LOCUS       sequence-236145-        7780 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 7780)
  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 7780)
  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: pGAD-Atg17(∆186-236)
FEATURES             Location/Qualifiers
     source          1..7780
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     promoter        1..402
                     /gene="S. cerevisiae ADH1"
                     /label=ADH1 promoter
                     /note="promoter for alcohol dehydrogenase 1"
     CDS             448..468
                     /codon_start=1
                     /product="nuclear localization signal of SV40 (simian virus
                     40) large T antigen"
                     /label=SV40 NLS
                     /translation="PKKKRKV"
     CDS             484..825
                     /codon_start=1
                     /gene="Saccharomyces cerevisiae GAL4 (truncated)"
                     /product="activation domain of the GAL4 transcriptional 
                     activator"
                     /label=GAL4 activation domain
                     /translation="ANFNQSGNIADSSLSFTFTNSSNGPNLITTQTNSQALSQPIASSN
                     VHDNFMNNEITASKIDDGNNSKPLSPGWTDQTAYNAFGITTGMFNTTTMDDVYNYLFDD
                     EDTPPNPKKE"
     RBS             1743..1754
                     /note="strong bacterial ribosome binding site (Elowitz and 
                     Leibler, 2000)"
     terminator      2354..2541
                     /gene="S. cerevisiae ADH1"
                     /label=ADH1 terminator
                     /note="transcription terminator for the S. cerevisiae 
                     alcohol dehydrogenase 1 (ADH1) gene"
     primer_bind     complement(2409..2433)
                     /label=pHybLex-R
                     /note="ADH terminator, reverse primer"
     CDS             complement(2662..3756)
                     /codon_start=1
                     /gene="S. cerevisiae LEU2"
                     /product="3-isopropylmalate dehydrogenase, required for 
                     leucine biosynthesis"
                     /label=LEU2
                     /note="yeast auxotrophic marker"
                     /translation="MSAPKKIVVLPGDHVGQEITAEAIKVLKAISDVRSNVKFDFENHL
                     IGGAAIEATGVPLPDEALEASKKADAVLLGAVGGPKWGTGSVRPEQGLLKIRKELQLYA
                     NLRPCNFASDSLLDLSPIKPQFAKGTDFVVVRELVGGIYFGKRKEDDGDGVAWDSEQYT
                     VPEVQRITRMAAFMALQHEPPLPIWSLDKANVLASSRLWRKTVEETIKNEFPTLKVQHQ
                     LIDSAAMILVRNPTHLNGIIITSNMFGDIVSDEASVIPGSLGLLPSASLASLPDKNTAF
                     GLYEPCHGSAPDLPKNKVNPIATILSAAMMLKLSLNLPEEGKAIEDAVKKVLDAGIRTG
                     DLGGSNSTTEVGDAVAEEVKKILA"
     promoter        complement(3757..4162)
                     /gene="S. cerevisiae LEU2"
                     /label=LEU2 promoter
     primer_bind     complement(4204..4220)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(4204..4220)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(4217..4239)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    4228..4244
                     /label=lac operator
                     /bound_moiety="lac repressor encoded by lacI"
                     /note="The lac repressor binds to the lac operator to 
                     inhibit transcription in E. coli. This inhibition can be 
                     relieved by adding lactose or 
                     isopropyl-beta-D-thiogalactopyranoside (IPTG)."
     promoter        complement(join(4252..4258,4259..4276,4277..4282))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    4297..4318
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(4435..4452)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(4606..5194)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(4686..4705)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(5365..6156,6157..6225))
                     /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     5988..6007
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(6226..6330)
                     /gene="bla"
                     /label=AmpR promoter
     primer_bind     6398..6416
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     primer_bind     complement(6454..6476)
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     primer_bind     6576..6595
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     rep_origin      6612..7776
                     /label=2u ori
                     /note="yeast 2u plasmid origin of replication"
ORIGIN
        1 gcatgcaact tcttttcttt ttttttcttt tctctctccc ccgttgttgt ctcaccatat
       61 ccgcaatgac aaaaaaatga tggaagacac taaaggaaaa aattaacgac aaagacagca
      121 ccaacagatg tcgttgttcc agagctgatg aggggtatct cgaagcacac gaaacttttt
      181 ccttccttca ttcacgcaca ctactctcta atgagcaacg gtatacggcc ttccttccag
      241 ttacttgaat ttgaaataaa aaaaagtttg ctgtcttgct atcaagtata aatagacctg
      301 caattattaa tcttttgttt cctcgtcatt gttctcgttc cctttcttcc ttgtttcttt
      361 ttctgcacaa tatttcaagc tataccaagc atacaatcaa ctccaagctt tgcaaagatg
      421 gataaagcgg aattaattcc cgagcctcca aaaaagaaga gaaaggtcga attgggtacc
      481 gccgccaatt ttaatcaaag tgggaatatt gctgatagct cattgtcctt cactttcact
      541 aacagtagca acggtccgaa cctcataaca actcaaacaa attctcaagc gctttcacaa
      601 ccaattgcct cctctaacgt tcatgataac ttcatgaata atgaaatcac ggctagtaaa
      661 attgatgatg gtaataattc aaaaccactg tcacctggtt ggacggacca aactgcgtat
      721 aacgcgtttg gaatcactac agggatgttt aataccacta caatggatga tgtatataac
      781 tatctattcg atgatgaaga taccccacca aacccaaaaa aagagatcga attccccggg
      841 ggatccatcg ataacgaagc agatgttaca aaatttgtta ataatgccag gaaaacgctg
      901 accgatgctc aacttttatg ttcaagtgct aatttaagga ttgtagatat taagaaaaaa
      961 ttgtcatctt ggcagttgag tatttcaaaa ctcaattttc taatagttgg cttgagacag
     1021 caaggaaagt ttctttacac tattttaaag gaaggcattg ggacaaagct aatccagaaa
     1081 caatggaatc aagctgtctt agtggtctta gttgatgaga tgaagtactg gcaatatgaa
     1141 atcacttcta aagtacaaag gttggatggt atagtaaatg aattgagcat atcagaaaaa
     1201 gatgataccg atccctctaa actaggagat tacatctcga gagacaacgt taacttattg
     1261 aatgacaaac tgaaagaagt gccagtaatt gagcgtcaaa ttgaaaacat taagcttcaa
     1321 tacgaaaata tggtcagaaa agttaacaaa gaactgattg ataccaagtt gacggacgta
     1381 actcaaaaat tccaaagtaa atttcgtgag gagctgttta aggtggtaca aggcgacgac
     1441 aaagaactat acaacatttt caaaactctg catgaggtaa ttgatgacgt ggacaaaaca
     1501 attcttaact tgggtcaatt tttgcaggca aaaataaagg aaaagacaga actacacagc
     1561 gaagtttctg aaataataaa cgatttcaat agaaatttgg aatatctatt aatctttaaa
     1621 gatatttcca atctgattga tagcttcaaa aattcctgta cacaagatat tcaaacaact
     1681 aaggaacttt gtgaatttta tgataatttt gaagaaagct acggtaactt agttctagaa
     1741 gcaaagagga gaaaggatgt ggcaaacaga atgaaaacta tattgaaaga ttgtgaaaag
     1801 cagttgcaga atttagatgc tcaggaccag gaagaacgtc agaattttat agcggaaaat
     1861 ggaacttatc ttcctgagac aatctggccc ggtaaaattg acgatttttc ttccctgtac
     1921 actttaaatt acaacgtgaa gaatccttag aaaaaaggaa aaaaaggctg cagagatcta
     1981 tgaatcgtag atactgaaaa accccgcaag ttcacttcaa ctgtgcatcg tgcaccatct
     2041 caatttcttt catttataca tcgttttgcc ttcttttatg taactatact cctctaagtt
     2101 tcaatcttgg ccatgtaacc tctgatctat agaatttttt aaatgactag aattaatgcc
     2161 catctttttt ttggacctaa attcttcatg aaaatatatt acgagggctt attcagaagc
     2221 tttggacttc ttcgccagag gtttggtcaa gtctccaatc aaggttgtcg gcttgtctac
     2281 cttgccagaa atttacgaaa agatggaaaa gggtcaaatc gttggtagat acgttgttga
     2341 cacttctaaa taagcgaatt tcttatgatt tatgattttt attattaaat aagttataaa
     2401 aaaaataagt gtatacaaat tttaaagtga ctcttaggtt ttaaaacgaa aattcttatt
     2461 cttgagtaac tctttcctgt aggtcaggtt gctttctcag gtatagcatg aggtcgctct
     2521 tattgaccac acctctaccg gcatgccggt cgaaattccc ctaccctatg aacatattcc
     2581 attttgtaat ttcgtgtcgt ttctattatg aatttcattt ataaagttta tgtacaaata
     2641 tcataaaaaa agagaatctt tttaagcaag gattttctta acctcttcgg cgacagcatc
     2701 accgacttcg gtggtactgt tggaaccacc taaatcacca gttctgatac ctgcatccaa
     2761 aaccttttta actgcatctt caatggcctt accttcttca ggcaagttca atgacaattt
     2821 caacatcatt gcagcagaca agatagtggc gatagggttg accttattct ttggcaaatc
     2881 tggagcagaa ccgtggcatg gttcgtacaa accaaatgcg gtgttcttgt ctggcaaaga
     2941 ggccaaggac gcagatggca acaaacccaa ggaacctggg ataacggagg cttcatcgga
     3001 gacgatatca ccaaacatgt tgctggtgat tataatacca tttaggtggg ttgggttcct
     3061 aactaggatc atggcggcag aatcaatcaa ttgatgttga accttcaatg taggaaattc
     3121 gttcttgatg gtttcctcca cagtttttct ccataatctt gaagaggcca aaacattagc
     3181 tttatccaag gaccaaatag gcaatggtgg ctcatgttgt agggccatga aagcggccat
     3241 tcttgtgatt ctttgcactt ctggaacggt gtattgttca ctatcccaag cgacaccatc
     3301 accatcgtct tcctttctct taccaaagta aatacctccc actaattctc tgacaacaac
     3361 gaagtcagta cctttagcaa attgtggctt gattggagat aagtctaaaa gagagtcgga
     3421 tgcaaagtta catggtctta agttggcgta caattgaagt tctttacgga tttttagtaa
     3481 accttgttca ggtctaacac tacctgtacc ccatttagga ccacccacag cacctaacaa
     3541 aacggcatca gccttcttgg aggcttccag cgcctcatct ggaagtggga cacctgtagc
     3601 ttcgatagca gcaccaccaa ttaaatgatt ttcgaaatcg aacttgacat tggaacgaac
     3661 atcagaaata gctttaagaa ccttaatggc ttcggctgtg atttcttgac caacgtggtc
     3721 acctggcaaa acgacgatct tcttaggggc agacattaga atggtatatc cttgaaatat
     3781 atatatatat tgctgaaatg taaaaggtaa gaaaagttag aaagtaagac gattgctaac
     3841 cacctattgg aaaaaacaat aggtccttaa ataatattgt caacttcaag tattgtgatg
     3901 caagcattta gtcatgaacg cttctctatt ctatatgaaa agccggttcc ggcgctctca
     3961 cctttccttt ttctcccaat ttttcagttg aaaaaggtat atgcgtcagg cgacctctga
     4021 aattaacaaa aaatttccag tcatcgaatt tgattctgtg cgatagcgcc cctgtgtgtt
     4081 ctcgttatgt tgaggaaaaa aataatggtt gctaagagat tcgaactctt gcatcttacg
     4141 atacctgagt attcccacag ttgggggatc tcgactctag ctagaggatc aattcgtaat
     4201 catgtcatag ctgtttcctg tgtgaaattg ttatccgctc acaattccac acaacatacg
     4261 agccggaagc ataaagtgta aagcctgggg tgcctaatga gtgagctaac tcacattaat
     4321 tgcgttgcgc tcactgcccg ctttccagtc gggaaacctg tcgtgccagc tgcattaatg
     4381 aatcggccaa cgcgcgggga gaggcggttt gcgtattggg cgctcttccg cttcctcgct
     4441 cactgactcg ctgcgctcgg tcgttcggct gcggcgagcg gtatcagctc actcaaaggc
     4501 ggtaatacgg ttatccacag aatcagggga taacgcagga aagaacatgt gagcaaaagg
     4561 ccagcaaaag gccaggaacc gtaaaaaggc cgcgttgctg gcgtttttcc ataggctccg
     4621 cccccctgac gagcatcaca aaaatcgacg ctcaagtcag aggtggcgaa acccgacagg
     4681 actataaaga taccaggcgt ttccccctgg aagctccctc gtgcgctctc ctgttccgac
     4741 cctgccgctt accggatacc tgtccgcctt tctcccttcg ggaagcgtgg cgctttctca
     4801 tagctcacgc tgtaggtatc tcagttcggt gtaggtcgtt cgctccaagc tgggctgtgt
     4861 gcacgaaccc cccgttcagc ccgaccgctg cgccttatcc ggtaactatc gtcttgagtc
     4921 caacccggta agacacgact tatcgccact ggcagcagcc actggtaaca ggattagcag
     4981 agcgaggtat gtaggcggtg ctacagagtt cttgaagtgg tggcctaact acggctacac
     5041 tagaagaaca gtatttggta tctgcgctct gctgaagcca gttaccttcg gaaaaagagt
     5101 tggtagctct tgatccggca aacaaaccac cgctggtagc ggtggttttt ttgtttgcaa
     5161 gcagcagatt acgcgcagaa aaaaaggatc tcaagaagat cctttgatct tttctacggg
     5221 gtctgacgct cagtggaacg aaaactcacg ttaagggatt ttggtcatga gattatcaaa
     5281 aaggatcttc acctagatcc ttttaaatta aaaatgaagt tttaaatcaa tctaaagtat
     5341 atatgagtaa acttggtctg acagttacca atgcttaatc agtgaggcac ctatctcagc
     5401 gatctgtcta tttcgttcat ccatagttgc ctgactcccc gtcgtgtaga taactacgat
     5461 acgggagggc ttaccatctg gccccagtgc tgcaatgata ccgcgagacc cacgctcacc
     5521 ggctccagat ttatcagcaa taaaccagcc agccggaagg gccgagcgca gaagtggtcc
     5581 tgcaacttta tccgcctcca tccagtctat taattgttgc cgggaagcta gagtaagtag
     5641 ttcgccagtt aatagtttgc gcaacgttgt tgccattgct acaggcatcg tggtgtcacg
     5701 ctcgtcgttt ggtatggctt cattcagctc cggttcccaa cgatcaaggc gagttacatg
     5761 atcccccatg ttgtgcaaaa aagcggttag ctccttcggt cctccgatcg ttgtcagaag
     5821 taagttggcc gcagtgttat cactcatggt tatggcagca ctgcataatt ctcttactgt
     5881 catgccatcc gtaagatgct tttctgtgac tggtgagtac tcaaccaagt cattctgaga
     5941 atagtgtatg cggcgaccga gttgctcttg cccggcgtca atacgggata ataccgcgcc
     6001 acatagcaga actttaaaag tgctcatcat tggaaaacgt tcttcggggc gaaaactctc
     6061 aaggatctta ccgctgttga gatccagttc gatgtaaccc actcgtgcac ccaactgatc
     6121 ttcagcatct tttactttca ccagcgtttc tgggtgagca aaaacaggaa ggcaaaatgc
     6181 cgcaaaaaag ggaataaggg cgacacggaa atgttgaata ctcatactct tcctttttca
     6241 atattattga agcatttatc agggttattg tctcatgagc ggatacatat ttgaatgtat
     6301 ttagaaaaat aaacaaatag gggttccgcg cacatttccc cgaaaagtgc cacctgacgt
     6361 ctaagaaacc attattatca tgacattaac ctataaaaat aggcgtatca cgaggccctt
     6421 tcgtctcgcg cgtttcggtg atgacggtga aaacctctga cacatgcagc tcccggagac
     6481 ggtcacagct tgtctgtaag cggatgccgg gagcagacaa gcccgtcagg gcgcgtcagc
     6541 gggtgttggc gggtgtcggg gctggcttaa ctatgcggca tcagagcaga ttgtactgag
     6601 agtgcaccat aacgcattta agcataaaca cgcactatgc cgttcttctc atgtatatat
     6661 atatacaggc aacacgcaga tataggtgcg acgtgaacag tgagctgtat gtgcgcagct
     6721 cgcgttgcat tttcggaagc gctcgttttc ggaaacgctt tgaagttcct attccgaagt
     6781 tcctattctc tagctagaaa gtataggaac ttcagagcgc ttttgaaaac caaaagcgct
     6841 ctgaagacgc actttcaaaa aaccaaaaac gcaccggact gtaacgagct actaaaatat
     6901 tgcgaatacc gcttccacaa acattgctca aaagtatctc tttgctatat atctctgtgc
     6961 tatatcccta tataacctac ccatccacct ttcgctcctt gaacttgcat ctaaactcga
     7021 cctctacatt ttttatgttt atctctagta ttactcttta gacaaaaaaa ttgtagtaag
     7081 aactattcat agagtgaatc gaaaacaata cgaaaatgta aacatttcct atacgtagta
     7141 tatagagaca aaatagaaga aaccgttcat aattttctga ccaatgaaga atcatcaacg
     7201 ctatcacttt ctgttcacaa agtatgcgca atccacatcg gtatagaata taatcgggga
     7261 tgcctttatc ttgaaaaaat gcacccgcag cttcgctagt aatcagtaaa cgcgggaagt
     7321 ggagtcaggc tttttttatg gaagagaaaa tagacaccaa agtagccttc ttctaacctt
     7381 aacggaccta cagtgcaaaa agttatcaag agactgcatt atagagcgca caaaggagaa
     7441 aaaaagtaat ctaagatgct ttgttagaaa aatagcgctc tcgggatgca tttttgtaga
     7501 acaaaaaaga agtatagatt ctttgttggt aaaatagcgc tctcgcgttg catttctgtt
     7561 ctgtaaaaat gcagctcaga ttctttgttt gaaaaattag cgctctcgcg ttgcattttt
     7621 gttttacaaa aatgaagcac agattcttcg ttggtaaaat agcgctttcg cgttgcattt
     7681 ctgttctgta aaaatgcagc tcagattctt tgtttgaaaa attagcgctc tcgcgttgca
     7741 tttttgttct acaaaatgaa gcacagatgc ttcgttgctt
//
