LOCUS       sequence-423101-        6657 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Expression of GFP-Vma13 under CPU1 promoter. .
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 6657)
  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 6657)
  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-GFP-Vma13-Ura
FEATURES             Location/Qualifiers
     source          1..6657
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     promoter        54..288
                     /gene="S. cerevisiae CUP1"
                     /label=CUP1 promoter
                     /note="inducible metallothionein promoter, regulated by 
                     Cu2+"
     CDS             310..1020
                     /codon_start=1
                     /product="S65T variant of Aequorea victoria green 
                     fluorescent protein (Heim et al., 1995)"
                     /label=GFP (S65T)
                     /note="excitable with blue light"
                     /translation="SKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTLKF
                     ICTTGKLPVPWPTLVTTFTYGVQCFSRYPDHMKRHDFFKSAMPEGYVQERTIFFKDDGN
                     YKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIKVN
                     FKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSALSKDPNEKRDHMVLLEF
                     VTAAGITHGMDELYK"
     primer_bind     complement(337..365)
                     /label=GFP-R
                     /note="GFP, reverse primer. Does NOT anneal to EGFP"
     primer_bind     960..980
                     /label=GFP-F
                     /note="GFP, forward primer. Does NOT anneal to EGFP"
     terminator      2484..2671
                     /gene="S. cerevisiae ADH1"
                     /label=ADH1 terminator
                     /note="transcription terminator for the S. cerevisiae 
                     alcohol dehydrogenase 1 (ADH1) gene"
     primer_bind     complement(2539..2563)
                     /label=pHybLex-R
                     /note="ADH terminator, reverse primer"
     protein_bind    2723..2756
                     /label=loxP
                     /bound_moiety="Cre recombinase"
                     /note="Cre-mediated recombination occurs in the 8-bp core 
                     sequence (ATGTATGC) (Shaw et al., 2021)."
     protein_bind    4208..4241
                     /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(4294..4313)
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        complement(4295..4313)
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     primer_bind     complement(4400..4417)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(4571..5159)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(4651..4670)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(5330..6121,6122..6190))
                     /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     5953..5972
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(6191..6295)
                     /gene="bla"
                     /label=AmpR promoter
     primer_bind     6363..6381
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     primer_bind     complement(6419..6441)
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     primer_bind     6541..6560
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     promoter        join(6641..6657,1..2)
                     /label=SP6 promoter
                     /note="promoter for bacteriophage SP6 RNA polymerase"
     primer_bind     join(6641..6657,1)
                     /label=SP6
                     /note="SP6 promoter, forward primer"
ORIGIN
        1 gaacgcggcc gccagctgaa gcttcgtacg ctgcaggtcg tgggattcct gggctagtta
       61 gaaaaagaca tttttgctgt cagtcactgt caagagattc ttttgctggc atttcttcta
      121 gaagcaaaaa gagcgatgcg tcttttccgc tgaaccgttc cagcaaaaaa gactaccaac
      181 gcaatatgga ttgtcagaat catataaaag agaagcaaat aactccttgt cttgtatcaa
      241 ttgcattata atatcttctt gttagtgcaa tatcatatag aagtcatcat gttaattaac
      301 atcgctagca gtaaaggaga agaacttttc actggagttg tcccaattct tgttgaatta
      361 gatggtgatg ttaatgggca caaattttct gtcagtggag agggtgaagg tgatgcaaca
      421 tacggaaaac ttacccttaa atttatttgc actactggaa aactacctgt tccatggcca
      481 acacttgtca ctactttcac ttatggtgtt caatgctttt caagataccc agatcatatg
      541 aaacggcatg actttttcaa gagtgccatg cccgaaggtt atgtacagga aagaactata
      601 tttttcaaag atgacgggaa ctacaagaca cgtgctgaag tcaagtttga aggtgatacc
      661 cttgttaata gaatcgagtt aaaaggtatt gattttaaag aagatggaaa cattcttgga
      721 cacaaattgg aatacaacta taactcacac aatgtataca tcatggcaga caaacaaaag
      781 aatggaatca aagttaactt caaaattaga cacaacattg aagatggaag cgttcaacta
      841 gcagaccatt atcaacaaaa tactccaatt ggcgatggcc ctgtcctttt accagacaac
      901 cattacctgt ccacacaatc tgccctttcg aaagatccca acgaaaagag agaccacatg
      961 gtccttcttg agtttgtaac agctgctggg attacacatg gcatggatga actatacaaa
     1021 cctaggatgg gcgcaaccaa aattttaatg gacagtactc atttcaatga gatccgtagt
     1081 ataatccgtt cgaggtcagt ggcatgggac gccttagcca gatctgagga attgagcgaa
     1141 attgatgcgt ctactgcaaa agcgttagaa tccattctgg tgaagaagaa cattggtgac
     1201 ggtttatcat cttcgaacaa tgcacattcc gggttcaaag tgaatggcaa gacgttgata
     1261 ccattaattc acttactttc cacctcagac aacgaagact gcaaaaaatc tgtgcagaac
     1321 ctaatagctg aattgttatc gtctgacaag tatggagacg ataccgtgaa gtttttccaa
     1381 gaagacccca agcaattgga acaattattt gatgtgtcac tcaagggaga cttccagact
     1441 gtgctaatct ctgggttcaa cgtggtctca ctcttagtgc aaaatgggtt gcacaatgtg
     1501 aaactagtgg aaaagctgtt gaaaaacaac aacttgatca atatcttgca aaacattgag
     1561 cagatggaca cttgttacgt gtgcatcaga ctattgcaag aactggccgt gataccagag
     1621 tatcgtgacg tgatatggtt gcatgagaag aagttcatgc ccaccttatt caagatcctg
     1681 caacgtgcca cggactctca attggccacg cggatagttg caacaaactc caaccacctg
     1741 ggtattcaat tgcagtacca ctctttacta ttgatatggt tgctgacctt taacccagtt
     1801 tttgcaaacg agctagtcca gaaatacttg agtgatttct tggacctctt gaaattggtt
     1861 aagataacca taaaggagaa agtgtccaga ttgtgcatat ccatcatcct gcaatgttgc
     1921 tccacgcgcg tcaagcagca caagaaggtg attaagcaac ttttgttgct cggcaacgcg
     1981 ttgcccaccg tacagagctt gagcgaaaga aagtattccg acgaagaatt gcgtcaagac
     2041 atcagcaacc tcaaggaaat cctagaaaac gagtaccaag aattgacctc cttcgatgaa
     2101 tacgtcgccg aattggactc caagttgctg tgctggtctc caccacatgt cgacaacggt
     2161 ttctggtccg ataacattga cgagttcaag aaggacaact acaagatctt tagacaattg
     2221 atcgaactct tgcaagcaaa ggtccgtaac ggcgacgtca acgcgaaaca agaaaagatc
     2281 attatccaag tcgccttgaa cgacatcact cacgtggtcg agcttctacc agagagcatc
     2341 gacgttctcg acaagactgg cggcaaagcc gacatcatgg agttgctgaa ccattcagat
     2401 tctagggtga aatacgaggc cctcaaggcc acgcaggcaa tcattggata taccttcaaa
     2461 taaggcgcgc cacttctaaa taagcgaatt tcttatgatt tatgattttt attattaaat
     2521 aagttataaa aaaaataagt gtatacaaat tttaaagtga ctcttaggtt ttaaaacgaa
     2581 aattcttatt cttgagtaac tctttcctgt aggtcaggtt gctttctcag gtatagtatg
     2641 aggtcgctct tattgaccac acctctaccg gcagatccgc tagggataac agggtaatat
     2701 ctgcaggtcg acaaccctta atataacttc gtataatgta tgctatacga agttattagg
     2761 tctagagatc ccaatacaac agatcacgtg atctttttgt aagatgaagt tgaagtagtg
     2821 ttgcaccgtg ccaatgcagg tggctattag attaaatatg tgatttgttc tattaagttt
     2881 cctgtataat taatggggag cgctgattct cttttggtac gcttcccatc cagcatttct
     2941 gtatctttca ccttcaacct taggatctct acccttggcg aaaagtcctc tgccaacaat
     3001 gatgatatct gatccaccac ttacaacttc gtcgacggtt ctgtactgct gacccaatgc
     3061 atcgcctttg tcgtctaaac ctacacctgg ggtcatgatt agccaatcaa acccttcttc
     3121 tcttcctccc atatcgttct gagcaatgaa cccaataacg aaatctttat cactctttgc
     3181 aatatcaacg gtacccttag tatattcacc gtgtgctaga gaacccttgg aagacaattc
     3241 agcaagcatc aataatcccc ttggttcttt ggtgacctct tgcgcacctt gtttcaagcc
     3301 agcaacaata ccagcaccag taaccccgtg ggcgttggtg atatcagacc attctgcgat
     3361 acggtaaacg cccgatgtat attgtaattt gactgtgtta ccgatatcgg cgaattttct
     3421 gtcctcaaat atcaagaact tgtatttctc tgccaatgct ttcaatggaa cgacagtacc
     3481 ctcataactg aaatcatcca agatatcaac gtgtgttttc aaaaggcaaa tgtatggacc
     3541 caacgtttca acaagtttca atagctcatc agtcgaacga acgtcaagag aagcacacaa
     3601 attggtcttc ttttcatcca ttaaacgtaa aagtttcgat gcaaccggac ttgcatgagt
     3661 ctcagctcta ctggtatatg attttgtgga catggtgcaa ctaattgacg ggagtgtatt
     3721 gacgctggcg tactggcttt cacaaaatgg cccaatcaca accacatctt agatagttga
     3781 aatgacttta gataacatca attgagatga gcttaatcat gtcaaagcta aaagtgtcac
     3841 catgaacgac aattcttaag caaatcacgt gatatagatc cacgaataac caccatttga
     3901 tgctcgaggc aagtaatgtg tgtaaaaaaa tgcgttacca ccatccaatg cagaccgatc
     3961 ttctacccag aatcacatat atttatgtac cgagtacctt ttttctatct tccaattgct
     4021 tctcccatat gattgtctcc gtaagctcga aatttctaag ttggatttta atcttcacgc
     4081 aggatgacag ttcgatgagc ttctgaggag tgtttagaac ataatcattt atccatggtc
     4141 tatctcttct tgtcgctttt tctcctcgat agaacctaaa taaaacgagc tctcgagaac
     4201 ccttaatata acttcgtata atgtatgcta tacgaagtta ttaggtgata tcagatccac
     4261 tagtggccta tgcggccgcg gatctgccgg tctccctata gtgagtcgta ttaatttcga
     4321 taagccaggt taacctgcat taatgaatcg gccaacgcgc ggggagaggc ggtttgcgta
     4381 ttgggcgctc ttccgcttcc tcgctcactg actcgctgcg ctcggtcgtt cggctgcggc
     4441 gagcggtatc agctcactca aaggcggtaa tacggttatc cacagaatca ggggataacg
     4501 caggaaagaa catgtgagca aaaggccagc aaaaggccag gaaccgtaaa aaggccgcgt
     4561 tgctggcgtt tttccatagg ctccgccccc ctgacgagca tcacaaaaat cgacgctcaa
     4621 gtcagaggtg gcgaaacccg acaggactat aaagatacca ggcgtttccc cctggaagct
     4681 ccctcgtgcg ctctcctgtt ccgaccctgc cgcttaccgg atacctgtcc gcctttctcc
     4741 cttcgggaag cgtggcgctt tctcatagct cacgctgtag gtatctcagt tcggtgtagg
     4801 tcgttcgctc caagctgggc tgtgtgcacg aaccccccgt tcagcccgac cgctgcgcct
     4861 tatccggtaa ctatcgtctt gagtccaacc cggtaagaca cgacttatcg ccactggcag
     4921 cagccactgg taacaggatt agcagagcga ggtatgtagg cggtgctaca gagttcttga
     4981 agtggtggcc taactacggc tacactagaa gaacagtatt tggtatctgc gctctgctga
     5041 agccagttac cttcggaaaa agagttggta gctcttgatc cggcaaacaa accaccgctg
     5101 gtagcggtgg tttttttgtt tgcaagcagc agattacgcg cagaaaaaaa ggatctcaag
     5161 aagatccttt gatcttttct acggggtctg acgctcagtg gaacgaaaac tcacgttaag
     5221 ggattttggt catgagatta tcaaaaagga tcttcaccta gatcctttta aattaaaaat
     5281 gaagttttaa atcaatctaa agtatatatg agtaaacttg gtctgacagt taccaatgct
     5341 taatcagtga ggcacctatc tcagcgatct gtctatttcg ttcatccata gttgcctgac
     5401 tccccgtcgt gtagataact acgatacggg agggcttacc atctggcccc agtgctgcaa
     5461 tgataccgcg agacccacgc tcaccggctc cagatttatc agcaataaac cagccagccg
     5521 gaagggccga gcgcagaagt ggtcctgcaa ctttatccgc ctccatccag tctattaatt
     5581 gttgccggga agctagagta agtagttcgc cagttaatag tttgcgcaac gttgttgcca
     5641 ttgctacagg catcgtggtg tcacgctcgt cgtttggtat ggcttcattc agctccggtt
     5701 cccaacgatc aaggcgagtt acatgatccc ccatgttgtg caaaaaagcg gttagctcct
     5761 tcggtcctcc gatcgttgtc agaagtaagt tggccgcagt gttatcactc atggttatgg
     5821 cagcactgca taattctctt actgtcatgc catccgtaag atgcttttct gtgactggtg
     5881 agtactcaac caagtcattc tgagaatagt gtatgcggcg accgagttgc tcttgcccgg
     5941 cgtcaatacg ggataatacc gcgccacata gcagaacttt aaaagtgctc atcattggaa
     6001 aacgttcttc ggggcgaaaa ctctcaagga tcttaccgct gttgagatcc agttcgatgt
     6061 aacccactcg tgcacccaac tgatcttcag catcttttac tttcaccagc gtttctgggt
     6121 gagcaaaaac aggaaggcaa aatgccgcaa aaaagggaat aagggcgaca cggaaatgtt
     6181 gaatactcat actcttcctt tttcaatatt attgaagcat ttatcagggt tattgtctca
     6241 tgagcggata catatttgaa tgtatttaga aaaataaaca aataggggtt ccgcgcacat
     6301 ttccccgaaa agtgccacct gacgtctaag aaaccattat tatcatgaca ttaacctata
     6361 aaaataggcg tatcacgagg ccctttcgtc tcgcgcgttt cggtgatgac ggtgaaaacc
     6421 tctgacacat gcagctcccg gagacggtca cagcttgtct gtaagcggat gccgggagca
     6481 gacaagcccg tcagggcgcg tcagcgggtg ttggcgggtg tcggggctgg cttaactatg
     6541 cggcatcaga gcagattgta ctgagagtgc accatatgga catattgtcg ttagaacgcg
     6601 gctacaatta atacataacc ttatgtatca tacacatacg atttaggtga cactata
//
