LOCUS       sequence-233423-        7921 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 7921)
  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 7921)
  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(F317Q)
FEATURES             Location/Qualifiers
     source          1..7921
                     /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             1903..1914
                     /note="strong bacterial ribosome binding site (Elowitz and 
                     Leibler, 2000)"
     terminator      2503..2690
                     /gene="S. cerevisiae ADH1"
                     /label=ADH1 terminator
                     /note="transcription terminator for the S. cerevisiae 
                     alcohol dehydrogenase 1 (ADH1) gene"
     primer_bind     complement(2558..2582)
                     /label=pHybLex-R
                     /note="ADH terminator, reverse primer"
     CDS             complement(2808..3902)
                     /codon_start=1
                     /gene="S. cerevisiae LEU2"
                     /product="3-isopropylmalate dehydrogenase, required for 
                     leucine biosynthesis"
                     /label=LEU2
                     /note="yeast auxotrophic marker"
                     /translation="MSAPKKIVVLPGDHVGQEITAEAIKVLKAISDVRSNVKFDFENHL
                     IGGAAFEATGVPLPDEALEASKKVDAVLLGAVGGPKWGTGSVRPEQGLLKIRKELQLYA
                     NLRPCNFASDSLLDLSPIKPQFAKGTDFVVVRELVGGIYFGKRKEDDGDGVAWDSEQYT
                     VPEVQRITRMAAFMALQHEPPLPIWSLDKANVLASSRLWRKTVEETIKNEFPTLKVQHQ
                     LIDSAAMILVKNPTHLNGIIITSNMFGDIISDEASVIPGSLGLLPSASLASLPDKNTAF
                     GLYEPCHGSAPDLPKNKVDPIATILSAAMMLKLSLNLPEEGKAIEDAVKKVLDAGIRTG
                     DLGGSNSTTEVGDAVAEEVKKILA"
     promoter        complement(3903..4307)
                     /gene="S. cerevisiae LEU2"
                     /label=LEU2 promoter
     primer_bind     complement(4349..4365)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(4349..4365)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(4362..4384)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    4373..4389
                     /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(4397..4403,4404..4421,4422..4427))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     primer_bind     complement(4580..4597)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(4751..5339)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(4831..4850)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(5510..6301,6302..6370))
                     /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     6133..6152
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(6371..6475)
                     /gene="bla"
                     /label=AmpR promoter
     primer_bind     6543..6561
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     primer_bind     complement(6599..6621)
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     primer_bind     6721..6740
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     rep_origin      6757..7921
                     /label=2u ori
                     /note="yeast 2u plasmid origin of replication"
ORIGIN
        1 gcttgcatgc aacttctttt cttttttttt cttttctctc tcccccgttg ttgtctcacc
       61 atatccgcaa tgacaaaaaa aatgatggaa gacactaaag gaaaaaatta acgacaaaga
      121 cagcaccaac agatgtcgtt gttccagagc tgatgagggg tatcttcgaa cacacgaaac
      181 tttttccttc cttcattcac gcacactact ctctaatgag caacggtata cggccttcct
      241 tccagttact tgaatttgaa ataaaaaaag tttgccgctt 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 atcgatatga acgaagcaga tgttacaaaa tttgttaata atgccaggaa
      901 aacgctgacc gatgctcaac ttttatgttc aagtgctaat ttaaggattg tagatattaa
      961 gaaaaaattg tcatcttggc agttgagtat ttcaaaactc aattttctaa tagttggctt
     1021 gagacagcaa ggaaagtttc tttacactat tttaaaggaa ggcattggga caaagctaat
     1081 ccagaaacaa tggaatcaag ctgtcttagt ggtcttagtt gatgagatga agtactggca
     1141 atatgaaatc acttctaaag tacaaaggtt ggatggtata gtaaatgaat tgagcatatc
     1201 agaaaaagat gataccgatc cctctaaact aggagattac atctcgagag acaacgttaa
     1261 cttattgaat gacaaactga aagaagtgcc agtaattgag cgtcaaattg aaaacattaa
     1321 gcttcaatac gaaaatatgg tcagaaaagt taacaaagaa ctgattgata ccaagttgac
     1381 ggacgtaact caaaaattcc aaagtaaatt tggtatagat aacctgatgg aaacaaatgt
     1441 tgcagagcag tttagcaggg aactaacgga ccttgaaaaa gatttagcag agataatgaa
     1501 ttcattgaca cagcactttg ataagacatt gctgttacaa gataaaaaaa ttgataacga
     1561 tgaacgtgag gagctgttta aggtggtaca aggcgacgac aaagaactat acaacatttt
     1621 caaaactctg catgaggtaa ttgatgacgt ggacaaaaca attcttaact tgggtcaatt
     1681 tttgcaggca aaaataaagg aaaagacaga actacacagc gaagtttctg aaataataaa
     1741 cgatttcaat agaaatttgg aatatctatt aatctttaaa gatatttcca atctgattga
     1801 tagccaaaaa aattcctgta cacaagatat tcaaacaact aaggaacttt gtgaatttta
     1861 tgataatttt gaagaaagct acggtaactt agttctagaa gcaaagagga gaaaggatgt
     1921 ggcaaacaga atgaaaacta tattgaaaga ttgtgaaaag cagttgcaga atttagatgc
     1981 tcaggaccag gaagaacgtc agaattttat agcggaaaat ggaacttatc ttcctgagac
     2041 aatctggccc ggtaaaattg acgatttttc ttccctgtac actttaaatt acaacgtgaa
     2101 gaatccttag gtcgacctgc agagatctat gaatcgtaga tactgaaaaa ccccgcaagt
     2161 tcacttcaac tgtgcatcgt gcaccatctc aatttctttc atttatacat cgttttgcct
     2221 tcttttatgt aactatactc ctctaagttt caatcttggc catgtaacct ctgatctata
     2281 gaatttttta aatgactaga attaatgccc atcttttttt tggacctaaa ttcttcatga
     2341 aaatatatta cgagggctta ttcagaagct ttggacttct tcgccagagg tttggtcaag
     2401 tctccaatca aggttgtcgg cttgtctacc ttgccagaaa tttacgaaaa gatggaaaag
     2461 ggtcaaatcg ttggtagata cgttgttgac acttctaaat aagcgaattt cttatgattt
     2521 atgattttta ttattaaata agttataaaa aaaataagtg tatacaaatt ttaaagtgac
     2581 tcttaggttt taaaacgaaa attcttgttc ttgagtaact ctttcctgta ggtcaggttg
     2641 ctttctcagg tatagcatga ggtcgctctt attgaccaca cctctaccgg catgcccgaa
     2701 attcccctac cctatgaaca tattccattt tgtaatttcg tgtcgtttct attatgaatt
     2761 tcatttataa agtttatgta caaatatcat aaaaaaagag aatcttttta agcaaggatt
     2821 ttcttaactt cttcggcgac agcatcaccg acttcggtgg tactgttgga accacctaaa
     2881 tcaccagttc tgatacctgc atccaaaacc tttttaactg catcttcaat ggccttacct
     2941 tcttcaggca agttcaatga caatttcaac atcattgcag cagacaagat agtggcgata
     3001 gggtcaacct tattctttgg caaatctgga gcagaaccgt ggcatggttc gtacaaacca
     3061 aatgcggtgt tcttgtctgg caaagaggcc aaggacgcag atggcaacaa acccaaggaa
     3121 cctgggataa cggaggcttc atcggagatg atatcaccaa acatgttgct ggtgattata
     3181 ataccattta ggtgggttgg gttcttaact aggatcatgg cggcagaatc aatcaattga
     3241 tgttgaacct tcaatgtagg aaattcgttc ttgatggttt cctccacagt ttttctccat
     3301 aatcttgaag aggccaaaac attagcttta tccaaggacc aaataggcaa tggtggctca
     3361 tgttgtaggg ccatgaaagc ggccattctt gtgattcttt gcacttctgg aacggtgtat
     3421 tgttcactat cccaagcgac accatcacca tcgtcttcct ttctcttacc aaagtaaata
     3481 cctcccacta attctctgac aacaacgaag tcagtacctt tagcaaattg tggcttgatt
     3541 ggagataagt ctaaaagaga gtcggatgca aagttacatg gtcttaagtt ggcgtacaat
     3601 tgaagttctt tacggatttt tagtaaacct tgttcaggtc taacactacc tgtaccccat
     3661 ttaggaccac ccacagcacc taacaaaacg gcatcaacct tcttggaggc ttccagcgcc
     3721 tcatctggaa gtgggacacc tgtagcttcg aaagcagcac caccaattaa atgattttcg
     3781 aaatcgaact tgacattgga acgaacatca gaaatagctt taagaacctt aatggcttcg
     3841 gctgtgattt cttgaccaac gtggtcacct ggcaaaacga cgatcttctt aggggcagac
     3901 attagaatgg tatatccttg aaatatatat atatattgct gaaatgtaaa aggtaagaaa
     3961 agttagaaag taagacgatt gctaaccacc tattggaaaa aacaataggt ccttaaataa
     4021 tattgtcaac ttcaagtatt gtgatgcaag catttagtca tgaacgcttc tctattctat
     4081 atgaaaagcc ggttccggcc tctcaccttt cctttttctc ccaatttttc agttgaaaaa
     4141 ggtatatgcg tcaggcgacc tctgaaatta acaaaaaatt tccagtcatc gaatttgatt
     4201 ctgtgcgata gcgcccctgt gtgttctcgt tatgttgagg aaaaaaataa tggttgctaa
     4261 gagattcgaa ctcttgcatc ttacgatacc tgagtattcc cacagttggg gatctcgact
     4321 ctagctagag gatcaattcg taatcatggt catagctgtt tcctgtgtga aattgttatc
     4381 cgctcacaat tccacacaac atacgagccg gaagcataaa gtgtaaagcc tggggtgcct
     4441 aatgagtgag gtaactcaca ttaattgcgt tgcgctcact gcccgctttc cagtcgggaa
     4501 acctgtcgtg ccagctggat taatgaatcg gccaacgcgc ggggagaggc ggtttgcgta
     4561 ttgggcgctc ttccgcttcc tcgctcactg actcgctgcg ctcggtcgtt cggctgcggc
     4621 gagcggtatc agctcactca aaggcggtaa tacggttatc cacagaatca ggggataacg
     4681 caggaaagaa catgtgagca aaaggccagc aaaaggccag gaaccgtaaa aaggccgcgt
     4741 tgctggcgtt tttccatagg ctccgccccc ctgacgagca tcacaaaaat cgacgctcaa
     4801 gtcagaggtg gcgaaacccg acaggactat aaagatacca ggcgtttccc cctggaagct
     4861 ccctcgtgcg ctctcctgtt ccgaccctgc cgcttaccgg atacctgtcc gcctttctcc
     4921 cttcgggaag cgtggcgctt tctcatagct cacgctgtag gtatctcagt tcggtgtagg
     4981 tcgttcgctc caagctgggc tgtgtgcacg aaccccccgt tcagcccgac cgctgcgcct
     5041 tatccggtaa ctatcgtctt gagtccaacc cggtaagaca cgacttatcg ccactggcag
     5101 cagccactgg taacaggatt agcagagcga ggtatgtagg cggtgctaca gagttcttga
     5161 agtggtggcc taactacggc tacactagaa ggacagtatt tggtatctgc gctctgctga
     5221 agccagttac cttcggaaaa agagttggta gctcttgatc cggcaaacaa accaccgctg
     5281 gtagcggtgg tttttttgtt tgcaagcagc agattacgcg cagaaaaaaa ggatctcaag
     5341 aagatccttt gatcttttct acggggtctg acgctcagtg gaacgaaaac tcacgttaag
     5401 ggattttggt catgagatta tcaaaaagga tcttcaccta gatcctttta aattaaaaat
     5461 gaagttttaa atcaatctaa agtatatatg agtaaacttg gtctgacagt taccaatgct
     5521 taatcagtga ggcacctatc tcagcgatct gtctatttcg ttcatccata gttgcctgac
     5581 tccccgtcgt gtagataact acgatacggg agggcttacc atctggcccc agtgctgcaa
     5641 tgataccgcg agacccacgc tcaccggctc cagatttatc agcaataaac cagccagccg
     5701 gaagggccga gcgcagaagt ggtcctgcaa ctttatccgc ctccatccag tctattaatt
     5761 gttgccggga agctagagta agtagttcgc cagttaatag tttgcgcaac gttgttgcca
     5821 ttgctacagg catcgtggtg tcacgctcgt cgtttggtat ggcttcattc agctccggtt
     5881 cccaacgatc aaggcgagtt acatgatccc ccatgttgtg caaaaaagcg gttagctcct
     5941 tcggtcctcc gatcgttgtc agaagtaagt tggccgcagt gttatcactc atggttatgg
     6001 cagcactgca taattctctt actgtcatgc catccgtaag atgcttttct gtgactggtg
     6061 agtactcaac caagtcattc tgagaatagt gtatgcggcg accgagttgc tcttgcccgg
     6121 cgtcaatacg ggataatacc gcgccacata gcagaacttt aaaagtgctc atcattggaa
     6181 aacgttcttc ggggcgaaaa ctctcaagga tcttaccgct gttgagatcc agttcgatgt
     6241 aacccactcg tgcacccaac tgatcttcag catcttttac tttcaccagc gtttctgggt
     6301 gagcaaaaac aggaaggcaa aatgccgcaa aaaagggaat aagggcgaca cggaaatgtt
     6361 gaatactcat actcttcctt tttcaatatt attgaagcat ttatcagggt tattgtctca
     6421 tgagcggata catatttgaa tgtatttaga aaaataaaca aataggggtt ccgcgcacat
     6481 ttccccgaaa agtgccacct gacgtctaag aaaccattat tatcatgaca ttaacctata
     6541 aaaataggcg tatcacgagg ccctttcgtc tcgcgcgttt cggtgatgac ggtgaaaacc
     6601 tctgacacat gcagctcccg gagacggtca cagcttgtct gtaagcggat gccgggagca
     6661 gacaagcccg tcagggcgcg tcagcgggtg ttggcgggtg tcggggctgg cttaactatg
     6721 cggcatcaga gcagattgta ctgagagtgc accataacgc atttaagcat aaacacgcac
     6781 tatgccgttc ttctcatgta tatatatata caggcaacac gcagatatag gtgcgacgtg
     6841 aacagtgagc tgtatgtgcg cagctcgcgt tgcattttcg gaagcgctcg ttttcggaaa
     6901 cgctttgaag ttcctattcc gaagttccta ttctctagct agaaagtata ggaacttcag
     6961 agcgcttttg aaaaccaaaa gcgctctgaa gacgcacttt caaaaaacca aaaacgcacc
     7021 ggactgtaac gagctactaa aatattgcga ataccgcttc cacaaacatt gctcaaaagt
     7081 atctctttgc tatatatctc tgtgctatat ccctatataa cctacccatc cacctttcgc
     7141 tccttgaact tgcatctaaa ctcgacctct acatttttta tgtttatctc tagtattact
     7201 ctttagacaa aaaaattgta gtaagaacta ttcatagagt gaatcgaaaa caatacgaaa
     7261 atgtaaacat ttcctatacg tagtatatag agacaaaata gaagaaaccg ttcataattt
     7321 tctgaccaat gaagaatcat caacgctatc actttctgtt cacaaagtat gcgcaatcca
     7381 catcggtata gaatataatc ggggatgcct ttatcttgaa aaaatgcacc cgcagcttcg
     7441 ctagtaatca gtaaacgcgg gaagtggagt caggcttttt ttatggaaga gaaaatagac
     7501 accaaagtag ccttcttcta accttaacgg acctacagtg caaaaagtta tcaagagact
     7561 gcattataga gcgcacaaag gagaaaaaaa gtaatctaag atgctttgtt agaaaaatag
     7621 cgctctcggg atgcattttt gtagaacaaa aaagaagtat agattctttg ttggtaaaat
     7681 agcgctctcg cgttgcattt ctgttctgta aaaatgcagc tcagattctt tgtttgaaaa
     7741 attagcgctc tcgcgttgca tttttgtttt acaaaaatga agcacagatt cttcgttggt
     7801 aaaatagcgc tttcgcgttg catttctgtt ctgtaaaaat gcagctcaga ttctttgttt
     7861 gaaaaattag cgctctcgcg ttgcattttt gttctacaaa atgaagcaca gatgcttcgt
     7921 t
//
