LOCUS       sequence-422542-        7299 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Expression of mRuby3-Pho8 under CPU1 promoter..
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 7299)
  AUTHORS   He CW, Cui XF, Ma SJ, Xu Q, Ran YP, Chen WZ, Mu JX, Li H, Zhu J, 
            Gong Q, Xie Z
  TITLE     Membrane recruitment of Atg8 by Hfl1 facilitates turnover of 
            vacuolar membrane proteins in yeast cells approaching stationary 
            phase.
  JOURNAL   BMC Biol. 2021 Jun 4;19(1):117. doi: 10.1186/s12915-021-01048-7.
  PUBMED    34088313
REFERENCE   2  (bases 1 to 7299)
  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: "BMC Biol.";
            date: "2021-06-4"; volume: "19(1):117. doi"; pages: " 
            10.1186/s12915-021-01048-7"
COMMENT     Sequence Label: PCUP1-mRuby3-Pho8-406
FEATURES             Location/Qualifiers
     source          1..7299
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     29..51
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     primer_bind     complement(89..107)
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     promoter        175..279
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(280..348,349..1140)
                     /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(498..517)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      1311..1899
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     1800..1819
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     2053..2070
                     /label=L4440
                     /note="L4440 vector, forward primer"
     protein_bind    2187..2208
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     promoter        join(2223..2228,2229..2246,2247..2253)
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    2261..2277
                     /label=lac operator
                     /bound_moiety="lac repressor encoded by lacI"
                     /note="The lac repressor binds to the lac operator to 
                     inhibit transcription in E. coli. This inhibition can be 
                     relieved by adding lactose or 
                     isopropyl-beta-D-thiogalactopyranoside (IPTG)."
     primer_bind     2266..2288
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     primer_bind     2285..2301
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     2285..2301
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     2320..2340
                     /label=T3
                     /note="T3 promoter, forward primer"
     promoter        2322..2340
                     /label=T3 promoter
                     /note="promoter for bacteriophage T3 RNA polymerase"
     promoter        2359..2593
                     /gene="S. cerevisiae CUP1"
                     /label=CUP1 promoter
                     /note="inducible metallothionein promoter, regulated by 
                     Cu2+"
     CDS             2604..3314
                     /codon_start=1
                     /product="monomeric red fluorescent protein derived from 
                     mRuby2, with improved brightness and photostability (Bajar 
                     et al., 2016)"
                     /label=mRuby3
                     /translation="MVSKGEELIKENMRMKVVMEGSVNGHQFKCTGEGEGRPYEGVQTM
                     RIKVIEGGPLPFAFDILATSFMYGSRTFIKYPADIPDFFKQSFPEGFTWERVTRYEDGG
                     VVTVTQDTSLEDGELVYNVKVRGVNFPSNGPVMQKKTKGWEPNTEMMYPADGGLRGYTD
                     IALKVDGGGHLHCNFVTTYRSKKTVGNIKMPGVHAVDHRLERIEESDNETYVVQREVAV
                     AKYSNLGGGMDELYK"
     primer_bind     complement(5394..5413)
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        complement(5395..5413)
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     primer_bind     complement(5423..5440)
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     complement(5423..5439)
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(5432..5454)
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     rep_origin      5580..6035
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(5667..5686)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     5877..5898
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     CDS             complement(6166..6969)
                     /codon_start=1
                     /gene="S. cerevisiae URA3"
                     /product="orotidine-5'-phosphate decarboxylase, required 
                     for uracil biosynthesis"
                     /label=URA3
                     /note="yeast auxotrophic marker, counterselectable with 
                     5-fluoroorotic acid (5-FOA)"
                     /translation="MSKATYKERAATHPSPVAAKLFNIMHEKQTNLCASLDVRTTKELL
                     ELVEALGPKICLLKTHVDILTDFSMEGTVKPLKALSAKYNFLLFEDRKFADIGNTVKLQ
                     YSAGVYRIAEWADITNAHGVVGPGIVSGLKQAAEEVTKEPRGLLMLAELSCKGSLSTGE
                     YTKGTVDIAKSDKDFVIGFIAQRDMGGRDEGYDWLIMTPGVGLDDKGDALGQQYRTVDD
                     VVSTGSDIIIVGRGLFAKGRDAKVEGERYRKAGWEAYLRRCGQQN"
     promoter        complement(6970..7185)
                     /gene="S. cerevisiae URA3"
                     /label=URA3 promoter
     primer_bind     complement(7209..7228)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
ORIGIN
        1 tccgcttaca gacaagctgt gaccgtctcc gggagctgca tgtgtcagag gttttcaccg
       61 tcatcaccga aacgcgcgag acgaaagggc ctcgtgatac gcctattttt ataggttaat
      121 gtcatgataa taatggtttc ttagacgtca ggtggcactt ttcggggaaa tgtgcgcgga
      181 acccctattt gtttattttt ctaaatacat tcaaatatgt atccgctcat gagacaataa
      241 ccctgataaa tgcttcaata atattgaaaa aggaagagta tgagtattca acatttccgt
      301 gtcgccctta ttcccttttt tgcggcattt tgccttcctg tttttgctca cccagaaacg
      361 ctggtgaaag taaaagatgc tgaagatcag ttgggtgcac gagtgggtta catcgaactg
      421 gatctcaaca gcggtaagat ccttgagagt tttcgccccg aagaacgttt tccaatgatg
      481 agcactttta aagttctgct atgtggcgcg gtattatccc gtattgacgc cgggcaagag
      541 caactcggtc gccgcataca ctattctcag aatgacttgg ttgagtactc accagtcaca
      601 gaaaagcatc ttacggatgg catgacagta agagaattat gcagtgctgc cataaccatg
      661 agtgataaca ctgcggccaa cttacttctg acaacgatcg gaggaccgaa ggagctaacc
      721 gcttttttgc acaacatggg ggatcatgta actcgccttg atcgttggga accggagctg
      781 aatgaagcca taccaaacga cgagcgtgac accacgatgc ctgtagcaat ggcaacaacg
      841 ttgcgcaaac tattaactgg cgaactactt actctagctt cccggcaaca attaatagac
      901 tggatggagg cggataaagt tgcaggacca cttctgcgct cggcccttcc ggctggctgg
      961 tttattgctg ataaatctgg agccggtgag cgtgggtctc gcggtatcat tgcagcactg
     1021 gggccagatg gtaagccctc ccgtatcgta gttatctaca cgacggggag tcaggcaact
     1081 atggatgaac gaaatagaca gatcgctgag ataggtgcct cactgattaa gcattggtaa
     1141 ctgtcagacc aagtttactc atatatactt tagattgatt taaaacttca tttttaattt
     1201 aaaaggatct aggtgaagat cctttttgat aatctcatga ccaaaatccc ttaacgtgag
     1261 ttttcgttcc actgagcgtc agaccccgta gaaaagatca aaggatcttc ttgagatcct
     1321 ttttttctgc gcgtaatctg ctgcttgcaa acaaaaaaac caccgctacc agcggtggtt
     1381 tgtttgccgg atcaagagct accaactctt tttccgaagg taactggctt cagcagagcg
     1441 cagataccaa atactgttct tctagtgtag ccgtagttag gccaccactt caagaactct
     1501 gtagcaccgc ctacatacct cgctctgcta atcctgttac cagtggctgc tgccagtggc
     1561 gataagtcgt gtcttaccgg gttggactca agacgatagt taccggataa ggcgcagcgg
     1621 tcgggctgaa cggggggttc gtgcacacag cccagcttgg agcgaacgac ctacaccgaa
     1681 ctgagatacc tacagcgtga gctatgagaa agcgccacgc ttcccgaagg gagaaaggcg
     1741 gacaggtatc cggtaagcgg cagggtcgga acaggagagc gcacgaggga gcttccaggg
     1801 ggaaacgcct ggtatcttta tagtcctgtc gggtttcgcc acctctgact tgagcgtcga
     1861 tttttgtgat gctcgtcagg ggggcggagc ctatggaaaa acgccagcaa cgcggccttt
     1921 ttacggttcc tggccttttg ctggcctttt gctcacatgt tctttcctgc gttatcccct
     1981 gattctgtgg ataaccgtat taccgccttt gagtgagctg ataccgctcg ccgcagccga
     2041 acgaccgagc gcagcgagtc agtgagcgag gaagcggaag agcgcccaat acgcaaaccg
     2101 cctctccccg cgcgttggcc gattcattaa tgcagctggc acgacaggtt tcccgactgg
     2161 aaagcgggca gtgagcgcaa cgcaattaat gtgagttagc tcactcatta ggcaccccag
     2221 gctttacact ttatgcttcc ggctcgtatg ttgtgtggaa ttgtgagcgg ataacaattt
     2281 cacacaggaa acagctatga ccatgattac gccaagcgcg caattaaccc tcactaaagg
     2341 gaacaaaagc tggagctcct agttagaaaa agacattttt gctgtcagtc actgtcaaga
     2401 gattcttttg ctggcatttc ttctagaagc aaaaagagcg atgcgtcttt tccgctgaac
     2461 cgttccagca aaaaagacta ccaacgcaat atggattgtc agaatcatat aaaagagaag
     2521 caaataactc cttgtcttgt atcaattgca ttataatatc ttcttgttag tgcaatatca
     2581 tatagaagtc atcgactagt aacatggtgt ctaagggcga agagctgatc aaggaaaata
     2641 tgcgtatgaa ggtggtcatg gaaggttcgg tcaacggcca ccaattcaaa tgcacaggtg
     2701 aaggagaagg cagaccgtac gagggagtgc aaaccatgag gatcaaagtc atcgagggag
     2761 gacccctgcc atttgccttt gacattcttg ccacgtcgtt catgtatggc agccgtacct
     2821 ttatcaagta cccggccgac atccctgatt tctttaaaca gtcctttcct gagggtttta
     2881 cttgggaaag agttacgaga tacgaagatg gtggagtcgt caccgtcacg caggacacca
     2941 gccttgagga tggcgagctc gtctacaacg tcaaggtcag aggggtaaac tttccctcca
     3001 atggtcccgt gatgcagaag aagaccaagg gttgggagcc taatacagag atgatgtatc
     3061 cagcagatgg tggtctgaga ggatacactg acatcgcact gaaagttgat ggtggtggcc
     3121 atctgcactg caacttcgtg acaacttaca ggtcaaaaaa gaccgtcggg aacatcaaga
     3181 tgcccggtgt ccatgccgtt gatcaccgcc tggaaaggat cgaggagagt gacaatgaaa
     3241 cctacgtagt gcaaagagaa gtggcagttg ccaaatacag caaccttggt ggtggcatgg
     3301 acgagctgta caagggagcc ggaacaagtg acatgatgac tcacacatta ccaagcgaac
     3361 agacacgtct tgttcctgga tctgactcga gctctcgtcc taaaaagaga cggatctcga
     3421 agagatcaaa gataatagta tccactgtgg tctgtattgg tttgttgctg gttttagtgc
     3481 aactggcatt tccaagcagt tttgcattac gttctgcatc acacaagaag aagaatgtca
     3541 tattcttcgt gacggatggg atgggacctg cgtctttgtc catggcaaga tcattcaacc
     3601 agcatgttaa tgatttacct atagacgaca tcctgacact tgacgagcat tttatcgggt
     3661 cttcgagaac aaggtcctcg gactcactag tcactgactc agctgcgggc gccaccgcat
     3721 ttgcatgtgc attaaagagc tataatgggg ccatcggtgt tgatccgcat cacagacctt
     3781 gtggtacagt gctcgaggcg gctaagttag caggttatct cactgggctt gttgttacga
     3841 caagaatcac agatgccact ccggcttcat tcagttcaca tgttgattac agatggcagg
     3901 aagatcttat cgctacgcac cagttaggcg aatatcctct ggggagagta gtagacttac
     3961 tcatgggtgg cggcaggagc catttctacc ctcaaggcga aaaggcttct ccatatggtc
     4021 accacggtgc tagaaaagat ggaagagatt taatcgatga ggctcaatct aatggctggc
     4081 agtacgtggg tgaccgtaaa aattttgatt ctttgttgaa aagccacggt gaaaacgtta
     4141 ctttaccttt cttgggtctc tttgcagata acgacattcc ttttgaaatt gacagagatg
     4201 aaaaggagta tccttccctt aaggaacaag tcaaggtggc tttgggtgcc ttggaaaaag
     4261 cttccaatga ggacaaagat tctaatgggt ttttcttaat ggtggaaggc tccagaattg
     4321 atcatgctgg tcaccaaaac gatccagctt cgcaggtaag ggaggtgttg gctttcgatg
     4381 aagcattcca atacgttttg gagtttgctg aaaattccga tacggagact gttttggtct
     4441 ctacgtcgga tcatgaaacg ggtggtttag tcacttcaag acaagtcact gctagctatc
     4501 ctcagtatgt ctggtatcca caggtgctcg ccaacgccac ccattcaggt gagtttttga
     4561 aaagaaaact agtagatttt gttcatgagc ataagggcgc aagcagcaaa atagaaaatt
     4621 tcatcaaaca tgagatcctg gaaaaagatt tgggtattta cgattacacg gatagcgatt
     4681 tagaaacatt aattcatttg gatgacaacg caaatgctat ccaagacaaa ctgaatgaca
     4741 tggtatcatt tagggcccaa attggctgga ccacgcatgg tcacagcgca gtcgacgtca
     4801 acatatatgc atacgccaat aagaaagcta catggtcata tgtcttgaac aatttacaag
     4861 gcaatcacga aaatacggaa gttggccagt tcttagaaaa ctttttagaa ttgaacctga
     4921 acgaagtcac tgatttgatt agggacacca aacacacatc agattttgat gctacagaaa
     4981 tagctagtga agtacaacat tatgacgaat actaccatga gttgaccaac tgatcctatc
     5041 taactctccc tcttttttca catattattt aatacgttat aatttatttg aatatatata
     5101 tatatatata tgtatatata ctaaccgcat gacacgtact gtatcgcatt tcgtttcgga
     5161 gtgcaggcga gttttaggtg cattctatag cgtgaagaag tgaaatctcg tcatttcttc
     5221 aagttatgcc attcagtcca gattgtttca catgctttca tatagtcatt tttgtcgtcg
     5281 cgaacttcaa atttgaaaac atcatcatac agagagattt ctccaataat gatcctttct
     5341 ttgtcgttgt catcatcggc atcgtcgtta agtgaagcgc ggtacccaat tcgccctata
     5401 gtgagtcgta ttacgcgcgc tcactggccg tcgttttaca acgtcgtgac tgggaaaacc
     5461 ctggcgttac ccaacttaat cgccttgcag cacatccccc tttcgccagc tggcgtaata
     5521 gcgaagaggc ccgcaccgat cgcccttccc aacagttgcg cagcctgaat ggcgaatgga
     5581 cgcgccctgt agcggcgcat taagcgcggc gggtgtggtg gttacgcgca gcgtgaccgc
     5641 tacacttgcc agcgccctag cgcccgctcc tttcgctttc ttcccttcct ttctcgccac
     5701 gttcgccggc tttccccgtc aagctctaaa tcgggggctc cctttagggt tccgatttag
     5761 tgctttacgg cacctcgacc ccaaaaaact tgattagggt gatggttcac gtagtgggcc
     5821 atcgccctga tagacggttt ttcgcccttt gacgttggag tccacgttct ttaatagtgg
     5881 actcttgttc caaactggaa caacactcaa ccctatctcg gtctattctt ttgatttata
     5941 agggattttg ccgatttcgg cctattggtt aaaaaatgag ctgatttaac aaaaatttaa
     6001 cgcgaatttt aacaaaatat taacgcttac aatttcctga tgcggtattt tctccttacg
     6061 catctgtgcg gtatttcaca ccgcataggg taataactga tataattaaa ttgaagctct
     6121 aatttgtgag tttagtatac atgcatttac ttataataca gttttttagt tttgctggcc
     6181 gcatcttctc aaatatgctt cccagcctgc ttttctgtaa cgttcaccct ctaccttagc
     6241 atcccttccc tttgcaaata gtcctcttcc aacaataata atgtcagatc ctgtagagac
     6301 cacatcatcc acggttctat actgttgacc caatgcgtct cccttgtcat ctaaacccac
     6361 accgggtgtc ataatcaacc aatcgtaacc ttcatctctt ccacccatgt ctctttgagc
     6421 aataaagccg ataacaaaat ctttgtcgct cttcgcaatg tcaacagtac ccttagtata
     6481 ttctccagta gatagggagc ccttgcatga caattctgct aacatcaaaa ggcctctagg
     6541 ttcctttgtt acttcttctg ccgcctgctt caaaccgcta acaatacctg ggcccaccac
     6601 accgtgtgca ttcgtaatgt ctgcccattc tgctattctg tatacacccg cagagtactg
     6661 caatttgact gtattaccaa tgtcagcaaa ttttctgtct tcgaagagta aaaaattgta
     6721 cttggcggat aatgccttta gcggcttaac tgtgccctcc atggaaaaat cagtcaagat
     6781 atccacatgt gtttttagta aacaaatttt gggacctaat gcttcaacta actccagtaa
     6841 ttccttggtg gtacgaacat ccaatgaagc acacaagttt gtttgctttt cgtgcatgat
     6901 attaaatagc ttggcagcaa caggactagg atgagtagca gcacgttcct tatatgtagc
     6961 tttcgacatg atttatcttc gtttcctgca ggtttttgtt ctgtgcagtt gggttaagaa
     7021 tactgggcaa tttcatgttt cttcaacact acatatgcgt atatatacca atctaagtct
     7081 gtgctccttc cttcgttctt ccttctgttc ggagattacc gaatcaaaaa aatttcaagg
     7141 aaaccgaaat caaaaaaaag aataaaaaaa aaatgatgaa ttgaaaaggt ggtatggtgc
     7201 actctcagta caatctgctc tgatgccgca tagttaagcc agccccgaca cccgccaaca
     7261 cccgctgacg cgccctgacg ggcttgtctg ctcccggca
//
