LOCUS       sequence-386219-        5859 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Plasmid used for the C-terminal tagging of Atp3 with mCherry and the
            auxin-inducible degron in yeast.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 5859)
  AUTHORS   Takhaveev V, Ozsezen S, Smith EN, Zylstra A, Chaillet ML, Chen H, 
            Papagiannakis A, Milias-Argeitis A, Heinemann M
  TITLE     Temporal segregation of biosynthetic processes is responsible for 
            metabolic oscillations during the budding yeast cell cycle.
  JOURNAL   Nat Metab. 2023 Feb;5(2):294-313. doi: 10.1038/s42255-023-00741-x. 
            Epub 2023 Feb 27.
  PUBMED    36849832
REFERENCE   2  (bases 1 to 5859)
  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"; doi: 
            "10.1038/s42255-023-00741-x"; journalName: "Nat Metab"; date: 
            "2023-02"; volume: "5"; issue: "2"; pages: "294-313"
COMMENT     Sequence Label: GA46
FEATURES             Location/Qualifiers
     source          1..5859
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     CDS             501..1208
                     /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(646..664)
                     /label=mCherry-R
                     /note="mCherry, reverse primer"
     primer_bind     914..933
                     /label=mCherry-F
                     /note="mCherry, forward primer"
     CDS             1251..1382
                     /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      1430..1619
                     /gene="S. cerevisiae CYC1"
                     /label=CYC1 terminator
                     /note="transcription terminator for CYC1"
     gene            complement(1674..2793)
                     /label=natMX6
                     /note="yeast selectable marker conferring nourseothricin 
                     resistance"
     terminator      complement(1674..1871)
                     /label=TEF terminator
                     /note="Ashbya gossypii TEF terminator"
     CDS             complement(1877..2449)
                     /codon_start=1
                     /gene="Streptomyces noursei nat1"
                     /product="nourseothricin acetyltransferase"
                     /label=NrsR
                     /note="confers resistance to nourseothricin"
                     /translation="MGTTLDDTAYRYRTSVPGDAEAIEALDGSFTTDTVFRVTATGDGF
                     TLREVPVDPPLTKVFPDDESDDESDDGEDGDPDSRTFVAYGDDGDLAGFVVVSYSGWNR
                     RLTVEDIEVAPEHRGHGVGRALMGLATEFARERGAGHLWLEVTNVNAPAIHAYRRMGFT
                     LCGLDTALYDGTASDGEQALYMSMPCP"
     promoter        complement(2450..2793)
                     /label=TEF promoter
                     /note="Ashbya gossypii TEF promoter"
     primer_bind     3334..3355
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     complement(3509..3528)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     3628..3650
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     primer_bind     complement(3688..3706)
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     promoter        3774..3878
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(3879..3947,3948..4739)
                     /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(4097..4116)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      4910..5498
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     5399..5418
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     5652..5669
                     /label=L4440
                     /note="L4440 vector, forward primer"
     protein_bind    5786..5807
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
ORIGIN
        1 ataaaattaa aatgcagcta ttgagaaccc atcctaacaa cattaaattg tctattaatg
       61 gaattggtaa agatgcccca actttccaag aatctgcttt gattgccgat aagttattga
      121 gtgtcatgaa ggccggcact tacccaaaga tttccatttt ctacaatgac ccagtgtctt
      181 ccctatcttt tgaaccatct gaaaaaccga tctttaacgc caagaccatt gaacaatccc
      241 catcattcgg caaatttgag atcgacacgg acgcaaacgt tccaagagat ttgtttgaat
      301 atactttggc taaccaaatg ttgacagcaa tggctcaagg ttatgctgct gaaatttccg
      361 ccagaagaaa cgctatggat aacgcttcca agaatgccgg tgatatgatc aatcgttact
      421 ctatcttgta caacagaaca agacaagctg tcattactaa tgaactggtt gatattatta
      481 ctggtgcttc ctctttggga atggtgagca agggcgagga ggataacatg gccatcatca
      541 aggagttcat gcgcttcaag gtgcacatgg agggctccgt gaacggccac gagttcgaga
      601 tcgagggcga gggcgagggc cgcccctacg agggcaccca gaccgccaag ctgaaggtga
      661 ccaagggtgg ccccctgccc ttcgcctggg acatcctgtc ccctcagttc atgtacggct
      721 ccaaggccta cgtgaagcac cccgccgaca tccccgacta cttgaagctg tccttccccg
      781 agggcttcaa gtgggagcgc gtgatgaact tcgaggacgg cggcgtggtg accgtgaccc
      841 aggactcctc cctgcaggac ggcgagttca tctacaaggt gaagctgcgc ggcaccaact
      901 tcccctccga cggccccgta atgcagaaga agaccatggg ctgggaggcc tcctccgagc
      961 ggatgtaccc cgaggacggc gccctgaagg gcgagatcaa gcagaggctg aagctgaagg
     1021 acggcggcca ctacgacgct gaggtcaaga ccacctacaa ggccaagaag cccgtgcagc
     1081 tgcccggcgc ctacaacgtc aacatcaagt tggacatcac ctcccacaac gaggactaca
     1141 ccatcgtgga acagtacgaa cgcgccgagg gccgccactc caccggcggc atggacgagc
     1201 tgtacaaggt cgacggtgca ggcgccgggg ctggtgctgg ggcaggtacc cctaaagatc
     1261 cagccaaacc tccggccaag gcacaagttg tgggatggcc accggtgaga tcataccgga
     1321 agaacgtgat ggtttcctgc caaaaatcaa gcggtggccc ggaggcggcg gcgttcgtga
     1381 aggtatcacc cgggtaactc gagtaagctt ggtaccgcgg ctagctaaga tccgctctaa
     1441 ccgaaaagga aggagttaga caacctgaag tctaggtccc tatttatttt tttatagtta
     1501 tgttagtatt aagaacgtta tttatatttc aaatttttct tttttttctg tacagacgcg
     1561 tgtacgcatg taacattata ctgaaaacct tgcttgagaa ggttttggga cgctcgaaga
     1621 tccagctcag cgtacgtcga cactggatgg cggcgttagt atcgaatcga cagcagtata
     1681 gcgaccagca ttcacatacg attgacgcat gatattactt tctgcgcact taacttcgca
     1741 tctgggcaga tgatgtcgag gcgaaaaaaa atataaatca cgctaacatt tgattaaaat
     1801 agaacaacta caatataaaa aaactataca aatgacaagt tcttgaaaac aagaatcttt
     1861 ttattgtcag tactgattag gggcagggca tgctcatgta gagcgcctgc tcgccgtccg
     1921 aggcggtgcc gtcgtacagg gcggtgtcca ggccgcagag ggtgaacccc atccgccggt
     1981 acgcgtggat cgccggtgcg ttgacgttgg tgacctccag ccagaggtgc ccggcgcccc
     2041 gctcgcgggc gaactccgtc gcgagcccca tcaacgcgcg cccgaccccg tgcccccggt
     2101 gctccggggc gacctcgatg tcctcgacgg tcagccggcg gttccagccg gagtacgaga
     2161 cgaccacgaa gcccgccagg tcgccgtcgt ccccgtacgc gacgaacgtc cgggagtccg
     2221 ggtcgccgtc ctccccgtcg tccgattcgt cgtccgattc gtcgtcgggg aacaccttgg
     2281 tcaggggcgg gtccaccggc acctcccgca gggtgaagcc gtccccggtg gcggtgacgc
     2341 ggaagacggt gtcggtggtg aaggacccat ccagtgcctc gatggcctcg gcgtcccccg
     2401 ggacactggt gcggtaccgg taagccgtgt cgtcaagagt ggtacccatg gttgtttatg
     2461 ttcggatgtg atgtgagaac tgtatcctag caagatttta aaaggaagta tatgaaagaa
     2521 gaacctcagt ggcaaatcct aaccttttat atttctctac aggggcgcgg cgtggggaca
     2581 attcaacgcg tctgtgaggg gagcgtttcc ctgctcgcag gtctgcagcg aggagccgta
     2641 atttttgctt cgcgccgtgc ggccatcaaa atgtatggat gcaaatgatt atacatgggg
     2701 atgtatgggc taaatgtacg ggcgacagtc acatcatgcc cctgagctgc gcacgtcaag
     2761 actgtcaagg agggtattct gggcctccat gtcgctggcc gggtgacccg gcggggacaa
     2821 ggcaagctta aaaaaatcac cctgcattgc ctctttattt gacgttgttt ttgtagaaca
     2881 tgaaacgaat tttgacttga tgagacgaag tatatatcca aagaatacct taaatagaaa
     2941 aggaaagata ataaatacta aacactacta tatattcagg taaaatacaa aaattataac
     3001 atttttaaaa cttttttttt ttgaaagtcc ttctcgcttt aggatttttt cccattaaga
     3061 ttatgatggt ttcatgtatg tgtcagttaa aaaaaatatg gctaagtagc aatgaaacga
     3121 accaagaaaa tgaaaacaat atatagtgct gttgaaataa caaagacatt ggtatatatt
     3181 tgtaaaatgt ctgttaaatg ttttttatct caggttcttg cttgtctttg gtagaagaaa
     3241 ttatagtaaa caaacaaaca aacaaacaaa aaaaaaaaaa aaaaaatggt cgcaatctca
     3301 atgatttggt tttttaccaa gcgtatgcta atagtggact cttgttccaa actggaacaa
     3361 cactcaactc tatctcgggc tattcttttg atttataagg gattttgccg atttcggtct
     3421 attggttaaa aaatgagctg atttaacaaa aatttaacgc gaattttaac aaaatattaa
     3481 cgtttacaat tttatggtgc actctcagta caatctgctc tgatgccgca tagttaagcc
     3541 agccccgaca cccgccaaca cccgctgacg cgccctgacg ggcttgtctg ctcccggcat
     3601 ccgcttacag acaagctgtg accgtctccg ggagctgcat gtgtcagagg ttttcaccgt
     3661 catcaccgaa acgcgcgaga cgaaagggcc tcgtgatacg cctattttta taggttaatg
     3721 tcatgataat aatggtttct tagacgtcag gtggcacttt tcggggaaat gtgcgcggaa
     3781 cccctatttg tttatttttc taaatacatt caaatatgta tccgctcatg agacaataac
     3841 cctgataaat gcttcaataa tattgaaaaa ggaagagtat gagtattcaa catttccgtg
     3901 tcgcccttat tccctttttt gcggcatttt gccttcctgt ttttgctcac ccagaaacgc
     3961 tggtgaaagt aaaagatgct gaagatcagt tgggtgcacg agtgggttac atcgaactgg
     4021 atctcaacag cggtaagatc cttgagagtt ttcgccccga agaacgtttt ccaatgatga
     4081 gcacttttaa agttctgcta tgtggcgcgg tattatcccg tattgacgcc gggcaagagc
     4141 aactcggtcg ccgcatacac tattctcaga atgacttggt tgagtactca ccagtcacag
     4201 aaaagcatct tacggatggc atgacagtaa gagaattatg cagtgctgcc ataaccatga
     4261 gtgataacac tgcggccaac ttacttctga caacgatcgg aggaccgaag gagctaaccg
     4321 cttttttgca caacatgggg gatcatgtaa ctcgccttga tcgttgggaa ccggagctga
     4381 atgaagccat accaaacgac gagcgtgaca ccacgatgcc tgtagcaatg gcaacaacgt
     4441 tgcgcaaact attaactggc gaactactta ctctagcttc ccggcaacaa ttaatagact
     4501 ggatggaggc ggataaagtt gcaggaccac ttctgcgctc ggcccttccg gctggctggt
     4561 ttattgctga taaatctgga gccggtgagc gtgggtctcg cggtatcatt gcagcactgg
     4621 ggccagatgg taagccctcc cgtatcgtag ttatctacac gacggggagt caggcaacta
     4681 tggatgaacg aaatagacag atcgctgaga taggtgcctc actgattaag cattggtaac
     4741 tgtcagacca agtttactca tatatacttt agattgattt aaaacttcat ttttaattta
     4801 aaaggatcta ggtgaagatc ctttttgata atctcatgac caaaatccct taacgtgagt
     4861 tttcgttcca ctgagcgtca gaccccgtag aaaagatcaa aggatcttct tgagatcctt
     4921 tttttctgcg cgtaatctgc tgcttgcaaa caaaaaaacc accgctacca gcggtggttt
     4981 gtttgccgga tcaagagcta ccaactcttt ttccgaaggt aactggcttc agcagagcgc
     5041 agataccaaa tactgttctt ctagtgtagc cgtagttagg ccaccacttc aagaactctg
     5101 tagcaccgcc tacatacctc gctctgctaa tcctgttacc agtggctgct gccagtggcg
     5161 ataagtcgtg tcttaccggg ttggactcaa gacgatagtt accggataag gcacagcggt
     5221 cgggctgaac ggggggttcg tgcacacagc ccagcttgga gcgaacgacc tacaccgaac
     5281 tgagatacct acagcgtgag ctatgagaaa gcgccacgct tcccgaaggg agaaaggcgg
     5341 acaggtatcc ggtaagcggc agggtcggaa caggagagcg cacgagggag cttccagggg
     5401 gaaacgcctg gtatctttat agtcctgtcg ggtttcgcca cctctgactt gagcgtcgat
     5461 ttttgtgatg ctcgtcaggg gggcggagcc tatggaaaaa cgccagcaac gcggcctttt
     5521 tacggttcct ggccttttgc tggccttttg ctcacatgtt ctttcctgcg ttatcccctg
     5581 attctgtgga taaccgtatt accgcctttg agtgagctga taccgctcgc cgcagccgaa
     5641 cgaccgagcg cagcgagtca gtgagcgagg aagcggaaga gcgcccaata cgcaaaccgc
     5701 ctctccccgc gcgttggccg attcattaat gcagctggca cgacaggttt cccgactgga
     5761 aagcgggcag tgagcgcaac gcaattaatg tgagttagct cactcattag gcaccccagg
     5821 ctttacactt tatgcttccg cggctcgtat gttgtgtgg
//
