LOCUS       sequence-255828-        5973 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Tagging the Saccharomyces cerevisiae Cdc20 gene with a mCherry and 
            auxin-degron tag, via homologous recombination.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 5973)
  AUTHORS   Ozsezen S, Papagiannakis A, Chen H, Niebel B, Milias-Argeitis A, 
            Heinemann M
  TITLE     Inference of the High-Level Interaction Topology between the 
            Metabolic and Cell-Cycle Oscillators from Single-Cell Dynamics.
  JOURNAL   Cell Syst. 2019 Oct 4. pii: S2405-4712(19)30311-4. doi: 
            10.1016/j.cels.2019.09.003.
  PUBMED    31606371
REFERENCE   2  (bases 1 to 5973)
  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: "Cell Syst. 
            2019 Oct 4. pii: S2405-4712(19)30311-4. doi: 
            10.1016/j.cels.2019.09.003."
COMMENT     Sequence Label: G30, Cdc20-mCherry-AID tagging
FEATURES             Location/Qualifiers
     source          1..5973
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     CDS             539..1246
                     /codon_start=1
                     /product="monomeric derivative of DsRed fluorescent protein
                     (Shaner et al., 2004)"
                     /label=mCherry
                     /note="mammalian codon-optimized"
                     /translation="MVSKGEEDNMAIIKEFMRFKVHMEGSVNGHEFEIEGEGEGRPYEG
                     TQTAKLKVTKGGPLPFAWDILSPQFMYGSKAYVKHPADIPDYLKLSFPEGFKWERVMNF
                     EDGGVVTVTQDSSLQDGEFIYKVKLRGTNFPSDGPVMQKKTMGWEASSERMYPEDGALK
                     GEIKQRLKLKDGGHYDAEVKTTYKAKKPVQLPGAYNVNIKLDITSHNEDYTIVEQYERA
                     EGRHSTGGMDELYK"
     primer_bind     complement(684..702)
                     /label=mCherry-R
                     /note="mCherry, reverse primer"
     primer_bind     952..971
                     /label=mCherry-F
                     /note="mCherry, forward primer"
     CDS             1289..1420
                     /codon_start=1
                     /product="minimal degron from the auxin-responsive IAA17 
                     protein of Arabidopsis thaliana (Morawska and Ulrich, 
                     2013)"
                     /label=AID*
                     /note="acts as an auxin-inducible degron (AID) in the 
                     presence of TIR1"
                     /translation="PKDPAKPPAKAQVVGWPPVRSYRKNVMVSCQKSSGGPEAAAFVK"
     terminator      1468..1657
                     /gene="S. cerevisiae CYC1"
                     /label=CYC1 terminator
                     /note="transcription terminator for CYC1"
     gene            complement(1712..2831)
                     /label=natMX6
                     /note="yeast selectable marker conferring nourseothricin 
                     resistance"
     terminator      complement(1712..1909)
                     /label=TEF terminator
                     /note="Ashbya gossypii TEF terminator"
     CDS             complement(1915..2487)
                     /codon_start=1
                     /gene="Streptomyces noursei nat1"
                     /product="nourseothricin acetyltransferase"
                     /label=NrsR
                     /note="confers resistance to nourseothricin"
                     /translation="MGTTLDDTAYRYRTSVPGDAEAIEALDGSFTTDTVFRVTATGDGF
                     TLREVPVDPPLTKVFPDDESDDESDDGEDGDPDSRTFVAYGDDGDLAGFVVVSYSGWNR
                     RLTVEDIEVAPEHRGHGVGRALMGLATEFARERGAGHLWLEVTNVNAPAIHAYRRMGFT
                     LCGLDTALYDGTASDGEQALYMSMPCP"
     promoter        complement(2488..2831)
                     /label=TEF promoter
                     /note="Ashbya gossypii TEF promoter"
     primer_bind     3448..3469
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     complement(3623..3642)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     3742..3764
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     primer_bind     complement(3802..3820)
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     promoter        3888..3992
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(3993..4061,4062..4853)
                     /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(4211..4230)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      5024..5612
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     5513..5532
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     5766..5783
                     /label=L4440
                     /note="L4440 vector, forward primer"
     protein_bind    5900..5921
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
ORIGIN
        1 agcactaagc tggtgtccat attcgccaaa tattctagcc tctggaggcg gacaaacaga
       61 taaacacatc catttttgga acagtatcac aggtgcacga gttggctcaa tcaataccgg
      121 atcccaggtg agctctttac attggggcca aagtcatacg tcaaccaatg gtggtatgat
      181 gaataaagag attgttgcca caggaggtaa tccagagaat gcaatctctg tttataatta
      241 tgaaacaaaa ttcaaagttg cagaagtagt tcatgctcat gaagcaagaa tatgctgttc
      301 tcaattgtcc cctgacggaa ccacattggc cacagtggga ggagatgaaa acttaaaatt
      361 ttataagata tttgatccaa gatgtacagg aagatcgaga gaagacggat tgatggatgg
      421 tatgctagga cttattggca aggaaggttg tcgaacaaat gataaggaga atcgttccaa
      481 aaattcgagt gagattcata caaggaggcc ctctagtacc agccaatatt tgatcaggat
      541 ggtgagcaag ggcgaggagg ataacatggc catcatcaag gagttcatgc gcttcaaggt
      601 gcacatggag ggctccgtga acggccacga gttcgagatc gagggcgagg gcgagggccg
      661 cccctacgag ggcacccaga ccgccaagct gaaggtgacc aagggtggcc ccctgccctt
      721 cgcctgggac atcctgtccc ctcagttcat gtacggctcc aaggcctacg tgaagcaccc
      781 cgccgacatc cccgactact tgaagctgtc cttccccgag ggcttcaagt gggagcgcgt
      841 gatgaacttc gaggacggcg gcgtggtgac cgtgacccag gactcctccc tgcaggacgg
      901 cgagttcatc tacaaggtga agctgcgcgg caccaacttc ccctccgacg gccccgtaat
      961 gcagaagaag accatgggct gggaggcctc ctccgagcgg atgtaccccg aggacggcgc
     1021 cctgaagggc gagatcaagc agaggctgaa gctgaaggac ggcggccact acgacgctga
     1081 ggtcaagacc acctacaagg ccaagaagcc cgtgcagctg cccggcgcct acaacgtcaa
     1141 catcaagttg gacatcacct cccacaacga ggactacacc atcgtggaac agtacgaacg
     1201 cgccgagggc cgccactcca ccggcggcat ggacgagctg tacaaggtcg acggtgcagg
     1261 cgccggggct ggtgctgggg caggtacccc taaagatcca gccaaacctc cggccaaggc
     1321 acaagttgtg ggatggccac cggtgagatc ataccggaag aacgtgatgg tttcctgcca
     1381 aaaatcaagc ggtggcccgg aggcggcggc gttcgtgaag gtatcacccg ggtaactcga
     1441 gtaagcttgg taccgcggct agctaagatc cgctctaacc gaaaaggaag gagttagaca
     1501 acctgaagtc taggtcccta tttatttttt tatagttatg ttagtattaa gaacgttatt
     1561 tatatttcaa atttttcttt tttttctgta cagacgcgtg tacgcatgta acattatact
     1621 gaaaaccttg cttgagaagg ttttgggacg ctcgaagatc cagctcagcg tacgtcgaca
     1681 ctggatggcg gcgttagtat cgaatcgaca gcagtatagc gaccagcatt cacatacgat
     1741 tgacgcatga tattactttc tgcgcactta acttcgcatc tgggcagatg atgtcgaggc
     1801 gaaaaaaaat ataaatcacg ctaacatttg attaaaatag aacaactaca atataaaaaa
     1861 actatacaaa tgacaagttc ttgaaaacaa gaatcttttt attgtcagta ctgattaggg
     1921 gcagggcatg ctcatgtaga gcgcctgctc gccgtccgag gcggtgccgt cgtacagggc
     1981 ggtgtccagg ccgcagaggg tgaaccccat ccgccggtac gcgtggatcg ccggtgcgtt
     2041 gacgttggtg acctccagcc agaggtgccc ggcgccccgc tcgcgggcga actccgtcgc
     2101 gagccccatc aacgcgcgcc cgaccccgtg cccccggtgc tccggggcga cctcgatgtc
     2161 ctcgacggtc agccggcggt tccagccgga gtacgagacg accacgaagc ccgccaggtc
     2221 gccgtcgtcc ccgtacgcga cgaacgtccg ggagtccggg tcgccgtcct ccccgtcgtc
     2281 cgattcgtcg tccgattcgt cgtcggggaa caccttggtc aggggcgggt ccaccggcac
     2341 ctcccgcagg gtgaagccgt ccccggtggc ggtgacgcgg aagacggtgt cggtggtgaa
     2401 ggacccatcc agtgcctcga tggcctcggc gtcccccggg acactggtgc ggtaccggta
     2461 agccgtgtcg tcaagagtgg tacccatggt tgtttatgtt cggatgtgat gtgagaactg
     2521 tatcctagca agattttaaa aggaagtata tgaaagaaga acctcagtgg caaatcctaa
     2581 ccttttatat ttctctacag gggcgcggcg tggggacaat tcaacgcgtc tgtgagggga
     2641 gcgtttccct gctcgcaggt ctgcagcgag gagccgtaat ttttgcttcg cgccgtgcgg
     2701 ccatcaaaat gtatggatgc aaatgattat acatggggat gtatgggcta aatgtacggg
     2761 cgacagtcac atcatgcccc tgagctgcgc acgtcaagac tgtcaaggag ggtattctgg
     2821 gcctccatgt cgctggccgg gtgacccggc ggggacaagg caagctgttc atgtcaaggc
     2881 atataatgaa atttccatac acgcatacat aatgtataaa cataaatgta ttcaagaagc
     2941 ttaagaaata aaagtaatga accttgagaa tacatcaaga tataactcat cacatgttaa
     3001 agaacccaca caccacacgc gcgagaactg ggagggagag aaatttagca aataacaaaa
     3061 tttaggcctt ggaagaaacc atagcaatgc aataatcgtt attaacataa tatctacatt
     3121 gagaacttct tgggctagtg gttttgctgc aaactggaaa attgtatcaa tcttttaacg
     3181 ttagctaaac atcttctgct gtgttagcat taggaggaag cgaaaaggaa aatacataat
     3241 gaaaccgaca caggattcac aagaaaaggt ttctattgaa cagcagttag ctgtagaatc
     3301 gataaggaag tttttgaact cgaaaacatc ttatgacgtg ttgcctgttt cttaccgttt
     3361 aattgtcttg gacacctcgt tgttagtgaa gaaatcactg aatgttcttt tgcaaaatag
     3421 cattgtctct gcgccattat ggtaatagtg gactcttgtt ccaaactgga acaacactca
     3481 actctatctc gggctattct tttgatttat aagggatttt gccgatttcg gtctattggt
     3541 taaaaaatga gctgatttaa caaaaattta acgcgaattt taacaaaata ttaacgttta
     3601 caattttatg gtgcactctc agtacaatct gctctgatgc cgcatagtta agccagcccc
     3661 gacacccgcc aacacccgct gacgcgccct gacgggcttg tctgctcccg gcatccgctt
     3721 acagacaagc tgtgaccgtc tccgggagct gcatgtgtca gaggttttca ccgtcatcac
     3781 cgaaacgcgc gagacgaaag ggcctcgtga tacgcctatt tttataggtt aatgtcatga
     3841 taataatggt ttcttagacg tcaggtggca cttttcgggg aaatgtgcgc ggaaccccta
     3901 tttgtttatt tttctaaata cattcaaata tgtatccgct catgagacaa taaccctgat
     3961 aaatgcttca ataatattga aaaaggaaga gtatgagtat tcaacatttc cgtgtcgccc
     4021 ttattccctt ttttgcggca ttttgccttc ctgtttttgc tcacccagaa acgctggtga
     4081 aagtaaaaga tgctgaagat cagttgggtg cacgagtggg ttacatcgaa ctggatctca
     4141 acagcggtaa gatccttgag agttttcgcc ccgaagaacg ttttccaatg atgagcactt
     4201 ttaaagttct gctatgtggc gcggtattat cccgtattga cgccgggcaa gagcaactcg
     4261 gtcgccgcat acactattct cagaatgact tggttgagta ctcaccagtc acagaaaagc
     4321 atcttacgga tggcatgaca gtaagagaat tatgcagtgc tgccataacc atgagtgata
     4381 acactgcggc caacttactt ctgacaacga tcggaggacc gaaggagcta accgcttttt
     4441 tgcacaacat gggggatcat gtaactcgcc ttgatcgttg ggaaccggag ctgaatgaag
     4501 ccataccaaa cgacgagcgt gacaccacga tgcctgtagc aatggcaaca acgttgcgca
     4561 aactattaac tggcgaacta cttactctag cttcccggca acaattaata gactggatgg
     4621 aggcggataa agttgcagga ccacttctgc gctcggccct tccggctggc tggtttattg
     4681 ctgataaatc tggagccggt gagcgtgggt ctcgcggtat cattgcagca ctggggccag
     4741 atggtaagcc ctcccgtatc gtagttatct acacgacggg gagtcaggca actatggatg
     4801 aacgaaatag acagatcgct gagataggtg cctcactgat taagcattgg taactgtcag
     4861 accaagttta ctcatatata ctttagattg atttaaaact tcatttttaa tttaaaagga
     4921 tctaggtgaa gatccttttt gataatctca tgaccaaaat cccttaacgt gagttttcgt
     4981 tccactgagc gtcagacccc gtagaaaaga tcaaaggatc ttcttgagat cctttttttc
     5041 tgcgcgtaat ctgctgcttg caaacaaaaa aaccaccgct accagcggtg gtttgtttgc
     5101 cggatcaaga gctaccaact ctttttccga aggtaactgg cttcagcaga gcgcagatac
     5161 caaatactgt tcttctagtg tagccgtagt taggccacca cttcaagaac tctgtagcac
     5221 cgcctacata cctcgctctg ctaatcctgt taccagtggc tgctgccagt ggcgataagt
     5281 cgtgtcttac cgggttggac tcaagacgat agttaccgga taaggcacag cggtcgggct
     5341 gaacgggggg ttcgtgcaca cagcccagct tggagcgaac gacctacacc gaactgagat
     5401 acctacagcg tgagctatga gaaagcgcca cgcttcccga agggagaaag gcggacaggt
     5461 atccggtaag cggcagggtc ggaacaggag agcgcacgag ggagcttcca gggggaaacg
     5521 cctggtatct ttatagtcct gtcgggtttc gccacctctg acttgagcgt cgatttttgt
     5581 gatgctcgtc aggggggcgg agcctatgga aaaacgccag caacgcggcc tttttacggt
     5641 tcctggcctt ttgctggcct tttgctcaca tgttctttcc tgcgttatcc cctgattctg
     5701 tggataaccg tattaccgcc tttgagtgag ctgataccgc tcgccgcagc cgaacgaccg
     5761 agcgcagcga gtcagtgagc gaggaagcgg aagagcgccc aatacgcaaa ccgcctctcc
     5821 ccgcgcgttg gccgattcat taatgcagct ggcacgacag gtttcccgac tggaaagcgg
     5881 gcagtgagcg caacgcaatt aatgtgagtt agctcactca ttaggcaccc caggctttac
     5941 actttatgct tccgcggctc gtatgttgtg tgg
//
