LOCUS       sequence-236917-        7933 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 7933)
  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 7933)
  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(I98E)
FEATURES             Location/Qualifiers
     source          1..7933
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     promoter        5..406
                     /gene="S. cerevisiae ADH1"
                     /label=ADH1 promoter
                     /note="promoter for alcohol dehydrogenase 1"
     CDS             452..472
                     /codon_start=1
                     /product="nuclear localization signal of SV40 (simian virus
                     40) large T antigen"
                     /label=SV40 NLS
                     /translation="PKKKRKV"
     CDS             488..829
                     /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             1900..1911
                     /note="strong bacterial ribosome binding site (Elowitz and 
                     Leibler, 2000)"
     terminator      2511..2698
                     /gene="S. cerevisiae ADH1"
                     /label=ADH1 terminator
                     /note="transcription terminator for the S. cerevisiae 
                     alcohol dehydrogenase 1 (ADH1) gene"
     primer_bind     complement(2566..2590)
                     /label=pHybLex-R
                     /note="ADH terminator, reverse primer"
     CDS             complement(2819..3913)
                     /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(3914..4319)
                     /gene="S. cerevisiae LEU2"
                     /label=LEU2 promoter
     primer_bind     complement(4361..4377)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(4361..4377)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(4374..4396)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    4385..4401
                     /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(4409..4415,4416..4433,4434..4439))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    4454..4475
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(4592..4609)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(4763..5351)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(4843..4862)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(5522..6313,6314..6382))
                     /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     6145..6164
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(6383..6487)
                     /gene="bla"
                     /label=AmpR promoter
     primer_bind     6555..6573
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     primer_bind     complement(6611..6633)
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     primer_bind     6733..6752
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     rep_origin      6769..7933
                     /label=2u ori
                     /note="yeast 2u plasmid origin of replication"
ORIGIN
        1 gcttgcatgc aacttctttt cttttttttt cttttctctc tcccccgttg ttgtctcacc
       61 atatccgcaa tgacaaaaaa atgatggaag acactaaagg aaaaaattaa cgacaaagac
      121 agcaccaaca gatgtcgttg ttccagagct gatgaggggt atctcgaagc acacgaaact
      181 ttttccttcc ttcattcacg cacactactc tctaatgagc aacggtatac ggccttcctt
      241 ccagttactt gaatttgaaa taaaaaaaag tttgctgtct tgctatcaag tataaataga
      301 cctgcaatta ttaatctttt gtttcctcgt cattgttctc gttccctttc ttccttgttt
      361 ctttttctgc acaatatttc aagctatacc aagcatacaa tcaactccaa gctttgcaaa
      421 gatggataaa gcggaattaa ttcccgagcc tccaaaaaag aagagaaagg tcgaattggg
      481 taccgccgcc aattttaatc aaagtgggaa tattgctgat agctcattgt ccttcacttt
      541 cactaacagt agcaacggtc cgaacctcat aacaactcaa acaaattctc aagcgctttc
      601 acaaccaatt gcctcctcta acgttcatga taacttcatg aataatgaaa tcacggctag
      661 taaaattgat gatggtaata attcaaaacc actgtcacct ggttggacgg accaaactgc
      721 gtataacgcg tttggaatca ctacagggat gtttaatacc actacaatgg atgatgtata
      781 taactatcta ttcgatgatg aagatacccc accaaaccca aaaaaagaga tcgaattccc
      841 cgggggatcc atcgataacg aagcagatgt tacaaaattt gttaataatg ccaggaaaac
      901 gctgaccgat gctcaacttt tatgttcaag tgctaattta aggattgtag atattaagaa
      961 aaaattgtca tcttggcagt tgagtatttc aaaactcaat tttctaatag ttggcttgag
     1021 acagcaagga aagtttcttt acactatttt aaaggaaggc attgggacaa agctaatcca
     1081 gaaacaatgg aatcaagctg tcttagtggt cttagttgat gagatgaagt actggcaata
     1141 tgaagagact tctaaagtac aaaggttgga tggtatagta aatgaattga gcatatcaga
     1201 aaaagatgat accgatccct ctaaactagg agattacatc tcgagagaca acgttaactt
     1261 attgaatgac aaactgaaag aagtgccagt aattgagcgt caaattgaaa acattaagct
     1321 tcaatacgaa aatatggtca gaaaagttaa caaagaactg attgatacca agttgacgga
     1381 cgtaactcaa aaattccaaa gtaaatttgg tatagataac ctgatggaaa caaatgttgc
     1441 agagcagttt agcagggaac taacggacct tgaaaaagat ttagcagaga taatgaattc
     1501 attgacacag cactttgata agacattgct gttacaagat aaaaaaattg ataacgatga
     1561 acgtgaggag ctgtttaagg tggtacaagg cgacgacaaa gaactataca acattttcaa
     1621 aactctgcat gaggtaattg atgacgtgga caaaacaatt cttaacttgg gtcaattttt
     1681 gcaggcaaaa ataaaggaaa agacagaact acacagcgaa gtttctgaaa taataaacga
     1741 tttcaataga aatttggaat atctattaat ctttaaagat atttccaatc tgattgatag
     1801 cttcaaaaat tcctgtacac aagatattca aacaactaag gaactttgtg aattttatga
     1861 taattttgaa gaaagctacg gtaacttagt tctagaagca aagaggagaa aggatgtggc
     1921 aaacagaatg aaaactatat tgaaagattg tgaaaagcag ttgcagaatt tagatgctca
     1981 ggaccaggaa gaacgtcaga attttatagc ggaaaatgga acttatcttc ctgagacaat
     2041 ctggcccggt aaaattgacg atttttcttc cctgtacact ttaaattaca acgtgaagaa
     2101 tccttagaaa aaaggaaaaa aaggctgcag agatctatga atcgtagata ctgaaaaacc
     2161 ccgcaagttc acttcaactg tgcatcgtgc accatctcaa tttctttcat ttatacatcg
     2221 ttttgccttc ttttatgtaa ctatactcct ctaagtttca atcttggcca tgtaacctct
     2281 gatctataga attttttaaa tgactagaat taatgcccat cttttttttg gacctaaatt
     2341 cttcatgaaa atatattacg agggcttatt cagaagcttt ggacttcttc gccagaggtt
     2401 tggtcaagtc tccaatcaag gttgtcggct tgtctacctt gccagaaatt tacgaaaaga
     2461 tggaaaaggg tcaaatcgtt ggtagatacg ttgttgacac ttctaaataa gcgaatttct
     2521 tatgatttat gatttttatt attaaataag ttataaaaaa aataagtgta tacaaatttt
     2581 aaagtgactc ttaggtttta aaacgaaaat tcttattctt gagtaactct ttcctgtagg
     2641 tcaggttgct ttctcaggta tagcatgagg tcgctcttat tgaccacacc tctaccggca
     2701 tgccggtcga aattccccta ccctatgaac atattccatt ttgtaatttc gtgtcgtttc
     2761 tattatgaat ttcatttata aagtttatgt acaaatatca taaaaaaaga gaatcttttt
     2821 aagcaaggat tttcttaacc tcttcggcga cagcatcacc gacttcggtg gtactgttgg
     2881 aaccacctaa atcaccagtt ctgatacctg catccaaaac ctttttaact gcatcttcaa
     2941 tggccttacc ttcttcaggc aagttcaatg acaatttcaa catcattgca gcagacaaga
     3001 tagtggcgat agggttgacc ttattctttg gcaaatctgg agcagaaccg tggcatggtt
     3061 cgtacaaacc aaatgcggtg ttcttgtctg gcaaagaggc caaggacgca gatggcaaca
     3121 aacccaagga acctgggata acggaggctt catcggagac gatatcacca aacatgttgc
     3181 tggtgattat aataccattt aggtgggttg ggttcctaac taggatcatg gcggcagaat
     3241 caatcaattg atgttgaacc ttcaatgtag gaaattcgtt cttgatggtt tcctccacag
     3301 tttttctcca taatcttgaa gaggccaaaa cattagcttt atccaaggac caaataggca
     3361 atggtggctc atgttgtagg gccatgaaag cggccattct tgtgattctt tgcacttctg
     3421 gaacggtgta ttgttcacta tcccaagcga caccatcacc atcgtcttcc tttctcttac
     3481 caaagtaaat acctcccact aattctctga caacaacgaa gtcagtacct ttagcaaatt
     3541 gtggcttgat tggagataag tctaaaagag agtcggatgc aaagttacat ggtcttaagt
     3601 tggcgtacaa ttgaagttct ttacggattt ttagtaaacc ttgttcaggt ctaacactac
     3661 ctgtacccca tttaggacca cccacagcac ctaacaaaac ggcatcagcc ttcttggagg
     3721 cttccagcgc ctcatctgga agtgggacac ctgtagcttc gatagcagca ccaccaatta
     3781 aatgattttc gaaatcgaac ttgacattgg aacgaacatc agaaatagct ttaagaacct
     3841 taatggcttc ggctgtgatt tcttgaccaa cgtggtcacc tggcaaaacg acgatcttct
     3901 taggggcaga cattagaatg gtatatcctt gaaatatata tatatattgc tgaaatgtaa
     3961 aaggtaagaa aagttagaaa gtaagacgat tgctaaccac ctattggaaa aaacaatagg
     4021 tccttaaata atattgtcaa cttcaagtat tgtgatgcaa gcatttagtc atgaacgctt
     4081 ctctattcta tatgaaaagc cggttccggc gctctcacct ttcctttttc tcccaatttt
     4141 tcagttgaaa aaggtatatg cgtcaggcga cctctgaaat taacaaaaaa tttccagtca
     4201 tcgaatttga ttctgtgcga tagcgcccct gtgtgttctc gttatgttga ggaaaaaaat
     4261 aatggttgct aagagattcg aactcttgca tcttacgata cctgagtatt cccacagttg
     4321 ggggatctcg actctagcta gaggatcaat tcgtaatcat gtcatagctg tttcctgtgt
     4381 gaaattgtta tccgctcaca attccacaca acatacgagc cggaagcata aagtgtaaag
     4441 cctggggtgc ctaatgagtg agctaactca cattaattgc gttgcgctca ctgcccgctt
     4501 tccagtcggg aaacctgtcg tgccagctgc attaatgaat cggccaacgc gcggggagag
     4561 gcggtttgcg tattgggcgc tcttccgctt cctcgctcac tgactcgctg cgctcggtcg
     4621 ttcggctgcg gcgagcggta tcagctcact caaaggcggt aatacggtta tccacagaat
     4681 caggggataa cgcaggaaag aacatgtgag caaaaggcca gcaaaaggcc aggaaccgta
     4741 aaaaggccgc gttgctggcg tttttccata ggctccgccc ccctgacgag catcacaaaa
     4801 atcgacgctc aagtcagagg tggcgaaacc cgacaggact ataaagatac caggcgtttc
     4861 cccctggaag ctccctcgtg cgctctcctg ttccgaccct gccgcttacc ggatacctgt
     4921 ccgcctttct cccttcggga agcgtggcgc tttctcatag ctcacgctgt aggtatctca
     4981 gttcggtgta ggtcgttcgc tccaagctgg gctgtgtgca cgaacccccc gttcagcccg
     5041 accgctgcgc cttatccggt aactatcgtc ttgagtccaa cccggtaaga cacgacttat
     5101 cgccactggc agcagccact ggtaacagga ttagcagagc gaggtatgta ggcggtgcta
     5161 cagagttctt gaagtggtgg cctaactacg gctacactag aagaacagta tttggtatct
     5221 gcgctctgct gaagccagtt accttcggaa aaagagttgg tagctcttga tccggcaaac
     5281 aaaccaccgc tggtagcggt ggtttttttg tttgcaagca gcagattacg cgcagaaaaa
     5341 aaggatctca agaagatcct ttgatctttt ctacggggtc tgacgctcag tggaacgaaa
     5401 actcacgtta agggattttg gtcatgagat tatcaaaaag gatcttcacc tagatccttt
     5461 taaattaaaa atgaagtttt aaatcaatct aaagtatata tgagtaaact tggtctgaca
     5521 gttaccaatg cttaatcagt gaggcaccta tctcagcgat ctgtctattt cgttcatcca
     5581 tagttgcctg actccccgtc gtgtagataa ctacgatacg ggagggctta ccatctggcc
     5641 ccagtgctgc aatgataccg cgagacccac gctcaccggc tccagattta tcagcaataa
     5701 accagccagc cggaagggcc gagcgcagaa gtggtcctgc aactttatcc gcctccatcc
     5761 agtctattaa ttgttgccgg gaagctagag taagtagttc gccagttaat agtttgcgca
     5821 acgttgttgc cattgctaca ggcatcgtgg tgtcacgctc gtcgtttggt atggcttcat
     5881 tcagctccgg ttcccaacga tcaaggcgag ttacatgatc ccccatgttg tgcaaaaaag
     5941 cggttagctc cttcggtcct ccgatcgttg tcagaagtaa gttggccgca gtgttatcac
     6001 tcatggttat ggcagcactg cataattctc ttactgtcat gccatccgta agatgctttt
     6061 ctgtgactgg tgagtactca accaagtcat tctgagaata gtgtatgcgg cgaccgagtt
     6121 gctcttgccc ggcgtcaata cgggataata ccgcgccaca tagcagaact ttaaaagtgc
     6181 tcatcattgg aaaacgttct tcggggcgaa aactctcaag gatcttaccg ctgttgagat
     6241 ccagttcgat gtaacccact cgtgcaccca actgatcttc agcatctttt actttcacca
     6301 gcgtttctgg gtgagcaaaa acaggaaggc aaaatgccgc aaaaaaggga ataagggcga
     6361 cacggaaatg ttgaatactc atactcttcc tttttcaata ttattgaagc atttatcagg
     6421 gttattgtct catgagcgga tacatatttg aatgtattta gaaaaataaa caaatagggg
     6481 ttccgcgcac atttccccga aaagtgccac ctgacgtcta agaaaccatt attatcatga
     6541 cattaaccta taaaaatagg cgtatcacga ggccctttcg tctcgcgcgt ttcggtgatg
     6601 acggtgaaaa cctctgacac atgcagctcc cggagacggt cacagcttgt ctgtaagcgg
     6661 atgccgggag cagacaagcc cgtcagggcg cgtcagcggg tgttggcggg tgtcggggct
     6721 ggcttaacta tgcggcatca gagcagattg tactgagagt gcaccataac gcatttaagc
     6781 ataaacacgc actatgccgt tcttctcatg tatatatata tacaggcaac acgcagatat
     6841 aggtgcgacg tgaacagtga gctgtatgtg cgcagctcgc gttgcatttt cggaagcgct
     6901 cgttttcgga aacgctttga agttcctatt ccgaagttcc tattctctag ctagaaagta
     6961 taggaacttc agagcgcttt tgaaaaccaa aagcgctctg aagacgcact ttcaaaaaac
     7021 caaaaacgca ccggactgta acgagctact aaaatattgc gaataccgct tccacaaaca
     7081 ttgctcaaaa gtatctcttt gctatatatc tctgtgctat atccctatat aacctaccca
     7141 tccacctttc gctccttgaa cttgcatcta aactcgacct ctacattttt tatgtttatc
     7201 tctagtatta ctctttagac aaaaaaattg tagtaagaac tattcataga gtgaatcgaa
     7261 aacaatacga aaatgtaaac atttcctata cgtagtatat agagacaaaa tagaagaaac
     7321 cgttcataat tttctgacca atgaagaatc atcaacgcta tcactttctg ttcacaaagt
     7381 atgcgcaatc cacatcggta tagaatataa tcggggatgc ctttatcttg aaaaaatgca
     7441 cccgcagctt cgctagtaat cagtaaacgc gggaagtgga gtcaggcttt ttttatggaa
     7501 gagaaaatag acaccaaagt agccttcttc taaccttaac ggacctacag tgcaaaaagt
     7561 tatcaagaga ctgcattata gagcgcacaa aggagaaaaa aagtaatcta agatgctttg
     7621 ttagaaaaat agcgctctcg ggatgcattt ttgtagaaca aaaaagaagt atagattctt
     7681 tgttggtaaa atagcgctct cgcgttgcat ttctgttctg taaaaatgca gctcagattc
     7741 tttgtttgaa aaattagcgc tctcgcgttg catttttgtt ttacaaaaat gaagcacaga
     7801 ttcttcgttg gtaaaatagc gctttcgcgt tgcatttctg ttctgtaaaa atgcagctca
     7861 gattctttgt ttgaaaaatt agcgctctcg cgttgcattt ttgttctaca aaatgaagca
     7921 cagatgcttc gtt
//
