LOCUS       sequence-423113-        6302 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Expression of 3HA-Atg8∆R. This variant bypasses the need of Atg4 for
            initial C-terminal processing..
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 6302)
  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 6302)
  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: P1K-3HA-Atg8∆R-MET15
FEATURES             Location/Qualifiers
     source          1..6302
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     CDS             1087..1176
                     /codon_start=1
                     /product="three tandem HA epitope tags"
                     /label=3xHA
                     /translation="YPYDVPDYAGYPYDVPDYAGSYPYDVPDYA"
     terminator      1560..1747
                     /gene="S. cerevisiae ADH1"
                     /label=ADH1 terminator
                     /note="transcription terminator for the S. cerevisiae 
                     alcohol dehydrogenase 1 (ADH1) gene"
     primer_bind     complement(1615..1639)
                     /label=pHybLex-R
                     /note="ADH terminator, reverse primer"
     protein_bind    1799..1832
                     /label=loxP
                     /bound_moiety="Cre recombinase"
                     /note="Cre-mediated recombination occurs in the 8-bp core 
                     sequence (ATGTATGC) (Shaw et al., 2021)."
     CDS             complement(2015..3349)
                     /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(3350..3700)
                     /gene="S. cerevisiae MET17"
                     /gene_synonym="MET15; MET25"
                     /label=MET17 promoter
                     /note="expression is repressed by methionine"
     protein_bind    3853..3886
                     /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(3939..3958)
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        complement(3940..3958)
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     primer_bind     complement(4045..4062)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(4216..4804)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(4296..4315)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(4975..5766,5767..5835))
                     /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     5598..5617
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(5836..5940)
                     /gene="bla"
                     /label=AmpR promoter
     primer_bind     6008..6026
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     primer_bind     complement(6064..6086)
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     primer_bind     6186..6205
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     promoter        join(6286..6302,1..2)
                     /label=SP6 promoter
                     /note="promoter for bacteriophage SP6 RNA polymerase"
     primer_bind     join(6286..6302,1)
                     /label=SP6
                     /note="SP6 promoter, forward primer"
ORIGIN
        1 gaacgcggcc gccagctgaa gcttcgtacg ctgcaggtcg tgggattcct gggttaatta
       61 acatcgctag cccttaaaaa ctccattgaa gactttggtc atccttcaca gtgatgaatc
      121 atacttgaag gatgtagaag ccttgaaaaa ctacattatc gaggaattaa atgttcgtga
      181 tgttgttatc acctccgatg aggcaaaata tggtgttgag tataaagcag ttgctgattg
      241 gcctgtattg ggtaagaagt tgaaaaaaga cgcaaagaag gttaaagatg ccctaccatc
      301 agtcacttct gagcaagtac gggaatattt ggaaagcgga aagttggagg ttgccggtat
      361 tgaactggtt aaaggagatt tgaatgctat tagaggcttg ccagaatctg ctgtccaagc
      421 tggacaagaa accagaaccg accaagacgt gttaatcatc atggatacaa atatttactc
      481 tgaactaaag agtgaaggtc tcgcaagaga gctggtcaac agaatccaaa aattgagaaa
      541 gaagtgtggt ttggaagcca ccgacgatgt tttagtggag tacgaattag ttaaagatac
      601 tatcgacttt gaagccattg tcaaagaaca ttttgatatg ttaagcaaga cctgtagatc
      661 cgacattgcc aaatatgacg gctcaaagac agacccaatt ggtgatgaag aacaatctat
      721 taatgacacc attttcaaat taaaagtgtt caaattatga aaacaactca tataaatacg
      781 tacaaatttt tctctactcg aagtgatata gatgtatatg tgtaagttta cgtttaagtt
      841 agagtcatgt aatgctaact gtctccaccg ataatgttgt ataatacccg tgaaatcata
      901 gcacatgata tatcatcacc cggaggccgg ttattttcgg cggcggcaaa aatatttggt
      961 ataattatgg aaatacaaaa aggggaacca ttaaaggttg aggaggggat tgataagaga
     1021 atctaataat tgtaaagttg agaaaatcat aataaaataa ttactagaga catgagatct
     1081 atcttttacc catacgatgt tcctgactat gcgggctatc cctatgacgt cccggactat
     1141 gcaggatcct atccatatga cgttccagat tacgctgctc agtgcagatc taagtctaca
     1201 tttaagtctg aatatccatt tgaaaaaagg aaggcggagt cggagaggat tgctgacagg
     1261 ttcaagaata ggatacctgt gatttgcgaa aaagctgaaa agtcagatat tccagagatt
     1321 gataagcgta aatatctagt tcctgctgac cttaccgtag ggcaatttgt ttatgttata
     1381 agaaagagaa ttatgctacc ccctgagaag gccatcttca tttttgtcaa tgatactttg
     1441 ccacctactg cggcgttgat gtctgccata tatcaagaac acaaggataa ggacgggttt
     1501 ttgtatgtca cttactcagg agaaaataca tttggctgag gcgcgccact tctaaataag
     1561 cgaatttctt atgatttatg atttttatta ttaaataagt tataaaaaaa ataagtgtat
     1621 acaaatttta aagtgactct taggttttaa aacgaaaatt cttattcttg agtaactctt
     1681 tcctgtaggt caggttgctt tctcaggtat agtatgaggt cgctcttatt gaccacacct
     1741 ctaccggcag atccgctagg gataacaggg taatatctgc aggtcgacaa cccttaatat
     1801 aacttcgtat aatgtatgct atacgaagtt attaggtcta gatgcgaatt aacactgttc
     1861 tttgatgtta gaacaattta ggttcaaagt acgagtcacg acatgtaaca agggaaaaaa
     1921 aggatattca tttcaataaa gttcgtttta taaaagtata gtacttgtga gagaaagtag
     1981 gtttatacat aattttacaa ctcattacgc acactcatgg tttttggcca gcgaaaacag
     2041 tttcaaaaga ttgctggaag tctgcaataa tgtcatcaat aaattcgata ccaacagaga
     2101 cacgaattaa gtccttggta acaccagatg ccaacttttc tttgtcattt aattgtttgt
     2161 gggtagtgaa gtatggagca atgactaagg tcttggcatc accaacattg gccaagttag
     2221 aggcaagctt taaattgtca acaacttgag caccagaaag tttgaatggg tcagtttcct
     2281 tgtcggcatt tggtaagtct tttacaccga aagataagac accaccgaaa ccgttagata
     2341 gatacttctt agcattttca tgatgagaat gagatgctaa accagggtat gaaacccaag
     2401 atacgtatgg ggattgttct aaccatttgg ctaacttcaa tgcattttca ccgtgtcttt
     2461 cagctctcaa agataatgtt tcaacacctt gtagtagcaa gaaagaggca aatgggttca
     2521 tcaatggacc caaatctctt aatagttcag ttctaacatg aacgatgtat gccaagttac
     2581 cgtaggcttc attgtagata gtaccgtgat atccttcggc aggttgagag aattgaggga
     2641 acttttctgg gtagtccttc catgggaact taccagagtc aacaataata ccaccgatag
     2701 tagtaccatg accaccaatc catttggtag cagaatgtgt tacaatatca gcaccgtatt
     2761 taattggctg acagaagtaa ccaccggcac caaatgtgtt gtcaacgaca actggaatac
     2821 cgtgtttgtg agcaattgca acaatttttt caaaatccgg aacattgtac tttggattac
     2881 caatggtttc caaataaaca gccttggttc tttcatcaaa gaccttttcg aattcttctg
     2941 gattgtcacc ttcaacaaat ctagcctcga taccaaatct tttgaacgag attttgaact
     3001 ggttataagt accaccgtat aagtaagaag tggaaacgat gttgtcacca gtgtgtgcca
     3061 aaccttggat ggcaagggtt tgagcggctt gaccggagga aacagccaaa gcagcagcac
     3121 caccttctaa agcagcaatt ctttcttcca aaacattact ggttgggttt tggaaacggg
     3181 aatagacgta acctggaact tctagaccaa acaattgcga accatgctta gagttttcga
     3241 aaacataaga agtggtggcg taaattggta cagctctgga tctgtgagca ttgtcaccag
     3301 ggttctcttg gccggcgtgt agttgaacag tatcgaaatg agatggcatt gtatggatgg
     3361 gggtaataga attgtatcta tgtatctgac gaccctgtat tacacgaagg aaatagtaga
     3421 tgaaaagaca agagagcaag aaaaaggaaa aacttccttt ctaacagacg ctttacttaa
     3481 ccttatatat atcttatttt tttcatcgag cgtgttcaat tggacaaggt gccacctttt
     3541 cgacacttcg gttattatgt tacacagttt tcatgaggat ggcgccttga ctaacttaat
     3601 tagtcatttg ccaaccacca cagttcccca atatcgacag cttcacgtgc catttgccat
     3661 tttgcgatca tgtgacctca agagaaaaag caaaaaataa tgagagctaa gggattcgaa
     3721 cccttgcatc cgaagatatc agagatttta gaggttaaat ccacctaaat ctgacgcctt
     3781 aaaccactcg gccaaactcc cattgtatgt tggaagaatt ggatttaaat ggagctctcg
     3841 agaaccctta atataacttc gtataatgta tgctatacga agttattagg tgatatcaga
     3901 tccactagtg gcctatgcgg ccgcggatct gccggtctcc ctatagtgag tcgtattaat
     3961 ttcgataagc caggttaacc tgcattaatg aatcggccaa cgcgcgggga gaggcggttt
     4021 gcgtattggg cgctcttccg cttcctcgct cactgactcg ctgcgctcgg tcgttcggct
     4081 gcggcgagcg gtatcagctc actcaaaggc ggtaatacgg ttatccacag aatcagggga
     4141 taacgcagga aagaacatgt gagcaaaagg ccagcaaaag gccaggaacc gtaaaaaggc
     4201 cgcgttgctg gcgtttttcc ataggctccg cccccctgac gagcatcaca aaaatcgacg
     4261 ctcaagtcag aggtggcgaa acccgacagg actataaaga taccaggcgt ttccccctgg
     4321 aagctccctc gtgcgctctc ctgttccgac cctgccgctt accggatacc tgtccgcctt
     4381 tctcccttcg ggaagcgtgg cgctttctca tagctcacgc tgtaggtatc tcagttcggt
     4441 gtaggtcgtt cgctccaagc tgggctgtgt gcacgaaccc cccgttcagc ccgaccgctg
     4501 cgccttatcc ggtaactatc gtcttgagtc caacccggta agacacgact tatcgccact
     4561 ggcagcagcc actggtaaca ggattagcag agcgaggtat gtaggcggtg ctacagagtt
     4621 cttgaagtgg tggcctaact acggctacac tagaagaaca gtatttggta tctgcgctct
     4681 gctgaagcca gttaccttcg gaaaaagagt tggtagctct tgatccggca aacaaaccac
     4741 cgctggtagc ggtggttttt ttgtttgcaa gcagcagatt acgcgcagaa aaaaaggatc
     4801 tcaagaagat cctttgatct tttctacggg gtctgacgct cagtggaacg aaaactcacg
     4861 ttaagggatt ttggtcatga gattatcaaa aaggatcttc acctagatcc ttttaaatta
     4921 aaaatgaagt tttaaatcaa tctaaagtat atatgagtaa acttggtctg acagttacca
     4981 atgcttaatc agtgaggcac ctatctcagc gatctgtcta tttcgttcat ccatagttgc
     5041 ctgactcccc gtcgtgtaga taactacgat acgggagggc ttaccatctg gccccagtgc
     5101 tgcaatgata ccgcgagacc cacgctcacc ggctccagat ttatcagcaa taaaccagcc
     5161 agccggaagg gccgagcgca gaagtggtcc tgcaacttta tccgcctcca tccagtctat
     5221 taattgttgc cgggaagcta gagtaagtag ttcgccagtt aatagtttgc gcaacgttgt
     5281 tgccattgct acaggcatcg tggtgtcacg ctcgtcgttt ggtatggctt cattcagctc
     5341 cggttcccaa cgatcaaggc gagttacatg atcccccatg ttgtgcaaaa aagcggttag
     5401 ctccttcggt cctccgatcg ttgtcagaag taagttggcc gcagtgttat cactcatggt
     5461 tatggcagca ctgcataatt ctcttactgt catgccatcc gtaagatgct tttctgtgac
     5521 tggtgagtac tcaaccaagt cattctgaga atagtgtatg cggcgaccga gttgctcttg
     5581 cccggcgtca atacgggata ataccgcgcc acatagcaga actttaaaag tgctcatcat
     5641 tggaaaacgt tcttcggggc gaaaactctc aaggatctta ccgctgttga gatccagttc
     5701 gatgtaaccc actcgtgcac ccaactgatc ttcagcatct tttactttca ccagcgtttc
     5761 tgggtgagca aaaacaggaa ggcaaaatgc cgcaaaaaag ggaataaggg cgacacggaa
     5821 atgttgaata ctcatactct tcctttttca atattattga agcatttatc agggttattg
     5881 tctcatgagc ggatacatat ttgaatgtat ttagaaaaat aaacaaatag gggttccgcg
     5941 cacatttccc cgaaaagtgc cacctgacgt ctaagaaacc attattatca tgacattaac
     6001 ctataaaaat aggcgtatca cgaggccctt tcgtctcgcg cgtttcggtg atgacggtga
     6061 aaacctctga cacatgcagc tcccggagac ggtcacagct tgtctgtaag cggatgccgg
     6121 gagcagacaa gcccgtcagg gcgcgtcagc gggtgttggc gggtgtcggg gctggcttaa
     6181 ctatgcggca tcagagcaga ttgtactgag agtgcaccat atggacatat tgtcgttaga
     6241 acgcggctac aattaataca taaccttatg tatcatacac atacgattta ggtgacacta
     6301 ta
//
