LOCUS       sequence-423111-        6988 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Expression of 4V5-Pho8 under CPU1 promoter. .
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 6988)
  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 6988)
  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: PCUP1-4V5-Pho8-Met
FEATURES             Location/Qualifiers
     source          1..6988
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     54..70
                     /label=KS primer
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     promoter        74..308
                     /gene="S. cerevisiae CUP1"
                     /label=CUP1 promoter
                     /note="inducible metallothionein promoter, regulated by 
                     Cu2+"
     CDS             318..359
                     /codon_start=1
                     /product="epitope tag from simian virus 5"
                     /label=V5 tag
                     /translation="GKPIPNPLLGLDST"
     CDS             366..407
                     /codon_start=1
                     /product="epitope tag from simian virus 5"
                     /label=V5 tag
                     /translation="GKPIPNPLLGLDST"
     CDS             414..455
                     /codon_start=1
                     /product="epitope tag from simian virus 5"
                     /label=V5 tag
                     /translation="GKPIPNPLLGLDST"
     CDS             462..503
                     /codon_start=1
                     /product="epitope tag from simian virus 5"
                     /label=V5 tag
                     /translation="GKPIPNPLLGLDST"
     terminator      2246..2433
                     /gene="S. cerevisiae ADH1"
                     /label=ADH1 terminator
                     /note="transcription terminator for the S. cerevisiae 
                     alcohol dehydrogenase 1 (ADH1) gene"
     primer_bind     complement(2301..2325)
                     /label=pHybLex-R
                     /note="ADH terminator, reverse primer"
     protein_bind    2485..2518
                     /label=loxP
                     /bound_moiety="Cre recombinase"
                     /note="Cre-mediated recombination occurs in the 8-bp core 
                     sequence (ATGTATGC) (Shaw et al., 2021)."
     CDS             complement(2701..4035)
                     /codon_start=1
                     /gene="S. cerevisiae MET17"
                     /gene_synonym="MET15; MET25"
                     /product="O-acetylserine and O-acetylhomoserine 
                     sulfhydrylase, required for sulfur amino acid synthesis"
                     /label=MET17
                     /translation="MPSHFDTVQLHAGQENPGDNAHRSRAVPIYATTSYVFENSKHGSQ
                     LFGLEVPGYVYSRFQNPTSNVLEERIAALEGGAAALAVSSGQAAQTLAIQGLAHTGDNI
                     VSTSYLYGGTYNQFKISFKRFGIEARFVEGDNPEEFEKVFDERTKAVYLETIGNPKYNV
                     PDFEKIVAIAHKHGIPVVVDNTFGAGGYFCQPIKYGADIVTHSATKWIGGHGTTIGGII
                     VDSGKFPWKDYPEKFPQFSQPAEGYHGTIYNEAYGNLAYIVHVRTELLRDLGPLMNPFA
                     SFLLLQGVETLSLRAERHGENALKLAKWLEQSPYVSWVSYPGLASHSHHENAKKYLSNG
                     FGGVLSFGVKDLPNADKETDPFKLSGAQVVDNLKLASNLANVGDAKTLVIAPYFTTHKQ
                     LNDKEKLASGVTKDLIRVSVGIEFIDDIIADFQQSFETVFAGQKP"
     promoter        complement(4036..4386)
                     /gene="S. cerevisiae MET17"
                     /gene_synonym="MET15; MET25"
                     /label=MET17 promoter
                     /note="expression is repressed by methionine"
     protein_bind    4539..4572
                     /label=loxP
                     /bound_moiety="Cre recombinase"
                     /note="Cre-mediated recombination occurs in the 8-bp core 
                     sequence (ATGTATGC) (Shaw et al., 2021)."
     primer_bind     complement(4625..4644)
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        complement(4626..4644)
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     primer_bind     complement(4731..4748)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(4902..5490)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(4982..5001)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(5661..6452,6453..6521))
                     /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     6284..6303
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(6522..6626)
                     /gene="bla"
                     /label=AmpR promoter
     primer_bind     6694..6712
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     primer_bind     complement(6750..6772)
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     primer_bind     6872..6891
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     promoter        join(6972..6988,1..2)
                     /label=SP6 promoter
                     /note="promoter for bacteriophage SP6 RNA polymerase"
     primer_bind     join(6972..6988,1)
                     /label=SP6
                     /note="SP6 promoter, forward primer"
ORIGIN
        1 gaacgcggcc gccagctgaa gcttcgtacg ctgcaggtcg tgggattcct gggcgaggtc
       61 gacggtatcg atactagtta gaaaaagaca tttttgctgt cagtcactgt caagagattc
      121 ttttgctggc atttcttcta gaagcaaaaa gagcgatgcg tcttttccgc tgaaccgttc
      181 cagcaaaaaa gactaccaac gcaatatgga ttgtcagaat catataaaag agaagcaaat
      241 aactccttgt cttgtatcaa ttgcattata atatcttctt gttagtgcaa tatcatatag
      301 aagtcatcgc tagcatgggt aaaccaattc caaatccatt gttgggtttg gattctactc
      361 ctagcggtaa accaattcca aatccattgt tgggtttgga ttctactcct agcggtaaac
      421 caattccaaa tccattgttg ggtttggatt ctactcctag cggtaaacca attccaaatc
      481 cattgttggg tttggattct actcctagga tgatgactca cacattacca agcgaacaga
      541 cacgtcttgt tcctggatct gactcgagct ctcgtcctaa aaagagacgg atctcgaaga
      601 gatcaaagat aatagtatcc actgtggtct gtattggttt gttgctggtt ttagtgcaac
      661 tggcatttcc aagcagtttt gcattacgtt ctgcatcaca caagaagaag aatgtcatat
      721 tcttcgtgac ggatgggatg ggacctgcgt ctttgtccat ggcaagatca ttcaaccagc
      781 atgttaatga tttacctata gacgacatcc tgacacttga cgagcatttt atcgggtctt
      841 cgagaacaag gtcctcggac tcactagtca ctgactcagc tgcgggcgcc accgcatttg
      901 catgtgcatt aaagagctat aatggggcca tcggtgttga tccgcatcac agaccttgtg
      961 gtacagtgct cgaggcggct aagttagcag gttatctcac tgggcttgtt gttacgacaa
     1021 gaatcacaga tgccactccg gcttcattca gttcacatgt tgattacaga tggcaggaag
     1081 atcttatcgc tacgcaccag ttaggcgaat atcctctggg gagagtagta gacttactca
     1141 tgggtggcgg caggagccat ttctaccctc aaggcgaaaa ggcttctcca tatggtcacc
     1201 acggtgctag aaaagatgga agagatttaa tcgatgaggc tcaatctaat ggctggcagt
     1261 acgtgggtga ccgtaaaaat tttgattctt tgttgaaaag ccacggtgaa aacgttactt
     1321 tacctttctt gggtctcttt gcagataacg acattccttt tgaaattgac agagatgaaa
     1381 aggagtatcc ttcccttaag gaacaagtca aggtggcttt gggtgccttg gaaaaagctt
     1441 ccaatgagga caaagattct aatgggtttt tcttaatggt ggaaggctcc agaattgatc
     1501 atgctggtca ccaaaacgat ccagcttcgc aggtaaggga ggtgttggct ttcgatgaag
     1561 cattccaata cgttttggag tttgctgaaa attccgatac ggagactgtt ttggtctcta
     1621 cgtcggatca tgaaacgggt ggtttagtca cttcaagaca agtcactgct agctatcctc
     1681 agtatgtctg gtatccacag gtgctcgcca acgccaccca ttcaggtgag tttttgaaaa
     1741 gaaaactagt agattttgtt catgagcata agggcgcaag cagcaaaata gaaaatttca
     1801 tcaaacatga gatcctggaa aaagatttgg gtatttacga ttacacggat agcgatttag
     1861 aaacattaat tcatttggat gacaacgcaa atgctatcca agacaaactg aatgacatgg
     1921 tatcatttag ggcccaaatt ggctggacca cgcatggtca cagcgcagtc gacgtcaaca
     1981 tatatgcata cgccaataag aaagctacat ggtcatatgt cttgaacaat ttacaaggca
     2041 atcacgaaaa tacggaagtt ggccagttct tagaaaactt tttagaattg aacctgaacg
     2101 aagtcactga tttgattagg gacaccaaac acacatcaga ttttgatgct acagaaatag
     2161 ctagtgaagt acaacattat gacgaatact accatgagtt gaccaactga tcctatctaa
     2221 ctctcggcgc gccacttcta aataagcgaa tttcttatga tttatgattt ttattattaa
     2281 ataagttata aaaaaaataa gtgtatacaa attttaaagt gactcttagg ttttaaaacg
     2341 aaaattctta ttcttgagta actctttcct gtaggtcagg ttgctttctc aggtatagta
     2401 tgaggtcgct cttattgacc acacctctac cggcagatcc gctagggata acagggtaat
     2461 atctgcaggt cgacaaccct taatataact tcgtataatg tatgctatac gaagttatta
     2521 ggtctagatg cgaattaaca ctgttctttg atgttagaac aatttaggtt caaagtacga
     2581 gtcacgacat gtaacaaggg aaaaaaagga tattcatttc aataaagttc gttttataaa
     2641 agtatagtac ttgtgagaga aagtaggttt atacataatt ttacaactca ttacgcacac
     2701 tcatggtttt tggccagcga aaacagtttc aaaagattgc tggaagtctg caataatgtc
     2761 atcaataaat tcgataccaa cagagacacg aattaagtcc ttggtaacac cagatgccaa
     2821 cttttctttg tcatttaatt gtttgtgggt agtgaagtat ggagcaatga ctaaggtctt
     2881 ggcatcacca acattggcca agttagaggc aagctttaaa ttgtcaacaa cttgagcacc
     2941 agaaagtttg aatgggtcag tttccttgtc ggcatttggt aagtctttta caccgaaaga
     3001 taagacacca ccgaaaccgt tagatagata cttcttagca ttttcatgat gagaatgaga
     3061 tgctaaacca gggtatgaaa cccaagatac gtatggggat tgttctaacc atttggctaa
     3121 cttcaatgca ttttcaccgt gtctttcagc tctcaaagat aatgtttcaa caccttgtag
     3181 tagcaagaaa gaggcaaatg ggttcatcaa tggacccaaa tctcttaata gttcagttct
     3241 aacatgaacg atgtatgcca agttaccgta ggcttcattg tagatagtac cgtgatatcc
     3301 ttcggcaggt tgagagaatt gagggaactt ttctgggtag tccttccatg ggaacttacc
     3361 agagtcaaca ataataccac cgatagtagt accatgacca ccaatccatt tggtagcaga
     3421 atgtgttaca atatcagcac cgtatttaat tggctgacag aagtaaccac cggcaccaaa
     3481 tgtgttgtca acgacaactg gaataccgtg tttgtgagca attgcaacaa ttttttcaaa
     3541 atccggaaca ttgtactttg gattaccaat ggtttccaaa taaacagcct tggttctttc
     3601 atcaaagacc ttttcgaatt cttctggatt gtcaccttca acaaatctag cctcgatacc
     3661 aaatcttttg aacgagattt tgaactggtt ataagtacca ccgtataagt aagaagtgga
     3721 aacgatgttg tcaccagtgt gtgccaaacc ttggatggca agggtttgag cggcttgacc
     3781 ggaggaaaca gccaaagcag cagcaccacc ttctaaagca gcaattcttt cttccaaaac
     3841 attactggtt gggttttgga aacgggaata gacgtaacct ggaacttcta gaccaaacaa
     3901 ttgcgaacca tgcttagagt tttcgaaaac ataagaagtg gtggcgtaaa ttggtacagc
     3961 tctggatctg tgagcattgt caccagggtt ctcttggccg gcgtgtagtt gaacagtatc
     4021 gaaatgagat ggcattgtat ggatgggggt aatagaattg tatctatgta tctgacgacc
     4081 ctgtattaca cgaaggaaat agtagatgaa aagacaagag agcaagaaaa aggaaaaact
     4141 tcctttctaa cagacgcttt acttaacctt atatatatct tatttttttc atcgagcgtg
     4201 ttcaattgga caaggtgcca ccttttcgac acttcggtta ttatgttaca cagttttcat
     4261 gaggatggcg ccttgactaa cttaattagt catttgccaa ccaccacagt tccccaatat
     4321 cgacagcttc acgtgccatt tgccattttg cgatcatgtg acctcaagag aaaaagcaaa
     4381 aaataatgag agctaaggga ttcgaaccct tgcatccgaa gatatcagag attttagagg
     4441 ttaaatccac ctaaatctga cgccttaaac cactcggcca aactcccatt gtatgttgga
     4501 agaattggat ttaaatggag ctctcgagaa cccttaatat aacttcgtat aatgtatgct
     4561 atacgaagtt attaggtgat atcagatcca ctagtggcct atgcggccgc ggatctgccg
     4621 gtctccctat agtgagtcgt attaatttcg ataagccagg ttaacctgca ttaatgaatc
     4681 ggccaacgcg cggggagagg cggtttgcgt attgggcgct cttccgcttc ctcgctcact
     4741 gactcgctgc gctcggtcgt tcggctgcgg cgagcggtat cagctcactc aaaggcggta
     4801 atacggttat ccacagaatc aggggataac gcaggaaaga acatgtgagc aaaaggccag
     4861 caaaaggcca ggaaccgtaa aaaggccgcg ttgctggcgt ttttccatag gctccgcccc
     4921 cctgacgagc atcacaaaaa tcgacgctca agtcagaggt ggcgaaaccc gacaggacta
     4981 taaagatacc aggcgtttcc ccctggaagc tccctcgtgc gctctcctgt tccgaccctg
     5041 ccgcttaccg gatacctgtc cgcctttctc ccttcgggaa gcgtggcgct ttctcatagc
     5101 tcacgctgta ggtatctcag ttcggtgtag gtcgttcgct ccaagctggg ctgtgtgcac
     5161 gaaccccccg ttcagcccga ccgctgcgcc ttatccggta actatcgtct tgagtccaac
     5221 ccggtaagac acgacttatc gccactggca gcagccactg gtaacaggat tagcagagcg
     5281 aggtatgtag gcggtgctac agagttcttg aagtggtggc ctaactacgg ctacactaga
     5341 agaacagtat ttggtatctg cgctctgctg aagccagtta ccttcggaaa aagagttggt
     5401 agctcttgat ccggcaaaca aaccaccgct ggtagcggtg gtttttttgt ttgcaagcag
     5461 cagattacgc gcagaaaaaa aggatctcaa gaagatcctt tgatcttttc tacggggtct
     5521 gacgctcagt ggaacgaaaa ctcacgttaa gggattttgg tcatgagatt atcaaaaagg
     5581 atcttcacct agatcctttt aaattaaaaa tgaagtttta aatcaatcta aagtatatat
     5641 gagtaaactt ggtctgacag ttaccaatgc ttaatcagtg aggcacctat ctcagcgatc
     5701 tgtctatttc gttcatccat agttgcctga ctccccgtcg tgtagataac tacgatacgg
     5761 gagggcttac catctggccc cagtgctgca atgataccgc gagacccacg ctcaccggct
     5821 ccagatttat cagcaataaa ccagccagcc ggaagggccg agcgcagaag tggtcctgca
     5881 actttatccg cctccatcca gtctattaat tgttgccggg aagctagagt aagtagttcg
     5941 ccagttaata gtttgcgcaa cgttgttgcc attgctacag gcatcgtggt gtcacgctcg
     6001 tcgtttggta tggcttcatt cagctccggt tcccaacgat caaggcgagt tacatgatcc
     6061 cccatgttgt gcaaaaaagc ggttagctcc ttcggtcctc cgatcgttgt cagaagtaag
     6121 ttggccgcag tgttatcact catggttatg gcagcactgc ataattctct tactgtcatg
     6181 ccatccgtaa gatgcttttc tgtgactggt gagtactcaa ccaagtcatt ctgagaatag
     6241 tgtatgcggc gaccgagttg ctcttgcccg gcgtcaatac gggataatac cgcgccacat
     6301 agcagaactt taaaagtgct catcattgga aaacgttctt cggggcgaaa actctcaagg
     6361 atcttaccgc tgttgagatc cagttcgatg taacccactc gtgcacccaa ctgatcttca
     6421 gcatctttta ctttcaccag cgtttctggg tgagcaaaaa caggaaggca aaatgccgca
     6481 aaaaagggaa taagggcgac acggaaatgt tgaatactca tactcttcct ttttcaatat
     6541 tattgaagca tttatcaggg ttattgtctc atgagcggat acatatttga atgtatttag
     6601 aaaaataaac aaataggggt tccgcgcaca tttccccgaa aagtgccacc tgacgtctaa
     6661 gaaaccatta ttatcatgac attaacctat aaaaataggc gtatcacgag gccctttcgt
     6721 ctcgcgcgtt tcggtgatga cggtgaaaac ctctgacaca tgcagctccc ggagacggtc
     6781 acagcttgtc tgtaagcgga tgccgggagc agacaagccc gtcagggcgc gtcagcgggt
     6841 gttggcgggt gtcggggctg gcttaactat gcggcatcag agcagattgt actgagagtg
     6901 caccatatgg acatattgtc gttagaacgc ggctacaatt aatacataac cttatgtatc
     6961 atacacatac gatttaggtg acactata
//
