LOCUS       sequence-424603-        6472 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Expression of 3VSV-Atg8 point mutant. Uses auxotrophic marker 
            MET15(Saccharomyces cerevisiae). .
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 6472)
  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 6472)
  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: ClhN-P1K-3VSV-Atg8(T56A R65A T87A)-MET15
FEATURES             Location/Qualifiers
     source          1..6472
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     CDS             1070..1102
                     /codon_start=1
                     /product="epitope tag from vesicular stomatitis virus G 
                     protein"
                     /label=VSV-G tag
                     /translation="YTDIEMNRLGK"
     CDS             1106..1138
                     /codon_start=1
                     /product="epitope tag from vesicular stomatitis virus G 
                     protein"
                     /label=VSV-G tag
                     /translation="YTDIEMNRLGK"
     CDS             1142..1174
                     /codon_start=1
                     /product="epitope tag from vesicular stomatitis virus G 
                     protein"
                     /label=VSV-G tag
                     /translation="YTDIEMNRLGK"
     terminator      1730..1917
                     /gene="S. cerevisiae ADH1"
                     /label=ADH1 terminator
                     /note="transcription terminator for the S. cerevisiae 
                     alcohol dehydrogenase 1 (ADH1) gene"
     primer_bind     complement(1785..1809)
                     /label=pHybLex-R
                     /note="ADH terminator, reverse primer"
     protein_bind    1969..2002
                     /label=loxP
                     /bound_moiety="Cre recombinase"
                     /note="Cre-mediated recombination occurs in the 8-bp core 
                     sequence (ATGTATGC) (Shaw et al., 2021)."
     CDS             complement(2185..3519)
                     /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(3520..3870)
                     /gene="S. cerevisiae MET17"
                     /gene_synonym="MET15; MET25"
                     /label=MET17 promoter
                     /note="expression is repressed by methionine"
     protein_bind    4023..4056
                     /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(4109..4128)
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        complement(4110..4128)
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     primer_bind     complement(4215..4232)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(4386..4974)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(4466..4485)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(5145..5936,5937..6005))
                     /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     5768..5787
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(6006..6110)
                     /gene="bla"
                     /label=AmpR promoter
     primer_bind     6178..6196
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     primer_bind     complement(6234..6256)
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     primer_bind     6356..6375
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     promoter        join(6456..6472,1..2)
                     /label=SP6 promoter
                     /note="promoter for bacteriophage SP6 RNA polymerase"
     primer_bind     join(6456..6472,1)
                     /label=SP6
                     /note="SP6 promoter, forward primer"
ORIGIN
        1 gaacgcggcc gccagctgaa gcttcgtacg ctgcaggtcg tgggattcct gggttaatta
       61 acatcgaatt cactttggtc atccttcaca gtgatgaatc atacttgaag gatgtagaag
      121 ccttgaaaaa ctacattatc gaggaattaa atgttcgtga tgttgttatc acctccgatg
      181 aggcaaaata tggtgttgag tataaagcag ttgctgattg gcctgtattg ggtaagaagt
      241 tgaaaaaaga cgcaaagaag gttaaagatg ccctaccatc agtcacttct gagcaagtac
      301 gggaatattt ggaaagcgga aagttggagg ttgccggtat tgaactggtt aaaggagatt
      361 tgaatgctat tagaggcttg ccagaatctg ctgtccaagc tggacaagaa accagaaccg
      421 accaagacgt gttaatcatc atggatacaa atatttactc tgaactaaag agtgaaggtc
      481 tcgcaagaga gctggtcaac agaatccaaa aattgagaaa gaagtgtggt ttggaagcca
      541 ccgacgatgt tttagtggag tacgaattag ttaaagatac tatcgacttt gaagccattg
      601 tcaaagaaca ttttgatatg ttaagcaaga cctgtagatc cgacattgcc aaatatgacg
      661 gctcaaagac agacccaatt ggtgatgaag aacaatctat taatgacacc attttcaaat
      721 taaaagtgtt caaattatga aaacaactca tataaatacg tacaaatttt tctctactcg
      781 aagtgatata gatgtatatg tgtaagttta cgtttaagtt agagtcatgt aatgctaact
      841 gtctccaccg ataatgttgt ataatacccg tgaaatcata gcacatgata tatcatcacc
      901 cggaggccgg ttattttcgg cggcggcaaa aatatttggt ataattatgg aaatacaaaa
      961 aggggaacca ttaaaggttg aggaggggat tgataagaga atctaataat tgtaaagttg
     1021 agaaaatcat aataaaataa ttactagaga catgaagtct gctagcggat acaccgatat
     1081 cgagatgaac cgtttgggta agggctacac tgacattgaa atgaatcgtt tgggtaaggg
     1141 ctacactgac attgaaatga atcgtttggg taagcctagg atgaagtcta catttaagtc
     1201 tgaatatcca tttgaaaaaa ggaaggcgga gtcggagagg attgctgaca ggttcaagaa
     1261 taggatacct gtgatttgcg aaaaagctga aaagtcagat attccagaga ttgataagcg
     1321 taaatatcta gttcctgctg accttgcggt agggcaattt gtttatgtta tagcgaagag
     1381 aattatgcta ccccctgaga aggccatctt catttttgtc aatgatactt tgccacctgc
     1441 tgcggcgttg atgtctgcca tatatcaaga acacaaggat aaggacgggt ttttgtatgt
     1501 cacttactca ggagaaaata catttggcag gtagtctttt atatgaaaag aaatgaagcg
     1561 ttaacatcta aaatcgaaat tatagggact cattagccat ctattgttcc agtataagaa
     1621 tgtttacata aaaaattcga aaaaaaaaga taaaataagg gaaaaatata tatatattca
     1681 ttaagaagtt ttgtacacaa cccgggtgag gcgcgccact tctaaataag cgaatttctt
     1741 atgatttatg atttttatta ttaaataagt tataaaaaaa ataagtgtat acaaatttta
     1801 aagtgactct taggttttaa aacgaaaatt cttattcttg agtaactctt tcctgtaggt
     1861 caggttgctt tctcaggtat agtatgaggt cgctcttatt gaccacacct ctaccggcag
     1921 atccgctagg gataacaggg taatatctgc aggtcgacaa cccttaatat aacttcgtat
     1981 aatgtatgct atacgaagtt attaggtcta gatgcgaatt aacactgttc tttgatgtta
     2041 gaacaattta ggttcaaagt acgagtcacg acatgtaaca agggaaaaaa aggatattca
     2101 tttcaataaa gttcgtttta taaaagtata gtacttgtga gagaaagtag gtttatacat
     2161 aattttacaa ctcattacgc acactcatgg tttttggcca gcgaaaacag tttcaaaaga
     2221 ttgctggaag tctgcaataa tgtcatcaat aaattcgata ccaacagaga cacgaattaa
     2281 gtccttggta acaccagatg ccaacttttc tttgtcattt aattgtttgt gggtagtgaa
     2341 gtatggagca atgactaagg tcttggcatc accaacattg gccaagttag aggcaagctt
     2401 taaattgtca acaacttgag caccagaaag tttgaatggg tcagtttcct tgtcggcatt
     2461 tggtaagtct tttacaccga aagataagac accaccgaaa ccgttagata gatacttctt
     2521 agcattttca tgatgagaat gagatgctaa accagggtat gaaacccaag atacgtatgg
     2581 ggattgttct aaccatttgg ctaacttcaa tgcattttca ccgtgtcttt cagctctcaa
     2641 agataatgtt tcaacacctt gtagtagcaa gaaagaggca aatgggttca tcaatggacc
     2701 caaatctctt aatagttcag ttctaacatg aacgatgtat gccaagttac cgtaggcttc
     2761 attgtagata gtaccgtgat atccttcggc aggttgagag aattgaggga acttttctgg
     2821 gtagtccttc catgggaact taccagagtc aacaataata ccaccgatag tagtaccatg
     2881 accaccaatc catttggtag cagaatgtgt tacaatatca gcaccgtatt taattggctg
     2941 acagaagtaa ccaccggcac caaatgtgtt gtcaacgaca actggaatac cgtgtttgtg
     3001 agcaattgca acaatttttt caaaatccgg aacattgtac tttggattac caatggtttc
     3061 caaataaaca gccttggttc tttcatcaaa gaccttttcg aattcttctg gattgtcacc
     3121 ttcaacaaat ctagcctcga taccaaatct tttgaacgag attttgaact ggttataagt
     3181 accaccgtat aagtaagaag tggaaacgat gttgtcacca gtgtgtgcca aaccttggat
     3241 ggcaagggtt tgagcggctt gaccggagga aacagccaaa gcagcagcac caccttctaa
     3301 agcagcaatt ctttcttcca aaacattact ggttgggttt tggaaacggg aatagacgta
     3361 acctggaact tctagaccaa acaattgcga accatgctta gagttttcga aaacataaga
     3421 agtggtggcg taaattggta cagctctgga tctgtgagca ttgtcaccag ggttctcttg
     3481 gccggcgtgt agttgaacag tatcgaaatg agatggcatt gtatggatgg gggtaataga
     3541 attgtatcta tgtatctgac gaccctgtat tacacgaagg aaatagtaga tgaaaagaca
     3601 agagagcaag aaaaaggaaa aacttccttt ctaacagacg ctttacttaa ccttatatat
     3661 atcttatttt tttcatcgag cgtgttcaat tggacaaggt gccacctttt cgacacttcg
     3721 gttattatgt tacacagttt tcatgaggat ggcgccttga ctaacttaat tagtcatttg
     3781 ccaaccacca cagttcccca atatcgacag cttcacgtgc catttgccat tttgcgatca
     3841 tgtgacctca agagaaaaag caaaaaataa tgagagctaa gggattcgaa cccttgcatc
     3901 cgaagatatc agagatttta gaggttaaat ccacctaaat ctgacgcctt aaaccactcg
     3961 gccaaactcc cattgtatgt tggaagaatt ggatttaaat ggagctctcg agaaccctta
     4021 atataacttc gtataatgta tgctatacga agttattagg tgatatcaga tccactagtg
     4081 gcctatgcgg ccgcggatct gccggtctcc ctatagtgag tcgtattaat ttcgataagc
     4141 caggttaacc tgcattaatg aatcggccaa cgcgcgggga gaggcggttt gcgtattggg
     4201 cgctcttccg cttcctcgct cactgactcg ctgcgctcgg tcgttcggct gcggcgagcg
     4261 gtatcagctc actcaaaggc ggtaatacgg ttatccacag aatcagggga taacgcagga
     4321 aagaacatgt gagcaaaagg ccagcaaaag gccaggaacc gtaaaaaggc cgcgttgctg
     4381 gcgtttttcc ataggctccg cccccctgac gagcatcaca aaaatcgacg ctcaagtcag
     4441 aggtggcgaa acccgacagg actataaaga taccaggcgt ttccccctgg aagctccctc
     4501 gtgcgctctc ctgttccgac cctgccgctt accggatacc tgtccgcctt tctcccttcg
     4561 ggaagcgtgg cgctttctca tagctcacgc tgtaggtatc tcagttcggt gtaggtcgtt
     4621 cgctccaagc tgggctgtgt gcacgaaccc cccgttcagc ccgaccgctg cgccttatcc
     4681 ggtaactatc gtcttgagtc caacccggta agacacgact tatcgccact ggcagcagcc
     4741 actggtaaca ggattagcag agcgaggtat gtaggcggtg ctacagagtt cttgaagtgg
     4801 tggcctaact acggctacac tagaagaaca gtatttggta tctgcgctct gctgaagcca
     4861 gttaccttcg gaaaaagagt tggtagctct tgatccggca aacaaaccac cgctggtagc
     4921 ggtggttttt ttgtttgcaa gcagcagatt acgcgcagaa aaaaaggatc tcaagaagat
     4981 cctttgatct tttctacggg gtctgacgct cagtggaacg aaaactcacg ttaagggatt
     5041 ttggtcatga gattatcaaa aaggatcttc acctagatcc ttttaaatta aaaatgaagt
     5101 tttaaatcaa tctaaagtat atatgagtaa acttggtctg acagttacca atgcttaatc
     5161 agtgaggcac ctatctcagc gatctgtcta tttcgttcat ccatagttgc ctgactcccc
     5221 gtcgtgtaga taactacgat acgggagggc ttaccatctg gccccagtgc tgcaatgata
     5281 ccgcgagacc cacgctcacc ggctccagat ttatcagcaa taaaccagcc agccggaagg
     5341 gccgagcgca gaagtggtcc tgcaacttta tccgcctcca tccagtctat taattgttgc
     5401 cgggaagcta gagtaagtag ttcgccagtt aatagtttgc gcaacgttgt tgccattgct
     5461 acaggcatcg tggtgtcacg ctcgtcgttt ggtatggctt cattcagctc cggttcccaa
     5521 cgatcaaggc gagttacatg atcccccatg ttgtgcaaaa aagcggttag ctccttcggt
     5581 cctccgatcg ttgtcagaag taagttggcc gcagtgttat cactcatggt tatggcagca
     5641 ctgcataatt ctcttactgt catgccatcc gtaagatgct tttctgtgac tggtgagtac
     5701 tcaaccaagt cattctgaga atagtgtatg cggcgaccga gttgctcttg cccggcgtca
     5761 atacgggata ataccgcgcc acatagcaga actttaaaag tgctcatcat tggaaaacgt
     5821 tcttcggggc gaaaactctc aaggatctta ccgctgttga gatccagttc gatgtaaccc
     5881 actcgtgcac ccaactgatc ttcagcatct tttactttca ccagcgtttc tgggtgagca
     5941 aaaacaggaa ggcaaaatgc cgcaaaaaag ggaataaggg cgacacggaa atgttgaata
     6001 ctcatactct tcctttttca atattattga agcatttatc agggttattg tctcatgagc
     6061 ggatacatat ttgaatgtat ttagaaaaat aaacaaatag gggttccgcg cacatttccc
     6121 cgaaaagtgc cacctgacgt ctaagaaacc attattatca tgacattaac ctataaaaat
     6181 aggcgtatca cgaggccctt tcgtctcgcg cgtttcggtg atgacggtga aaacctctga
     6241 cacatgcagc tcccggagac ggtcacagct tgtctgtaag cggatgccgg gagcagacaa
     6301 gcccgtcagg gcgcgtcagc gggtgttggc gggtgtcggg gctggcttaa ctatgcggca
     6361 tcagagcaga ttgtactgag agtgcaccat atggacatat tgtcgttaga acgcggctac
     6421 aattaataca taaccttatg tatcatacac atacgattta ggtgacacta ta
//
