LOCUS       sequence-239638-        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(F317D, I325A)
FEATURES             Location/Qualifiers
     source          1..7933
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     promoter        113..514
                     /gene="S. cerevisiae ADH1"
                     /label=ADH1 promoter
                     /note="promoter for alcohol dehydrogenase 1"
     CDS             560..580
                     /codon_start=1
                     /product="nuclear localization signal of SV40 (simian virus
                     40) large T antigen"
                     /label=SV40 NLS
                     /translation="PKKKRKV"
     CDS             596..937
                     /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             2008..2019
                     /note="strong bacterial ribosome binding site (Elowitz and 
                     Leibler, 2000)"
     terminator      2619..2806
                     /gene="S. cerevisiae ADH1"
                     /label=ADH1 terminator
                     /note="transcription terminator for the S. cerevisiae 
                     alcohol dehydrogenase 1 (ADH1) gene"
     primer_bind     complement(2674..2698)
                     /label=pHybLex-R
                     /note="ADH terminator, reverse primer"
     CDS             complement(2927..4021)
                     /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(4022..4427)
                     /gene="S. cerevisiae LEU2"
                     /label=LEU2 promoter
     primer_bind     complement(4469..4485)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(4469..4485)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(4482..4504)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    4493..4509
                     /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(4517..4523,4524..4541,4542..4547))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    4562..4583
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(4700..4717)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(4871..5459)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(4951..4970)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(5630..6421,6422..6490))
                     /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     6253..6272
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(6491..6595)
                     /gene="bla"
                     /label=AmpR promoter
     primer_bind     6663..6681
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     primer_bind     complement(6719..6741)
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     primer_bind     6841..6860
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     rep_origin      join(6877..7933,1..108)
                     /label=2u ori
                     /note="yeast 2u plasmid origin of replication"
ORIGIN
        1 cgcgttgcat ttctgttctg taaaaatgca gctcagattc tttgtttgaa aaattagcgc
       61 tctcgcgttg catttttgtt ctacaaaatg aagcacagat gcttcgttgc ttgcatgcaa
      121 cttcttttct ttttttttct tttctctctc ccccgttgtt gtctcaccat atccgcaatg
      181 acaaaaaaat gatggaagac actaaaggaa aaaattaacg acaaagacag caccaacaga
      241 tgtcgttgtt ccagagctga tgaggggtat ctcgaagcac acgaaacttt ttccttcctt
      301 cattcacgca cactactctc taatgagcaa cggtatacgg ccttccttcc agttacttga
      361 atttgaaata aaaaaaagtt tgctgtcttg ctatcaagta taaatagacc tgcaattatt
      421 aatcttttgt ttcctcgtca ttgttctcgt tccctttctt ccttgtttct ttttctgcac
      481 aatatttcaa gctataccaa gcatacaatc aactccaagc tttgcaaaga tggataaagc
      541 ggaattaatt cccgagcctc caaaaaagaa gagaaaggtc gaattgggta ccgccgccaa
      601 ttttaatcaa agtgggaata ttgctgatag ctcattgtcc ttcactttca ctaacagtag
      661 caacggtccg aacctcataa caactcaaac aaattctcaa gcgctttcac aaccaattgc
      721 ctcctctaac gttcatgata acttcatgaa taatgaaatc acggctagta aaattgatga
      781 tggtaataat tcaaaaccac tgtcacctgg ttggacggac caaactgcgt ataacgcgtt
      841 tggaatcact acagggatgt ttaataccac tacaatggat gatgtatata actatctatt
      901 cgatgatgaa gataccccac caaacccaaa aaaagagatc gaattccccg ggggatccat
      961 cgataacgaa gcagatgtta caaaatttgt taataatgcc aggaaaacgc tgaccgatgc
     1021 tcaactttta tgttcaagtg ctaatttaag gattgtagat attaagaaaa aattgtcatc
     1081 ttggcagttg agtatttcaa aactcaattt tctaatagtt ggcttgagac agcaaggaaa
     1141 gtttctttac actattttaa aggaaggcat tgggacaaag ctaatccaga aacaatggaa
     1201 tcaagctgtc ttagtggtct tagttgatga gatgaagtac tggcaatatg aaatcacttc
     1261 taaagtacaa aggttggatg gtatagtaaa tgaattgagc atatcagaaa aagatgatac
     1321 cgatccctct aaactaggag attacatctc gagagacaac gttaacttat tgaatgacaa
     1381 actgaaagaa gtgccagtaa ttgagcgtca aattgaaaac attaagcttc aatacgaaaa
     1441 tatggtcaga aaagttaaca aagaactgat tgataccaag ttgacggacg taactcaaaa
     1501 attccaaagt aaatttggta tagataacct gatggaaaca aatgttgcag agcagtttag
     1561 cagggaacta acggaccttg aaaaagattt agcagagata atgaattcat tgacacagca
     1621 ctttgataag acattgctgt tacaagataa aaaaattgat aacgatgaac gtgaggagct
     1681 gtttaaggtg gtacaaggcg acgacaaaga actatacaac attttcaaaa ctctgcatga
     1741 ggtaattgat gacgtggaca aaacaattct taacttgggt caatttttgc aggcaaaaat
     1801 aaaggaaaag acagaactac acagcgaagt ttctgaaata ataaacgatt tcaatagaaa
     1861 tttggaatat ctattaatct ttaaagatat ttccaatctg attgatagcg acaaaaattc
     1921 ctgtacacaa gatgctcaaa caactaagga actttgtgaa ttttatgata attttgaaga
     1981 aagctacggt aacttagttc tagaagcaaa gaggagaaag gatgtggcaa acagaatgaa
     2041 aactatattg aaagattgtg aaaagcagtt gcagaattta gatgctcagg accaggaaga
     2101 acgtcagaat tttatagcgg aaaatggaac ttatcttcct gagacaatct ggcccggtaa
     2161 aattgacgat ttttcttccc tgtacacttt aaattacaac gtgaagaatc cttagaaaaa
     2221 aggaaaaaaa ggctgcagag atctatgaat cgtagatact gaaaaacccc gcaagttcac
     2281 ttcaactgtg catcgtgcac catctcaatt tctttcattt atacatcgtt ttgccttctt
     2341 ttatgtaact atactcctct aagtttcaat cttggccatg taacctctga tctatagaat
     2401 tttttaaatg actagaatta atgcccatct tttttttgga cctaaattct tcatgaaaat
     2461 atattacgag ggcttattca gaagctttgg acttcttcgc cagaggtttg gtcaagtctc
     2521 caatcaaggt tgtcggcttg tctaccttgc cagaaattta cgaaaagatg gaaaagggtc
     2581 aaatcgttgg tagatacgtt gttgacactt ctaaataagc gaatttctta tgatttatga
     2641 tttttattat taaataagtt ataaaaaaaa taagtgtata caaattttaa agtgactctt
     2701 aggttttaaa acgaaaattc ttattcttga gtaactcttt cctgtaggtc aggttgcttt
     2761 ctcaggtata gcatgaggtc gctcttattg accacacctc taccggcatg ccggtcgaaa
     2821 ttcccctacc ctatgaacat attccatttt gtaatttcgt gtcgtttcta ttatgaattt
     2881 catttataaa gtttatgtac aaatatcata aaaaaagaga atctttttaa gcaaggattt
     2941 tcttaacctc ttcggcgaca gcatcaccga cttcggtggt actgttggaa ccacctaaat
     3001 caccagttct gatacctgca tccaaaacct ttttaactgc atcttcaatg gccttacctt
     3061 cttcaggcaa gttcaatgac aatttcaaca tcattgcagc agacaagata gtggcgatag
     3121 ggttgacctt attctttggc aaatctggag cagaaccgtg gcatggttcg tacaaaccaa
     3181 atgcggtgtt cttgtctggc aaagaggcca aggacgcaga tggcaacaaa cccaaggaac
     3241 ctgggataac ggaggcttca tcggagacga tatcaccaaa catgttgctg gtgattataa
     3301 taccatttag gtgggttggg ttcctaacta ggatcatggc ggcagaatca atcaattgat
     3361 gttgaacctt caatgtagga aattcgttct tgatggtttc ctccacagtt tttctccata
     3421 atcttgaaga ggccaaaaca ttagctttat ccaaggacca aataggcaat ggtggctcat
     3481 gttgtagggc catgaaagcg gccattcttg tgattctttg cacttctgga acggtgtatt
     3541 gttcactatc ccaagcgaca ccatcaccat cgtcttcctt tctcttacca aagtaaatac
     3601 ctcccactaa ttctctgaca acaacgaagt cagtaccttt agcaaattgt ggcttgattg
     3661 gagataagtc taaaagagag tcggatgcaa agttacatgg tcttaagttg gcgtacaatt
     3721 gaagttcttt acggattttt agtaaacctt gttcaggtct aacactacct gtaccccatt
     3781 taggaccacc cacagcacct aacaaaacgg catcagcctt cttggaggct tccagcgcct
     3841 catctggaag tgggacacct gtagcttcga tagcagcacc accaattaaa tgattttcga
     3901 aatcgaactt gacattggaa cgaacatcag aaatagcttt aagaacctta atggcttcgg
     3961 ctgtgatttc ttgaccaacg tggtcacctg gcaaaacgac gatcttctta ggggcagaca
     4021 ttagaatggt atatccttga aatatatata tatattgctg aaatgtaaaa ggtaagaaaa
     4081 gttagaaagt aagacgattg ctaaccacct attggaaaaa acaataggtc cttaaataat
     4141 attgtcaact tcaagtattg tgatgcaagc atttagtcat gaacgcttct ctattctata
     4201 tgaaaagccg gttccggcgc tctcaccttt cctttttctc ccaatttttc agttgaaaaa
     4261 ggtatatgcg tcaggcgacc tctgaaatta acaaaaaatt tccagtcatc gaatttgatt
     4321 ctgtgcgata gcgcccctgt gtgttctcgt tatgttgagg aaaaaaataa tggttgctaa
     4381 gagattcgaa ctcttgcatc ttacgatacc tgagtattcc cacagttggg ggatctcgac
     4441 tctagctaga ggatcaattc gtaatcatgt catagctgtt tcctgtgtga aattgttatc
     4501 cgctcacaat tccacacaac atacgagccg gaagcataaa gtgtaaagcc tggggtgcct
     4561 aatgagtgag ctaactcaca ttaattgcgt tgcgctcact gcccgctttc cagtcgggaa
     4621 acctgtcgtg ccagctgcat taatgaatcg gccaacgcgc ggggagaggc ggtttgcgta
     4681 ttgggcgctc ttccgcttcc tcgctcactg actcgctgcg ctcggtcgtt cggctgcggc
     4741 gagcggtatc agctcactca aaggcggtaa tacggttatc cacagaatca ggggataacg
     4801 caggaaagaa catgtgagca aaaggccagc aaaaggccag gaaccgtaaa aaggccgcgt
     4861 tgctggcgtt tttccatagg ctccgccccc ctgacgagca tcacaaaaat cgacgctcaa
     4921 gtcagaggtg gcgaaacccg acaggactat aaagatacca ggcgtttccc cctggaagct
     4981 ccctcgtgcg ctctcctgtt ccgaccctgc cgcttaccgg atacctgtcc gcctttctcc
     5041 cttcgggaag cgtggcgctt tctcatagct cacgctgtag gtatctcagt tcggtgtagg
     5101 tcgttcgctc caagctgggc tgtgtgcacg aaccccccgt tcagcccgac cgctgcgcct
     5161 tatccggtaa ctatcgtctt gagtccaacc cggtaagaca cgacttatcg ccactggcag
     5221 cagccactgg taacaggatt agcagagcga ggtatgtagg cggtgctaca gagttcttga
     5281 agtggtggcc taactacggc tacactagaa gaacagtatt tggtatctgc gctctgctga
     5341 agccagttac cttcggaaaa agagttggta gctcttgatc cggcaaacaa accaccgctg
     5401 gtagcggtgg tttttttgtt tgcaagcagc agattacgcg cagaaaaaaa ggatctcaag
     5461 aagatccttt aatcttttct acggggtctg acgctcagtg gaacgaaaac tcacgttaag
     5521 ggattttggt catgagatta tcaaaaagga tcttcaccta gatcctttta aattaaaaat
     5581 gaagttttaa atcaatctaa agtatatatg agtaaacttg gtctgacagt taccaatgct
     5641 taatcagtga ggcacctatc tcagcgatct gtctatttcg ttcatccata gttgcctgac
     5701 tccccgtcgt gtagataact acgatacggg agggcttacc atctggcccc agtgctgcaa
     5761 tgataccgcg agacccacgc tcaccggctc cagatttatc agcaataaac cagccagccg
     5821 gaagggccga gcgcagaagt ggtcctgcaa ctttatccgc ctccatccag tctattaatt
     5881 gttgccggga agctagagta agtagttcgc cagttaatag tttgcgcaac gttgttgcca
     5941 ttgctacagg catcgtggtg tcacgctcgt cgtttggtat ggcttcattc agctccggtt
     6001 cccaacgatc aaggcgagtt acatgatccc ccatgttgtg caaaaaagcg gttagctcct
     6061 tcggtcctcc gatcgttgtc agaagtaagt tggccgcagt gttatcactc atggttatgg
     6121 cagcactgca taattctctt actgtcatgc catccgtaag atgcttttct gtgactggtg
     6181 agtactcaac caagtcattc tgagaatagt gtatgcggcg accgagttgc tcttgcccgg
     6241 cgtcaatacg ggataatacc gcgccacata gcagaacttt aaaagtgctc atcattggaa
     6301 aacgttcttc ggggcgaaaa ctctcaagga tcttaccgct gttgagatcc agttcgatgt
     6361 aacccactcg tgcacccaac tgatcttcag catcttttac tttcaccagc gtttctgggt
     6421 gagcaaaaac aggaaggcaa aatgccgcaa aaaagggaat aagggcgaca cggaaatgtt
     6481 gaatactcat actcttcctt tttcaatatt attgaagcat ttatcagggt tattgtctca
     6541 tgagcggata catatttgaa tgtatttaga aaaataaaca aataggggtt ccgcgcacat
     6601 ttccccgaaa agtgccacct gacgtctaag aaaccattat tatcatgaca ttaacctata
     6661 aaaataggcg tatcacgagg ccctttcgtc tcgcgcgttt cggtgatgac ggtgaaaacc
     6721 tctgacacat gcagctcccg gagacggtca cagcttgtct gtaagcggat gccgggagca
     6781 gacaagcccg tcagggcgcg tcagcgggtg ttggcgggtg tcggggctgg cttaactatg
     6841 cggcatcaga gcagattgta ctgagagtgc accataacgc atttaagcat aaacacgcac
     6901 tatgccgttc ttctcatgta tatatatata caggcaacac gcagatatag gtgcgacgtg
     6961 aacagtgagc tgtatgtgcg cagctcgcgt tgcattttcg gaagcgctcg ttttcggaaa
     7021 cgctttgaag ttcctattcc gaagttccta ttctctagct agaaagtata ggaacttcag
     7081 agcgcttttg aaaaccaaaa gcgctctgaa gacgcacttt caaaaaacca aaaacgcacc
     7141 ggactgtaac gagctactaa aatattgcga ataccgcttc cacaaacatt gctcaaaagt
     7201 atctctttgc tatatatctc tgtgctatat ccctatataa cctacccatc cacctttcgc
     7261 tccttgaact tgcatctaaa ctcgacctct acatttttta tgtttatctc tagtattact
     7321 ctttagacaa aaaaattgta gtaagaacta ttcatagagt gaatcgaaaa caatacgaaa
     7381 atgtaaacat ttcctatacg tagtatatag agacaaaata gaagaaaccg ttcataattt
     7441 tctgaccaat gaagaatcat caacgctatc actttctgtt cacaaagtat gcgcaatcca
     7501 catcggtata gaatataatc ggggatgcct ttatcttgaa aaaatgcacc cgcagcttcg
     7561 ctagtaatca gtaaacgcgg gaagtggagt caggcttttt ttatggaaga gaaaatagac
     7621 accaaagtag ccttcttcta accttaacgg acctacagtg caaaaagtta tcaagagact
     7681 gcattataga gcgcacaaag gagaaaaaaa gtaatctaag atgctttgtt agaaaaatag
     7741 cgctctcggg atgcattttt gtagaacaaa aaagaagtat agattctttg ttggtaaaat
     7801 agcgctctcg cgttgcattt ctgttctgta aaaatgcagc tcagattctt tgtttgaaaa
     7861 attagcgctc tcgcgttgca tttttgtttt acaaaaatga agcacagatt cttcgttggt
     7921 aaaatagcgc ttt
//
