LOCUS       sequence-255827-        5265 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Tagging the Saccharomyces cerevisiae Cdc20 gene with a auxin-degron 
            tag, via homologous recombination.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 5265)
  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 5265)
  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: G31, Cdc20-AID tagging
FEATURES             Location/Qualifiers
     source          1..5265
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     CDS             581..712
                     /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      760..949
                     /gene="S. cerevisiae CYC1"
                     /label=CYC1 terminator
                     /note="transcription terminator for CYC1"
     gene            complement(1004..2123)
                     /label=natMX6
                     /note="yeast selectable marker conferring nourseothricin 
                     resistance"
     terminator      complement(1004..1201)
                     /label=TEF terminator
                     /note="Ashbya gossypii TEF terminator"
     CDS             complement(1207..1779)
                     /codon_start=1
                     /gene="Streptomyces noursei nat1"
                     /product="nourseothricin acetyltransferase"
                     /label=NrsR
                     /note="confers resistance to nourseothricin"
                     /translation="MGTTLDDTAYRYRTSVPGDAEAIEALDGSFTTDTVFRVTATGDGF
                     TLREVPVDPPLTKVFPDDESDDESDDGEDGDPDSRTFVAYGDDGDLAGFVVVSYSGWNR
                     RLTVEDIEVAPEHRGHGVGRALMGLATEFARERGAGHLWLEVTNVNAPAIHAYRRMGFT
                     LCGLDTALYDGTASDGEQALYMSMPCP"
     promoter        complement(1780..2123)
                     /label=TEF promoter
                     /note="Ashbya gossypii TEF promoter"
     primer_bind     2740..2761
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     complement(2915..2934)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     3034..3056
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     primer_bind     complement(3094..3112)
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     promoter        3180..3284
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(3285..3353,3354..4145)
                     /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(3503..3522)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      4316..4904
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     4805..4824
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     5058..5075
                     /label=L4440
                     /note="L4440 vector, forward primer"
     protein_bind    5192..5213
                     /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 tgatcagggt
      541 cgacggtgca ggcgccgggg ctggtgctgg ggcaggtacc cctaaagatc cagccaaacc
      601 tccggccaag gcacaagttg tgggatggcc accggtgaga tcataccgga agaacgtgat
      661 ggtttcctgc caaaaatcaa gcggtggccc ggaggcggcg gcgttcgtga aggtatcacc
      721 cgggtaactc gagtaagctt ggtaccgcgg ctagctaaga tccgctctaa ccgaaaagga
      781 aggagttaga caacctgaag tctaggtccc tatttatttt tttatagtta tgttagtatt
      841 aagaacgtta tttatatttc aaatttttct tttttttctg tacagacgcg tgtacgcatg
      901 taacattata ctgaaaacct tgcttgagaa ggttttggga cgctcgaaga tccagctcag
      961 cgtacgtcga cactggatgg cggcgttagt atcgaatcga cagcagtata gcgaccagca
     1021 ttcacatacg attgacgcat gatattactt tctgcgcact taacttcgca tctgggcaga
     1081 tgatgtcgag gcgaaaaaaa atataaatca cgctaacatt tgattaaaat agaacaacta
     1141 caatataaaa aaactataca aatgacaagt tcttgaaaac aagaatcttt ttattgtcag
     1201 tactgattag gggcagggca tgctcatgta gagcgcctgc tcgccgtccg aggcggtgcc
     1261 gtcgtacagg gcggtgtcca ggccgcagag ggtgaacccc atccgccggt acgcgtggat
     1321 cgccggtgcg ttgacgttgg tgacctccag ccagaggtgc ccggcgcccc gctcgcgggc
     1381 gaactccgtc gcgagcccca tcaacgcgcg cccgaccccg tgcccccggt gctccggggc
     1441 gacctcgatg tcctcgacgg tcagccggcg gttccagccg gagtacgaga cgaccacgaa
     1501 gcccgccagg tcgccgtcgt ccccgtacgc gacgaacgtc cgggagtccg ggtcgccgtc
     1561 ctccccgtcg tccgattcgt cgtccgattc gtcgtcgggg aacaccttgg tcaggggcgg
     1621 gtccaccggc acctcccgca gggtgaagcc gtccccggtg gcggtgacgc ggaagacggt
     1681 gtcggtggtg aaggacccat ccagtgcctc gatggcctcg gcgtcccccg ggacactggt
     1741 gcggtaccgg taagccgtgt cgtcaagagt ggtacccatg gttgtttatg ttcggatgtg
     1801 atgtgagaac tgtatcctag caagatttta aaaggaagta tatgaaagaa gaacctcagt
     1861 ggcaaatcct aaccttttat atttctctac aggggcgcgg cgtggggaca attcaacgcg
     1921 tctgtgaggg gagcgtttcc ctgctcgcag gtctgcagcg aggagccgta atttttgctt
     1981 cgcgccgtgc ggccatcaaa atgtatggat gcaaatgatt atacatgggg atgtatgggc
     2041 taaatgtacg ggcgacagtc acatcatgcc cctgagctgc gcacgtcaag actgtcaagg
     2101 agggtattct gggcctccat gtcgctggcc gggtgacccg gcggggacaa ggcaagctgt
     2161 tcatgtcaag gcatataatg aaatttccat acacgcatac ataatgtata aacataaatg
     2221 tattcaagaa gcttaagaaa taaaagtaat gaaccttgag aatacatcaa gatataactc
     2281 atcacatgtt aaagaaccca cacaccacac gcgcgagaac tgggagggag agaaatttag
     2341 caaataacaa aatttaggcc ttggaagaaa ccatagcaat gcaataatcg ttattaacat
     2401 aatatctaca ttgagaactt cttgggctag tggttttgct gcaaactgga aaattgtatc
     2461 aatcttttaa cgttagctaa acatcttctg ctgtgttagc attaggagga agcgaaaagg
     2521 aaaatacata atgaaaccga cacaggattc acaagaaaag gtttctattg aacagcagtt
     2581 agctgtagaa tcgataagga agtttttgaa ctcgaaaaca tcttatgacg tgttgcctgt
     2641 ttcttaccgt ttaattgtct tggacacctc gttgttagtg aagaaatcac tgaatgttct
     2701 tttgcaaaat agcattgtct ctgcgccatt atggtaatag tggactcttg ttccaaactg
     2761 gaacaacact caactctatc tcgggctatt cttttgattt ataagggatt ttgccgattt
     2821 cggtctattg gttaaaaaat gagctgattt aacaaaaatt taacgcgaat tttaacaaaa
     2881 tattaacgtt tacaatttta tggtgcactc tcagtacaat ctgctctgat gccgcatagt
     2941 taagccagcc ccgacacccg ccaacacccg ctgacgcgcc ctgacgggct tgtctgctcc
     3001 cggcatccgc ttacagacaa gctgtgaccg tctccgggag ctgcatgtgt cagaggtttt
     3061 caccgtcatc accgaaacgc gcgagacgaa agggcctcgt gatacgccta tttttatagg
     3121 ttaatgtcat gataataatg gtttcttaga cgtcaggtgg cacttttcgg ggaaatgtgc
     3181 gcggaacccc tatttgttta tttttctaaa tacattcaaa tatgtatccg ctcatgagac
     3241 aataaccctg ataaatgctt caataatatt gaaaaaggaa gagtatgagt attcaacatt
     3301 tccgtgtcgc ccttattccc ttttttgcgg cattttgcct tcctgttttt gctcacccag
     3361 aaacgctggt gaaagtaaaa gatgctgaag atcagttggg tgcacgagtg ggttacatcg
     3421 aactggatct caacagcggt aagatccttg agagttttcg ccccgaagaa cgttttccaa
     3481 tgatgagcac ttttaaagtt ctgctatgtg gcgcggtatt atcccgtatt gacgccgggc
     3541 aagagcaact cggtcgccgc atacactatt ctcagaatga cttggttgag tactcaccag
     3601 tcacagaaaa gcatcttacg gatggcatga cagtaagaga attatgcagt gctgccataa
     3661 ccatgagtga taacactgcg gccaacttac ttctgacaac gatcggagga ccgaaggagc
     3721 taaccgcttt tttgcacaac atgggggatc atgtaactcg ccttgatcgt tgggaaccgg
     3781 agctgaatga agccatacca aacgacgagc gtgacaccac gatgcctgta gcaatggcaa
     3841 caacgttgcg caaactatta actggcgaac tacttactct agcttcccgg caacaattaa
     3901 tagactggat ggaggcggat aaagttgcag gaccacttct gcgctcggcc cttccggctg
     3961 gctggtttat tgctgataaa tctggagccg gtgagcgtgg gtctcgcggt atcattgcag
     4021 cactggggcc agatggtaag ccctcccgta tcgtagttat ctacacgacg gggagtcagg
     4081 caactatgga tgaacgaaat agacagatcg ctgagatagg tgcctcactg attaagcatt
     4141 ggtaactgtc agaccaagtt tactcatata tactttagat tgatttaaaa cttcattttt
     4201 aatttaaaag gatctaggtg aagatccttt ttgataatct catgaccaaa atcccttaac
     4261 gtgagttttc gttccactga gcgtcagacc ccgtagaaaa gatcaaagga tcttcttgag
     4321 atcctttttt tctgcgcgta atctgctgct tgcaaacaaa aaaaccaccg ctaccagcgg
     4381 tggtttgttt gccggatcaa gagctaccaa ctctttttcc gaaggtaact ggcttcagca
     4441 gagcgcagat accaaatact gttcttctag tgtagccgta gttaggccac cacttcaaga
     4501 actctgtagc accgcctaca tacctcgctc tgctaatcct gttaccagtg gctgctgcca
     4561 gtggcgataa gtcgtgtctt accgggttgg actcaagacg atagttaccg gataaggcac
     4621 agcggtcggg ctgaacgggg ggttcgtgca cacagcccag cttggagcga acgacctaca
     4681 ccgaactgag atacctacag cgtgagctat gagaaagcgc cacgcttccc gaagggagaa
     4741 aggcggacag gtatccggta agcggcaggg tcggaacagg agagcgcacg agggagcttc
     4801 cagggggaaa cgcctggtat ctttatagtc ctgtcgggtt tcgccacctc tgacttgagc
     4861 gtcgattttt gtgatgctcg tcaggggggc ggagcctatg gaaaaacgcc agcaacgcgg
     4921 cctttttacg gttcctggcc ttttgctggc cttttgctca catgttcttt cctgcgttat
     4981 cccctgattc tgtggataac cgtattaccg cctttgagtg agctgatacc gctcgccgca
     5041 gccgaacgac cgagcgcagc gagtcagtga gcgaggaagc ggaagagcgc ccaatacgca
     5101 aaccgcctct ccccgcgcgt tggccgattc attaatgcag ctggcacgac aggtttcccg
     5161 actggaaagc gggcagtgag cgcaacgcaa ttaatgtgag ttagctcact cattaggcac
     5221 cccaggcttt acactttatg cttccgcggc tcgtatgttg tgtgg
//
