LOCUS       sequence-240225-        7255 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  For Yeast-two-hybrid analysis.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 7255)
  AUTHORS   Liu X, Mao K, Yu AYH, Omairi-Nasser A, Austin J 2nd, Glick BS, Yip 
            CK, Klionsky DJ
  TITLE     The Atg17-Atg31-Atg29 Complex Coordinates with Atg11 to Recruit the 
            Vam7 SNARE and Mediate Autophagosome-Vacuole Fusion.
  JOURNAL   Curr Biol. 2016 Jan 25;26(2):150-160. doi: 
            10.1016/j.cub.2015.11.054. Epub 2016 Jan 7.
  PUBMED    26774783
REFERENCE   2  (bases 1 to 7255)
  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"; doi: 
            "10.1016/j.cub.2015.11.054"; journalName: "Curr Biol"; date: 
            "2016-01-25- 25"; volume: "26"; issue: "2"; pages: "150-160"
COMMENT     Sequence Label: pGBDU-Atg17(I325A)
FEATURES             Location/Qualifiers
     source          1..7255
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     CDS             325..765
                     /codon_start=1
                     /gene="Saccharomyces cerevisiae GAL4 (truncated)"
                     /product="DNA binding domain of the GAL4 transcriptional 
                     activator"
                     /label=GAL4 DNA binding domain
                     /translation="MKLLSSIEQACDICRLKKLKCSKEKPKCAKCLKNNWECRYSPKTK
                     RSPLTRAHLTEVESRLERLEQLFLLIFPREDLDMILKMDSLQDIKALLTGLFVQDNVNK
                     DAVTDRLASVETDMPLTLRQHRISATSSSEESSNKGQRQLTVS"
     RBS             1836..1847
                     /note="strong bacterial ribosome binding site (Elowitz and 
                     Leibler, 2000)"
     terminator      2447..2634
                     /gene="S. cerevisiae ADH1"
                     /label=ADH1 terminator
                     /note="transcription terminator for the S. cerevisiae 
                     alcohol dehydrogenase 1 (ADH1) gene"
     primer_bind     complement(2502..2526)
                     /label=pHybLex-R
                     /note="ADH terminator, reverse primer"
     promoter        2646..2857
                     /gene="S. cerevisiae URA3"
                     /label=URA3 promoter
     CDS             2858..3661
                     /codon_start=1
                     /gene="S. cerevisiae URA3"
                     /product="orotidine-5'-phosphate decarboxylase, required 
                     for uracil biosynthesis"
                     /label=URA3
                     /note="yeast auxotrophic marker, counterselectable with 
                     5-fluoroorotic acid (5-FOA)"
                     /translation="MSKATYKERAATHPSPVAAKLFNIMHEKQTNLCASLDVRTTKELL
                     ELVEALGPKICLLKTHVDILTDFSMEGTVKPLKALSAKYNFLLFEDRKFADIGNTVKLQ
                     YSAGVYRIAEWADITNAHGVVGPGIVSGLKQAAEEVTKEPRGLLMLAELSCKGSLSTGE
                     YTKGTVDIAKSDKDFVIGFIAQRDMGGRDEGYDWLIMTPGVGLDDKGDALGQQYRTVDD
                     VVSTGSDIIIVGRGLFAKGRDAKVEGERYRKAGWEAYLRRCGQQN"
     primer_bind     complement(3805..3822)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(3976..4564)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(4056..4075)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(4735..5526,5527..5595))
                     /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     5358..5377
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(5596..5700)
                     /gene="bla"
                     /label=AmpR promoter
     primer_bind     5768..5786
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     primer_bind     complement(5824..5846)
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     primer_bind     5946..5965
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     rep_origin      5982..7146
                     /label=2u ori
                     /note="yeast 2u plasmid origin of replication"
     promoter        join(7151..7255,1..297)
                     /gene="S. cerevisiae ADH1"
                     /label=ADH1 promoter
                     /note="promoter for alcohol dehydrogenase 1"
ORIGIN
        1 acgacaaaga cagcaccaac agatgtcgtt gttccagagc tgatgagggg tatctcgaag
       61 cacacgaaac tttttccttc cttcattcac gcacactact ctctaatgag caacggtata
      121 cggccttcct tccagttact tgaatttgaa ataaaaaaaa gtttgctgtc ttgctatcaa
      181 gtataaatag acctgcaatt attaatcttt tgtttcctcg tcattgttct cgttcccttt
      241 cttccttgtt tctttttctg cacaatattt caagctatac caagcataca atcaactcca
      301 agcttgaagc aagcctcctg aaagatgaag ctactgtctt ctatcgaaca agcatgcgat
      361 atttgccgac ttaaaaagct caagtgctcc aaagaaaaac cgaagtgcgc caagtgtctg
      421 aagaacaact gggagtgtcg ctactctccc aaaaccaaaa ggtctccgct gactagggca
      481 catctgacag aagtggaatc aaggctagaa agactggaac agctatttct actgattttt
      541 cctcgagaag accttgacat gattttgaaa atggattctt tacaggatat aaaagcattg
      601 ttaacaggat tatttgtaca agataatgtg aataaagatg ccgtcacaga tagattggct
      661 tcagtggaga ctgatatgcc tctaacattg agacagcata gaataagtgc gacatcatca
      721 tcggaagaga gtagtaacaa aggtcaaaga cagttgactg tatcgccgga attccccggg
      781 ggatccatcg ataacgaagc agatgttaca aaatttgtta ataatgccag gaaaacgctg
      841 accgatgctc aacttttatg ttcaagtgct aatttaagga ttgtagatat taagaaaaaa
      901 ttgtcatctt ggcagttgag tatttcaaaa ctcaattttc taatagttgg cttgagacag
      961 caaggaaagt ttctttacac tattttaaag gaaggcattg ggacaaagct aatccagaaa
     1021 caatggaatc aagctgtctt agtggtctta gttgatgaga tgaagtactg gcaatatgaa
     1081 atcacttcta aagtacaaag gttggatggt atagtaaatg aattgagcat atcagaaaaa
     1141 gatgataccg atccctctaa actaggagat tacatctcga gagacaacgt taacttattg
     1201 aatgacaaac tgaaagaagt gccagtaatt gagcgtcaaa ttgaaaacat taagcttcaa
     1261 tacgaaaata tggtcagaaa agttaacaaa gaactgattg ataccaagtt gacggacgta
     1321 actcaaaaat tccaaagtaa atttggtata gataacctga tggaaacaaa tgttgcagag
     1381 cagtttagca gggaactaac ggaccttgaa aaagatttag cagagataat gaattcattg
     1441 acacagcact ttgataagac attgctgtta caagataaaa aaattgataa cgatgaacgt
     1501 gaggagctgt ttaaggtggt acaaggcgac gacaaagaac tatacaacat tttcaaaact
     1561 ctgcatgagg taattgatga cgtggacaaa acaattctta acttgggtca atttttgcag
     1621 gcaaaaataa aggaaaagac agaactacac agcgaagttt ctgaaataat aaacgatttc
     1681 aatagaaatt tggaatatct attaatcttt aaagatattt ccaatctgat tgatagcttc
     1741 aaaaattcct gtacacaaga tgctcaaaca actaaggaac tttgtgaatt ttatgataat
     1801 tttgaagaaa gctacggtaa cttagttcta gaagcaaaga ggagaaagga tgtggcaaac
     1861 agaatgaaaa ctatattgaa agattgtgaa aagcagttgc agaatttaga tgctcaggac
     1921 caggaagaac gtcagaattt tatagcggaa aatggaactt atcttcctga gacaatctgg
     1981 cccggtaaaa ttgacgattt ttcttccctg tacactttaa attacaacgt gaagaatcct
     2041 tagaaaaaag gaaaaaaagg ctgcagagat ctatgaatcg tagatactga aaaaccccgc
     2101 aagttcactt caactgtgca tcgtgcacca tctcaatttc tttcatttat acatcgtttt
     2161 gccttctttt atgtaactat actcctctaa gtttcaatct tggccatgta acctctgatc
     2221 tatagaattt tttaaatgac tagaattaat gcccatcttt tttttggacc taaattcttc
     2281 atgaaaatat attacgaggg cttattcaga agctttggac ttcttcgcca gaggtttggt
     2341 caagtctcca atcaaggttg tcggcttgtc taccttgcca gaaatttacg aaaagatgga
     2401 aaagggtcaa atcgttggta gatacgttgt tgacacttct aaataagcga atttcttatg
     2461 atttatgatt tttattatta aataagttat aaaaaaaata agtgtataca aattttaaag
     2521 tgactcttag gttttaaaac gaaaattctt attcttgagt aactctttcc tgtaggtcag
     2581 gttgctttct caggtatagc atgaggtcgc tcttattgac cacacctcta ccggcatgcc
     2641 agcttttcaa ttcatcattt tttttttatt cttttttttg atttcggttt ccttgaaatt
     2701 tttttgattc ggtaatctcc gaacagaagg aagaacgaag gaaggagcac agacttagat
     2761 tggtatatat acgcatatgt agtgttgaag aaacatgaaa ttgcccagta ttcttaaccc
     2821 aactgcacag aacaaaaacc ggaaacgaag ataaatcatg tcgaaagcta catataagga
     2881 acgtgctgct actcatccta gtcctgttgc tgccaagcta tttaatatca tgcacgaaaa
     2941 gcaaacaaac ttgtgtgctt cattggatgt tcgtaccacc aaggaattac tggagttagt
     3001 tgaagcatta ggtcccaaaa tttgtttact aaaaacacat gtggatatct tgactgattt
     3061 ttccatggag ggcacagtta agccgctaaa ggcattatcc gccaagtaca attttttact
     3121 cttcgaagac agaaaatttg ctgacattgg taatacagtc aaattgcagt actctgcggg
     3181 tgtatacaga atagcagaat gggcagacat tacgaatgca cacggtgtgg tgggcccagg
     3241 tattgttagc ggtttgaagc aggcggcaga agaagtaaca aaggaaccta gaggcctttt
     3301 gatgttagca gaattgtcat gcaagggctc cctatctact ggagaatata ctaagggtac
     3361 tgttgacatt gcgaagagcg acaaagattt tgttatcggc tttattgctc aaagagacat
     3421 gggtggaaga gatgaaggtt acgattggtt gattatgaca cccggtgtgg gtttagatga
     3481 caagggagac gcattgggtc aacagtatag aaccgtggat gatgtggtct ctacaggatc
     3541 tgacattatt attgttggaa gaggactatt tgcaaaggga agggatgcta aggtagaggg
     3601 tgaacgttac agaaaagcag gctgggaagc atatttgaga agatgcggcc agcaaaacta
     3661 aaaaactgta ttataagtaa atgcatgtat actaaactca caaattagag cttcaattta
     3721 attatatcag ttattacccc tgcattaatg aatcggccaa cgcgcgggga gaggcggttt
     3781 gcgtattggg cgctcttccg cttcctcgct cactgactcg ctgcgctcgg tcgttcggct
     3841 gcggcgagcg gtatcagctc actcaaaggc ggtaatacgg ttatccacag aatcagggga
     3901 taacgcagga aagaacatgt gagcaaaagg ccagcaaaag gccaggaacc gtaaaaaggc
     3961 cgcgttgctg gcgtttttcc ataggctccg cccccctgac gagcatcaca aaaatcgacg
     4021 ctcaagtcag aggtggcgaa acccgacagg actataaaga taccaggcgt ttccccctgg
     4081 aagctccctc gtgcgctctc ctgttccgac cctgccgctt accggatacc tgtccgcctt
     4141 tctcccttcg ggaagcgtgg cgctttctca tagctcacgc tgtaggtatc tcagttcggt
     4201 gtaggtcgtt cgctccaagc tgggctgtgt gcacgaaccc cccgttcagc ccgaccgctg
     4261 cgccttatcc ggtaactatc gtcttgagtc caacccggta agacacgact tatcgccact
     4321 ggcagcagcc actggtaaca ggattagcag agcgaggtat gtaggcggtg ctacagagtt
     4381 cttgaagtgg tggcctaact acggctacac tagaagaaca gtatttggta tctgcgctct
     4441 gctgaagcca gttaccttcg gaaaaagagt tggtagctct tgatccggca aacaaaccac
     4501 cgctggtagc ggtggttttt ttgtttgcaa gcagcagatt acgcgcagaa aaaaaggatc
     4561 tcaagaagat cctttgatct tttctacggg gtctgacgct cagtggaacg aaaactcacg
     4621 ttaagggatt ttggtcatga gattatcaaa aaggatcttc acctagatcc ttttaaatta
     4681 aaaatgaagt tttaaatcaa tctaaagtat atatgagtaa acttggtctg acagttacca
     4741 atgcttaatc agtgaggcac ctatctcagc gatctgtcta tttcgttcat ccatagttgc
     4801 ctgactcccc gtcgtgtaga taactacgat acgggagggc ttaccatctg gccccagtgc
     4861 tgcaatgata ccgcgagacc cacgctcacc ggctccagat ttatcagcaa taaaccagcc
     4921 agccggaagg gccgagcgca gaagtggtcc tgcaacttta tccgcctcca tccagtctat
     4981 taattgttgc cgggaagcta gagtaagtag ttcgccagtt aatagtttgc gcaacgttgt
     5041 tgccattgct acaggcatcg tggtgtcacg ctcgtcgttt ggtatggctt cattcagctc
     5101 cggttcccaa cgatcaaggc gagttacatg atcccccatg ttgtgcaaaa aagcggttag
     5161 ctccttcggt cctccgatcg ttgtcagaag taagttggcc gcagtgttat cactcatggt
     5221 tatggcagca ctgcataatt ctcttactgt catgccatcc gtaagatgct tttctgtgac
     5281 tggtgagtac tcaaccaagt cattctgaga atagtgtatg cggcgaccga gttgctcttg
     5341 cccggcgtca atacgggata ataccgcgcc acatagcaga actttaaaag tgctcatcat
     5401 tggaaaacgt tcttcggggc gaaaactctc aaggatctta ccgctgttga gatccagttc
     5461 gatgtaaccc actcgtgcac ccaactgatc ttcagcatct tttactttca ccagcgtttc
     5521 tgggtgagca aaaacaggaa ggcaaaatgc cgcaaaaaag ggaataaggg cgacacggaa
     5581 atgttgaata ctcatactct tcctttttca atattattga agcatttatc agggttattg
     5641 tctcatgagc ggatacatat ttgaatgtat ttagaaaaat aaacaaatag gggttccgcg
     5701 cacatttccc cgaaaagtgc cacctgacgt ctaagaaacc attattatca tgacattaac
     5761 ctataaaaat aggcgtatca cgaggccctt tcgtctcgcg cgtttcggtg atgacggtga
     5821 aaacctctga cacatgcagc tcccggagac ggtcacagct tgtctgtaag cggatgccgg
     5881 gagcagacaa gcccgtcagg gcgcgtcagc gggtgttggc gggtgtcggg gctggcttaa
     5941 ctatgcggca tcagagcaga ttgtactgag agtgcaccat aacgcattta agcataaaca
     6001 cgcactatgc cgttcttctc atgtatatat atatacaggc aacacgcaga tataggtgcg
     6061 acgtgaacag tgagctgtat gtgcgcagct cgcgttgcat tttcggaagc gctcgttttc
     6121 ggaaacgctt tgaagttcct attccgaagt tcctattctc tagctagaaa gtataggaac
     6181 ttcagagcgc ttttgaaaac caaaagcgct ctgaagacgc actttcaaaa aaccaaaaac
     6241 gcaccggact gtaacgagct actaaaatat tgcgaatacc gcttccacaa acattgctca
     6301 aaagtatctc tttgctatat atctctgtgc tatatcccta tataacctac ccatccacct
     6361 ttcgctcctt gaacttgcat ctaaactcga cctctacatt ttttatgttt atctctagta
     6421 ttactcttta gacaaaaaaa ttgtagtaag aactattcat agagtgaatc gaaaacaata
     6481 cgaaaatgta aacatttcct atacgtagta tatagagaca aaatagaaga aaccgttcat
     6541 aattttctga ccaatgaaga atcatcaacg ctatcacttt ctgttcacaa agtatgcgca
     6601 atccacatcg gtatagaata taatcgggga tgcctttatc ttgaaaaaat gcacccgcag
     6661 cttcgctagt aatcagtaaa cgcgggaagt ggagtcaggc tttttttatg gaagagaaaa
     6721 tagacaccaa agtagccttc ttctaacctt aacggaccta cagtgcaaaa agttatcaag
     6781 agactgcatt atagagcgca caaaggagaa aaaaagtaat ctaagatgct ttgttagaaa
     6841 aatagcgctc tcgggatgca tttttgtaga acaaaaaaga agtatagatt ctttgttggt
     6901 aaaatagcgc tctcgcgttg catttctgtt ctgtaaaaat gcagctcaga ttctttgttt
     6961 gaaaaattag cgctctcgcg ttgcattttt gttttacaaa aatgaagcac agattcttcg
     7021 ttggtaaaat agcgctttcg cgttgcattt ctgttctgta aaaatgcagc tcagattctt
     7081 tgtttgaaaa attagcgctc tcgcgttgca tttttgttct acaaaatgaa gcacagatgc
     7141 ttcgttgctt gcatgcaact tcttttcttt ttttttcttt tctctctccc ccgttgttgt
     7201 ctcaccatat ccgcaatgac aaaaaaatga tggaagacac taaaggaaaa aatta
//
