctgataaatc
     1021 tggagccggt gagcgtgggt ctcgcggtat cattgcagca ctggggccag atggtaagcc
     1081 ctcccgtatc gtagttatct acacgacggg gagtcaggca actatggatg aacgaaatag
     1141 acagatcgct gagataggtg cctcactgat taagcattgg taactgtcag accaagttta
     1201 ctcatatata ctttagattg atttaaaact tcatttttaa tttaaaagga tctaggtgaa
     1261 gatccttttt gataatctca tgaccaaaat cccttaacgt gagttttcgt tccactgagc
     1321 gtcagacccc gtagaaaaga tcaaaggatc ttcttgagat cctttttttc tgcgcgtaat
     1381 ctgctgcttg caaacaaaaa aaccaccgct accagcggtg gtttgtttgc cggatcaaga
     1441 gctaccaact ctttttccga aggtaactgg cttcagcaga gcgcagatac caaatactgt
     1501 tcttctagtg tagccgtagt taggccacca cttcaagaac tctgtagcac cgcctacata
     1561 cctcgctctg ctaatcctgt taccagtggc tgctgccagt ggcgataagt cgtgtcttac
     1621 cgggttggac tcaagacgat agttaccgga taaggcgcag cggtcgggct gaacgggggg
     1681 ttcgtgcaca cagcccagct tggagcgaac gacctacacc gaactgagat acctacagcg
     1741 tgagctatga gaaagcgcca cgcttcccga agggagaaag gcggacaggt atccggtaag
     1801 cggcagggtc ggaacaggag agcgcacgag ggagcttcca gggggaaacg cctggtatct
     1861 ttatagtcct gtcgggtttc gccacctctg acttgagcgt cgatttttgt gatgctcgtc
     1921 aggggggcgg agcctatgga aaaacgccag caacgcggcc tttttacggt tcctggcctt
     1981 ttgctggcct tttgctcaca tgttctttcc tgcgttatcc cctgattctg tggataaccg
     2041 tattaccgcc tttgagtgag ctgataccgc tcgccgcagc cgaacgaccg agcgcagcga
     2101 gtcagtgagc gaggaagcgg aagagcgccc aatacgcaaa ccgcctctcc ccgcgcgttg
     2161 gccgattcat taatgcaggt taacctggct tatcgaaatt aatacgactc actataggga
     2221 gaccggcaga tccgcggccg cataggccac tagttaacta tgcggcatca gagcagattg
     2281 tactgagagt gcaccatatc gactacgtcg ttaaggccgt ttctgacaga gtaaaattct
     2341 tgagggaact ttcaccatta tgggaaatgg ttcaagaagg tattgactta aactccatca
     2401 aatggtcagg tcattgagtg ttttttattt gttgtatttt ttttttttta gagaaaatcc
     2461 tccaatatat aaattaggaa tcatagtttc atgattttct gttacaccta actttttgtg
     2521 tggtgccctc ctccttgtca atattaatgt taaagtgcaa ttctttttcc ttatcacgtt
     2581 gagccattag tatcaatttg cttacctgta ttcctttaca tcctcctttt tctccttctt
     2641 gataaatgta tgtagattgc gtatatagtt tcgtctaccc tatgaacata ttccattttg
     2701 taatttcgtg tcgtttctat tatgaatttc atttataaag tttatgtaca aatatcataa
     2761 aaaaagagaa tctttttaag caaggatttt cttaacttct tcggcgacag catcaccgac
     2821 ttcggtggta ctgttggaac cacctaaatc accagttctg atacctgcat ccaaaacctt
     2881 tttaactgca tcttcaatgg ccttaccttc ttcaggcaag ttcaatgaca atttcaacat
     2941 cattgcagca gacaagatag tggcgatagg gttgacctta ttctttggca aatctggagc
     3001 agaaccgtgg catggttcgt acaaaccaaa tgcggtgttc ttgtctggca aagaggccaa
     3061 ggacgcagat ggcaacaaac ccaaggaacc tgggataacg gaggcttcat cggagatgat
     3121 atcaccaaac atgttgctgg tgattataat accatttagg tgggttgggt tcttaactag
     3181 gatcatggcg gcagaatcaa tcaattgatg ttgaaccttc aatgtaggga attcgttctt
     3241 gatggtttcc tccacagttt ttctccataa tcttgaagag gccaaaacat tagctttatc
     3301 caaggaccaa ataggcaatg gtggctcatg ttgtagggcc atgaaagcgg ccattcttgt
     3361 gattctttgc acttctggaa cggtgtattg ttcactatcc caagcgacac catcaccatc
     3421 gtcttccttt ctcttaccaa agtaaatacc tcccactaat tctctgacaa caacgaagtc
     3481 agtaccttta gcaaattgtg gcttgattgg agataagtct aaaagagagt cggatgcaaa
     3541 gttacatggt cttaagttgg cgtacaattg aagttcttta cggattttta gtaaaccttg
     3601 ttcaggtcta acactaccgg taccccattt aggaccaccc acagcaccta acaaaacggc
     3661 atcagccttc ttggaggctt ccagcgcctc atctggaagt ggaacacctg tagcatcgat
     3721 agcagcacca ccaattaaat gattttcgaa atcgaacttg acattggaac gaacatcaga
     3781 aatagcttta agaaccttaa tggcttcggc tgtgatttct tgaccaacgt ggtcacctgg
     3841 caaaacgacg atcttcttag gggcagacat tagaatggta tatccttgaa atatatatat
     3901 atatattgct gaaatgtaaa aggtaagaaa agttagaaag taagacgatt gctaaccacc
     3961 tattggaaaa aacaataggt ccttaaataa tattgtcaac ttcaagtatt gtgatgcaag
     4021 catttagtca tgaacgcttc tctattctat atgaaaagcc ggttccggcg ctctcacctt
     4081 tcctttttct cccaattttt cagttgaaaa aggtatatgc gtcaggcgac ctctgaaatt
     4141 aacaaaaaat ttccagtcat cgaatttgat tctgtgcgat agcgcccctg tgtgttctcg
     4201 ttatgttgag gaaaaaaata atggttgcta agagattcga actcttgcat cttacgatac
     4261 ctgagtattc ccacagttaa ctgcggtcaa gatatttctt gaatcaggcg ccttagaccg
     4321 ctcggccaaa caaccaatta cttgttgaga aatagagtat aattatccta taaatataac
     4381 gtttttgaac acacatgaac aaggaagtac aggacaattg attttgaaga gaatgtggat
     4441 tttgatgtaa ttgttgggat tccattttta ataaggcaat aatattaggt atgtagatat
     4501 actagaagtt ctcctcgacc ggtcgatatg cggtgtgaaa taccgcacag atgcgtaagg
     4561 agaaaatacc gcatcagggg cgcgccttaa ttaaccacta aagggaacaa aagctggtac
     4621 cctattactc ttggcctcct ctagtacact ctatattttt ttatgcctcg gtaatgattt
     4681 tcattttttt tttccaccta gcggatgact cttttttttt cttagcgatt ggcattatca
     4741 cataatgaat tatacattat ataaagtaat gtgatttctt cgaagaatat actaaaaaat
     4801 gagcaggcaa gataaacgaa ggcaaagcga gctcatggtg tccaaaggag aggagttaat
     4861 caaggaaaac atgagaatga aagttgtcat ggagggctcc gttaatggtc accaattcaa
     4921 gtgtacaggg gaaggtgaag gtcgtcctta cgaaggtgtg caaactatga gaattaaagt
     4981 aattgaaggc ggaccactac catttgcatt tgacattctg gcaacgtcat tcatgtacgg
     5041 atcacgaact ttcatcaagt accctgcgga tataccagac tttttcaagc aatcttttcc
     5101 agagggtttt acatgggaaa gggttacaag atacgaagat gggggtgtcg tcacagttac
     5161 ccaagatact tcattagaag atggcgaact tgtctataat gtgaaagtaa gaggggtgaa
     5221 ttttccttct aacggacctg tgatgcagaa aaagaccaaa ggttgggaac caaatactga
     5281 aatgatgtac ccagctgatg gaggtttgag aggctacaca gatattgcgc ttaaagttga
     5341 tggtggaggt catttgcatt gtaattttgt taccacttat cgttctaaaa agactgttgg
     5401 caatatcaaa atgccaggag tgcatgctgt agaccacaga ctagaaagaa ttgaagagag
     5461 cgataacgaa acctatgttg tacagagaga ggtggccgta gccaaatatt ctaacttagg
     5521 cggtggtatg gatgaattgt ataagaggcc tatgagagaa gttattagta ttaatggtat
     5581 gttcgatttg cccgtccagg ctagatcttt tttttgttag ttcattttgg cgtttcatct
     5641 ttcattacta acttcaacgc aaaatttttt tttttttgat ttctctttac agtcggtcaa
     5701 gctggttgtc agattggtaa tgcctgttgg gaattatatt cccttgagca cggtattaag
     5761 ccggatggac atctagaaga tggcctttca aagccgaagg gaggagaaga gggtttctcc
     5821 acgtttttcc atgaaaccgg ctacggtaag ttcgttccaa gggctattta cgtggattta
     5881 gagcccaacg ttattgacga agtccgtaac ggtccttaca aggacttgtt ccatccagaa
     5941 cagttgatca gtggtaagga ggacgcggct aataattatg caagaggcca ttacaccgtt
     6001 ggtagagaaa ttttgggcga tgttctggat aggattagaa aactggcaga ccaatgtgat
     6061 gggttacaag ggttcttgtt tacccattct cttggtggtg gtactggttc cggtctaggg
     6121 tccttactgt tggaagaact atccgctgaa tacggtaaga aatccaagct ggaatttgcc
     6181 gtataccctg ctccacaagt gtctacttct gtggttgagc cttacaacac agttttaact
     6241 acacatacta cattggaaca tgcagattgt actttcatgg tcgataatga ggctatttac
     6301 gacatgtgca aaagaaactt ggatatccca agaccaagct ttgcaaactt aaacaaccta
     6361 attgctcaag tggtatcatc tgttacagca tcattgagat tcgacggttc attaaatgta
     6421 gatttgaacg aatttcaaac caatttggtt ccatatccaa gaattcattt ccccttagtt
     6481 tcatattctc cagtcttatc caaatcaaag gcattccatg agtccaactc tgtgtcagaa
     6541 attacaaacg cttgttttga acctggtaac cagatggtca agtgtgatcc aagagatggt
     6601 aaatacatgg ctacttgtct gttatacagg ggtgatgtgg taacaagaga tgttcaaaga
     6661 gctgtcgagc aggtgaaaaa caagaagacc gtccaattgg ttgattggtg tccaactggt
     6721 ttcaagatcg gtatttgcta cgaacctcca actgccacac caaactcaca attggccact
     6781 gtggataggg ccgtctgtat gttgtcaaat accacatcca ttgctgaggc ttggaagaga
     6841 atcgatagaa aattcgattt aatgtatgcc aaacgtgctt tcgtccactg gtatgtcggt
     6901 gaaggtatgg aagaaggtga attcaccgaa gctagagaag atttggctgc tttagaaaga
     6961 gattacatcg aagtgggtgc cgactcatac gctgaggaag aggaatttta aggttcactc
     7021 tcgcccccaa cctccttatc ctttctatat ttcaagtata ttgtaaaaaa aaaaattccc
     7081 cctactgtta tatctctttg gtgatatctt atctataatc tacttattat atttctctaa
     7141 tatctatgta caaaaaataa aaaaaaaaac cattctcctt ctctaacctg catttgatga
     7201 aagcttagta agtctcatcg tagttgatgg gccaaagaca tttgtacttg tttcatcaga
     7261 ttcttacatc acccatttcc atgattattt cacggatttc attagtaatt gcatttttgt
     7321 atccctagtt ccttttctcc cgccttgccg tcgaattgaa aaaaaaaatg gaaatgagga
     7381 catcttccat tgatatccag gacaaagcaa gagtagaagt aactattagc aaggagaatt
     7441 ccaaaaatag tgaacttcaa ggtaagcaga tctatagttt caatatctga ttttctgctt
     7501 ttcaaaactt aaaattcatt agaaggtaaa acaagaaccc gtaaaattaa agttaccgat
     7561 attatcaggt ttttcacccc atgtctacta tcccatttag aaagaactat gtgttcaaaa
     7621 actgggccgg tcgacctgca gcgtacgaag cttcagctgg cggccgcgtt ctatagtgtc
     7681 acctaaatcg tatgtgtatg atacataagg ttatgtatta attgtagccg cgttctaacg
     7741 acaatatgtc catatggtgc actctcagta caatctgctc tgatgccgca tagttaagcc
     7801 agccccgaca cccgcc
//
