LOCUS       sequence-422763-        6299 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Expression of 3HA-Atg8∆GR. This variant cannot be conjugated to PE..
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 6299)
  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 6299)
  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∆GR-MET15
FEATURES             Location/Qualifiers
     source          1..6299
                     /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      1557..1744
                     /gene="S. cerevisiae ADH1"
                     /label=ADH1 terminator
                     /note="transcription terminator for the S. cerevisiae 
                     alcohol dehydrogenase 1 (ADH1) gene"
     primer_bind     complement(1612..1636)
                     /label=pHybLex-R
                     /note="ADH terminator, reverse primer"
     protein_bind    1796..1829
                     /label=loxP
                     /bound_moiety="Cre recombinase"
                     /note="Cre-mediated recombination occurs in the 8-bp core 
                     sequence (ATGTATGC) (Shaw et al., 2021)."
     CDS             complement(2012..3346)
                     /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(3347..3697)
                     /gene="S. cerevisiae MET17"
                     /gene_synonym="MET15; MET25"
                     /label=MET17 promoter
                     /note="expression is repressed by methionine"
     protein_bind    3850..3883
                     /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(3936..3955)
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        complement(3937..3955)
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     primer_bind     complement(4042..4059)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(4213..4801)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(4293..4312)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(4972..5763,5764..5832))
                     /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     5595..5614
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(5833..5937)
                     /gene="bla"
                     /label=AmpR promoter
     primer_bind     6005..6023
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     primer_bind     complement(6061..6083)
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     primer_bind     6183..6202
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     promoter        join(6283..6299,1..2)
                     /label=SP6 promoter
                     /note="promoter for bacteriophage SP6 RNA polymerase"
     primer_bind     join(6283..6299,1)
                     /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 ttttgaggcg cgccacttct aaataagcga
     1561 atttcttatg atttatgatt tttattatta aataagttat aaaaaaaata agtgtataca
     1621 aattttaaag tgactcttag gttttaaaac gaaaattctt attcttgagt aactctttcc
     1681 tgtaggtcag gttgctttct caggtatagt atgaggtcgc tcttattgac cacacctcta
     1741 ccggcagatc cgctagggat aacagggtaa tatctgcagg tcgacaaccc ttaatataac
     1801 ttcgtataat gtatgctata cgaagttatt aggtctagat gcgaattaac actgttcttt
     1861 gatgttagaa caatttaggt tcaaagtacg agtcacgaca tgtaacaagg gaaaaaaagg
     1921 atattcattt caataaagtt cgttttataa aagtatagta cttgtgagag aaagtaggtt
     1981 tatacataat tttacaactc attacgcaca ctcatggttt ttggccagcg aaaacagttt
     2041 caaaagattg ctggaagtct gcaataatgt catcaataaa ttcgatacca acagagacac
     2101 gaattaagtc cttggtaaca ccagatgcca acttttcttt gtcatttaat tgtttgtggg
     2161 tagtgaagta tggagcaatg actaaggtct tggcatcacc aacattggcc aagttagagg
     2221 caagctttaa attgtcaaca acttgagcac cagaaagttt gaatgggtca gtttccttgt
     2281 cggcatttgg taagtctttt acaccgaaag ataagacacc accgaaaccg ttagatagat
     2341 acttcttagc attttcatga tgagaatgag atgctaaacc agggtatgaa acccaagata
     2401 cgtatgggga ttgttctaac catttggcta acttcaatgc attttcaccg tgtctttcag
     2461 ctctcaaaga taatgtttca acaccttgta gtagcaagaa agaggcaaat gggttcatca
     2521 atggacccaa atctcttaat agttcagttc taacatgaac gatgtatgcc aagttaccgt
     2581 aggcttcatt gtagatagta ccgtgatatc cttcggcagg ttgagagaat tgagggaact
     2641 tttctgggta gtccttccat gggaacttac cagagtcaac aataatacca ccgatagtag
     2701 taccatgacc accaatccat ttggtagcag aatgtgttac aatatcagca ccgtatttaa
     2761 ttggctgaca gaagtaacca ccggcaccaa atgtgttgtc aacgacaact ggaataccgt
     2821 gtttgtgagc aattgcaaca attttttcaa aatccggaac attgtacttt ggattaccaa
     2881 tggtttccaa ataaacagcc ttggttcttt catcaaagac cttttcgaat tcttctggat
     2941 tgtcaccttc aacaaatcta gcctcgatac caaatctttt gaacgagatt ttgaactggt
     3001 tataagtacc accgtataag taagaagtgg aaacgatgtt gtcaccagtg tgtgccaaac
     3061 cttggatggc aagggtttga gcggcttgac cggaggaaac agccaaagca gcagcaccac
     3121 cttctaaagc agcaattctt tcttccaaaa cattactggt tgggttttgg aaacgggaat
     3181 agacgtaacc tggaacttct agaccaaaca attgcgaacc atgcttagag ttttcgaaaa
     3241 cataagaagt ggtggcgtaa attggtacag ctctggatct gtgagcattg tcaccagggt
     3301 tctcttggcc ggcgtgtagt tgaacagtat cgaaatgaga tggcattgta tggatggggg
     3361 taatagaatt gtatctatgt atctgacgac cctgtattac acgaaggaaa tagtagatga
     3421 aaagacaaga gagcaagaaa aaggaaaaac ttcctttcta acagacgctt tacttaacct
     3481 tatatatatc ttattttttt catcgagcgt gttcaattgg acaaggtgcc accttttcga
     3541 cacttcggtt attatgttac acagttttca tgaggatggc gccttgacta acttaattag
     3601 tcatttgcca accaccacag ttccccaata tcgacagctt cacgtgccat ttgccatttt
     3661 gcgatcatgt gacctcaaga gaaaaagcaa aaaataatga gagctaaggg attcgaaccc
     3721 ttgcatccga agatatcaga gattttagag gttaaatcca cctaaatctg acgccttaaa
     3781 ccactcggcc aaactcccat tgtatgttgg aagaattgga tttaaatgga gctctcgaga
     3841 acccttaata taacttcgta taatgtatgc tatacgaagt tattaggtga tatcagatcc
     3901 actagtggcc tatgcggccg cggatctgcc ggtctcccta tagtgagtcg tattaatttc
     3961 gataagccag gttaacctgc attaatgaat cggccaacgc gcggggagag gcggtttgcg
     4021 tattgggcgc tcttccgctt cctcgctcac tgactcgctg cgctcggtcg ttcggctgcg
     4081 gcgagcggta tcagctcact caaaggcggt aatacggtta tccacagaat caggggataa
     4141 cgcaggaaag aacatgtgag caaaaggcca gcaaaaggcc aggaaccgta aaaaggccgc
     4201 gttgctggcg tttttccata ggctccgccc ccctgacgag catcacaaaa atcgacgctc
     4261 aagtcagagg tggcgaaacc cgacaggact ataaagatac caggcgtttc cccctggaag
     4321 ctccctcgtg cgctctcctg ttccgaccct gccgcttacc ggatacctgt ccgcctttct
     4381 cccttcggga agcgtggcgc tttctcatag ctcacgctgt aggtatctca gttcggtgta
     4441 ggtcgttcgc tccaagctgg gctgtgtgca cgaacccccc gttcagcccg accgctgcgc
     4501 cttatccggt aactatcgtc ttgagtccaa cccggtaaga cacgacttat cgccactggc
     4561 agcagccact ggtaacagga ttagcagagc gaggtatgta ggcggtgcta cagagttctt
     4621 gaagtggtgg cctaactacg gctacactag aagaacagta tttggtatct gcgctctgct
     4681 gaagccagtt accttcggaa aaagagttgg tagctcttga tccggcaaac aaaccaccgc
     4741 tggtagcggt ggtttttttg tttgcaagca gcagattacg cgcagaaaaa aaggatctca
     4801 agaagatcct ttgatctttt ctacggggtc tgacgctcag tggaacgaaa actcacgtta
     4861 agggattttg gtcatgagat tatcaaaaag gatcttcacc tagatccttt taaattaaaa
     4921 atgaagtttt aaatcaatct aaagtatata tgagtaaact tggtctgaca gttaccaatg
     4981 cttaatcagt gaggcaccta tctcagcgat ctgtctattt cgttcatcca tagttgcctg
     5041 actccccgtc gtgtagataa ctacgatacg ggagggctta ccatctggcc ccagtgctgc
     5101 aatgataccg cgagacccac gctcaccggc tccagattta tcagcaataa accagccagc
     5161 cggaagggcc gagcgcagaa gtggtcctgc aactttatcc gcctccatcc agtctattaa
     5221 ttgttgccgg gaagctagag taagtagttc gccagttaat agtttgcgca acgttgttgc
     5281 cattgctaca ggcatcgtgg tgtcacgctc gtcgtttggt atggcttcat tcagctccgg
     5341 ttcccaacga tcaaggcgag ttacatgatc ccccatgttg tgcaaaaaag cggttagctc
     5401 cttcggtcct ccgatcgttg tcagaagtaa gttggccgca gtgttatcac tcatggttat
     5461 ggcagcactg cataattctc ttactgtcat gccatccgta agatgctttt ctgtgactgg
     5521 tgagtactca accaagtcat tctgagaata gtgtatgcgg cgaccgagtt gctcttgccc
     5581 ggcgtcaata cgggataata ccgcgccaca tagcagaact ttaaaagtgc tcatcattgg
     5641 aaaacgttct tcggggcgaa aactctcaag gatcttaccg ctgttgagat ccagttcgat
     5701 gtaacccact cgtgcaccca actgatcttc agcatctttt actttcacca gcgtttctgg
     5761 gtgagcaaaa acaggaaggc aaaatgccgc aaaaaaggga ataagggcga cacggaaatg
     5821 ttgaatactc atactcttcc tttttcaata ttattgaagc atttatcagg gttattgtct
     5881 catgagcgga tacatatttg aatgtattta gaaaaataaa caaatagggg ttccgcgcac
     5941 atttccccga aaagtgccac ctgacgtcta agaaaccatt attatcatga cattaaccta
     6001 taaaaatagg cgtatcacga ggccctttcg tctcgcgcgt ttcggtgatg acggtgaaaa
     6061 cctctgacac atgcagctcc cggagacggt cacagcttgt ctgtaagcgg atgccgggag
     6121 cagacaagcc cgtcagggcg cgtcagcggg tgttggcggg tgtcggggct ggcttaacta
     6181 tgcggcatca gagcagattg tactgagagt gcaccatatg gacatattgt cgttagaacg
     6241 cggctacaat taatacataa ccttatgtat catacacata cgatttaggt gacactata
//
