LOCUS       sequence-236912-        7867 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 7867)
  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 7867)
  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(∆313-334)
FEATURES             Location/Qualifiers
     source          1..7867
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     promoter        107..508
                     /gene="S. cerevisiae ADH1"
                     /label=ADH1 promoter
                     /note="promoter for alcohol dehydrogenase 1"
     CDS             554..574
                     /codon_start=1
                     /product="nuclear localization signal of SV40 (simian virus
                     40) large T antigen"
                     /label=SV40 NLS
                     /translation="PKKKRKV"
     CDS             590..931
                     /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             1936..1947
                     /note="strong bacterial ribosome binding site (Elowitz and 
                     Leibler, 2000)"
     terminator      2547..2734
                     /gene="S. cerevisiae ADH1"
                     /label=ADH1 terminator
                     /note="transcription terminator for the S. cerevisiae 
                     alcohol dehydrogenase 1 (ADH1) gene"
     primer_bind     complement(2602..2626)
                     /label=pHybLex-R
                     /note="ADH terminator, reverse primer"
     CDS             complement(2855..3949)
                     /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(3950..4355)
                     /gene="S. cerevisiae LEU2"
                     /label=LEU2 promoter
     primer_bind     complement(4397..4413)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(4397..4413)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(4410..4432)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    4421..4437
                     /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(4445..4451,4452..4469,4470..4475))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    4490..4511
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(4628..4645)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(4799..5387)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(4879..4898)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(5558..6349,6350..6418))
                     /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     6181..6200
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(6419..6523)
                     /gene="bla"
                     /label=AmpR promoter
     primer_bind     6591..6609
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     primer_bind     complement(6647..6669)
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     primer_bind     6769..6788
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     rep_origin      join(6805..7867,1..102)
                     /label=2u ori
                     /note="yeast 2u plasmid origin of replication"
ORIGIN
        1 gcatttctgt tctgtaaaaa tgcagctcag attctttgtt tgaaaaatta gcgctctcgc
       61 gttgcatttt tgttctacaa aatgaagcac agatgcttcg ttgcttgcat gcaacttctt
      121 ttcttttttt ttcttttctc tctcccccgt tgttgtctca ccatatccgc aatgacaaaa
      181 aaatgatgga agacactaaa ggaaaaaatt aacgacaaag acagcaccaa cagatgtcgt
      241 tgttccagag ctgatgaggg gtatctcgaa gcacacgaaa ctttttcctt ccttcattca
      301 cgcacactac tctctaatga gcaacggtat acggccttcc ttccagttac ttgaatttga
      361 aataaaaaaa agtttgctgt cttgctatca agtataaata gacctgcaat tattaatctt
      421 ttgtttcctc gtcattgttc tcgttccctt tcttccttgt ttctttttct gcacaatatt
      481 tcaagctata ccaagcatac aatcaactcc aagctttgca aagatggata aagcggaatt
      541 aattcccgag cctccaaaaa agaagagaaa ggtcgaattg ggtaccgccg ccaattttaa
      601 tcaaagtggg aatattgctg atagctcatt gtccttcact ttcactaaca gtagcaacgg
      661 tccgaacctc ataacaactc aaacaaattc tcaagcgctt tcacaaccaa ttgcctcctc
      721 taacgttcat gataacttca tgaataatga aatcacggct agtaaaattg atgatggtaa
      781 taattcaaaa ccactgtcac ctggttggac ggaccaaact gcgtataacg cgtttggaat
      841 cactacaggg atgtttaata ccactacaat ggatgatgta tataactatc tattcgatga
      901 tgaagatacc ccaccaaacc caaaaaaaga gatcgaattc cccgggggat ccatcgataa
      961 cgaagcagat gttacaaaat ttgttaataa tgccaggaaa acgctgaccg atgctcaact
     1021 tttatgttca agtgctaatt taaggattgt agatattaag aaaaaattgt catcttggca
     1081 gttgagtatt tcaaaactca attttctaat agttggcttg agacagcaag gaaagtttct
     1141 ttacactatt ttaaaggaag gcattgggac aaagctaatc cagaaacaat ggaatcaagc
     1201 tgtcttagtg gtcttagttg atgagatgaa gtactggcaa tatgaaatca cttctaaagt
     1261 acaaaggttg gatggtatag taaatgaatt gagcatatca gaaaaagatg ataccgatcc
     1321 ctctaaacta ggagattaca tctcgagaga caacgttaac ttattgaatg acaaactgaa
     1381 agaagtgcca gtaattgagc gtcaaattga aaacattaag cttcaatacg aaaatatggt
     1441 cagaaaagtt aacaaagaac tgattgatac caagttgacg gacgtaactc aaaaattcca
     1501 aagtaaattt ggtatagata acctgatgga aacaaatgtt gcagagcagt ttagcaggga
     1561 actaacggac cttgaaaaag atttagcaga gataatgaat tcattgacac agcactttga
     1621 taagacattg ctgttacaag ataaaaaaat tgataacgat gaacgtgagg agctgtttaa
     1681 ggtggtacaa ggcgacgaca aagaactata caacattttc aaaactctgc atgaggtaat
     1741 tgatgacgtg gacaaaacaa ttcttaactt gggtcaattt ttgcaggcaa aaataaagga
     1801 aaagacagaa ctacacagcg aagtttctga aataataaac gatttcaata gaaatttgga
     1861 atatctatta atctttaaag atatttccaa ttatgataat tttgaagaaa gctacggtaa
     1921 cttagttcta gaagcaaaga ggagaaagga tgtggcaaac agaatgaaaa ctatattgaa
     1981 agattgtgaa aagcagttgc agaatttaga tgctcaggac caggaagaac gtcagaattt
     2041 tatagcggaa aatggaactt atcttcctga gacaatctgg cccggtaaaa ttgacgattt
     2101 ttcttccctg tacactttaa attacaacgt gaagaatcct tagaaaaaag gaaaaaaagg
     2161 ctgcagagat ctatgaatcg tagatactga aaaaccccgc aagttcactt caactgtgca
     2221 tcgtgcacca tctcaatttc tttcatttat acatcgtttt gccttctttt atgtaactat
     2281 actcctctaa gtttcaatct tggccatgta acctctgatc tatagaattt tttaaatgac
     2341 tagaattaat gcccatcttt tttttggacc taaattcttc atgaaaatat attacgaggg
     2401 cttattcaga agctttggac ttcttcgcca gaggtttggt caagtctcca atcaaggttg
     2461 tcggcttgtc taccttgcca gaaatttacg aaaagatgga aaagggtcaa atcgttggta
     2521 gatacgttgt tgacacttct aaataagcga atttcttatg atttatgatt tttattatta
     2581 aataagttat aaaaaaaata agtgtataca aattttaaag tgactcttag gttttaaaac
     2641 gaaaattctt attcttgagt aactctttcc tgtaggtcag gttgctttct caggtatagc
     2701 atgaggtcgc tcttattgac cacacctcta ccggcatgcc ggtcgaaatt cccctaccct
     2761 atgaacatat tccattttgt aatttcgtgt cgtttctatt atgaatttca tttataaagt
     2821 ttatgtacaa atatcataaa aaaagagaat ctttttaagc aaggattttc ttaacctctt
     2881 cggcgacagc atcaccgact tcggtggtac tgttggaacc acctaaatca ccagttctga
     2941 tacctgcatc caaaaccttt ttaactgcat cttcaatggc cttaccttct tcaggcaagt
     3001 tcaatgacaa tttcaacatc attgcagcag acaagatagt ggcgataggg ttgaccttat
     3061 tctttggcaa atctggagca gaaccgtggc atggttcgta caaaccaaat gcggtgttct
     3121 tgtctggcaa agaggccaag gacgcagatg gcaacaaacc caaggaacct gggataacgg
     3181 aggcttcatc ggagacgata tcaccaaaca tgttgctggt gattataata ccatttaggt
     3241 gggttgggtt cctaactagg atcatggcgg cagaatcaat caattgatgt tgaaccttca
     3301 atgtaggaaa ttcgttcttg atggtttcct ccacagtttt tctccataat cttgaagagg
     3361 ccaaaacatt agctttatcc aaggaccaaa taggcaatgg tggctcatgt tgtagggcca
     3421 tgaaagcggc cattcttgtg attctttgca cttctggaac ggtgtattgt tcactatccc
     3481 aagcgacacc atcaccatcg tcttcctttc tcttaccaaa gtaaatacct cccactaatt
     3541 ctctgacaac aacgaagtca gtacctttag caaattgtgg cttgattgga gataagtcta
     3601 aaagagagtc ggatgcaaag ttacatggtc ttaagttggc gtacaattga agttctttac
     3661 ggatttttag taaaccttgt tcaggtctaa cactacctgt accccattta ggaccaccca
     3721 cagcacctaa caaaacggca tcagccttct tggaggcttc cagcgcctca tctggaagtg
     3781 ggacacctgt agcttcgata gcagcaccac caattaaatg attttcgaaa tcgaacttga
     3841 cattggaacg aacatcagaa atagctttaa gaaccttaat ggcttcggct gtgatttctt
     3901 gaccaacgtg gtcacctggc aaaacgacga tcttcttagg ggcagacatt agaatggtat
     3961 atccttgaaa tatatatata tattgctgaa atgtaaaagg taagaaaagt tagaaagtaa
     4021 gacgattgct aaccacctat tggaaaaaac aataggtcct taaataatat tgtcaacttc
     4081 aagtattgtg atgcaagcat ttagtcatga acgcttctct attctatatg aaaagccggt
     4141 tccggcgctc tcacctttcc tttttctccc aatttttcag ttgaaaaagg tatatgcgtc
     4201 aggcgacctc tgaaattaac aaaaaatttc cagtcatcga atttgattct gtgcgatagc
     4261 gcccctgtgt gttctcgtta tgttgaggaa aaaaataatg gttgctaaga gattcgaact
     4321 cttgcatctt acgatacctg agtattccca cagttggggg atctcgactc tagctagagg
     4381 atcaattcgt aatcatgtca tagctgtttc ctgtgtgaaa ttgttatccg ctcacaattc
     4441 cacacaacat acgagccgga agcataaagt gtaaagcctg gggtgcctaa tgagtgagct
     4501 aactcacatt aattgcgttg cgctcactgc ccgctttcca gtcgggaaac ctgtcgtgcc
     4561 agctgcatta atgaatcggc caacgcgcgg ggagaggcgg tttgcgtatt gggcgctctt
     4621 ccgcttcctc gctcactgac tcgctgcgct cggtcgttcg gctgcggcga gcggtatcag
     4681 ctcactcaaa ggcggtaata cggttatcca cagaatcagg ggataacgca ggaaagaaca
     4741 tgtgagcaaa aggccagcaa aaggccagga accgtaaaaa ggccgcgttg ctggcgtttt
     4801 tccataggct ccgcccccct gacgagcatc acaaaaatcg acgctcaagt cagaggtggc
     4861 gaaacccgac aggactataa agataccagg cgtttccccc tggaagctcc ctcgtgcgct
     4921 ctcctgttcc gaccctgccg cttaccggat acctgtccgc ctttctccct tcgggaagcg
     4981 tggcgctttc tcatagctca cgctgtaggt atctcagttc ggtgtaggtc gttcgctcca
     5041 agctgggctg tgtgcacgaa ccccccgttc agcccgaccg ctgcgcctta tccggtaact
     5101 atcgtcttga gtccaacccg gtaagacacg acttatcgcc actggcagca gccactggta
     5161 acaggattag cagagcgagg tatgtaggcg gtgctacaga gttcttgaag tggtggccta
     5221 actacggcta cactagaaga acagtatttg gtatctgcgc tctgctgaag ccagttacct
     5281 tcggaaaaag agttggtagc tcttgatccg gcaaacaaac caccgctggt agcggtggtt
     5341 tttttgtttg caagcagcag attacgcgca gaaaaaaagg atctcaagaa gatcctttga
     5401 tcttttctac ggggtctgac gctcagtgga acgaaaactc acgttaaggg attttggtca
     5461 tgagattatc aaaaaggatc ttcacctaga tccttttaaa ttaaaaatga agttttaaat
     5521 caatctaaag tatatatgag taaacttggt ctgacagtta ccaatgctta atcagtgagg
     5581 cacctatctc agcgatctgt ctatttcgtt catccatagt tgcctgactc cccgtcgtgt
     5641 agataactac gatacgggag ggcttaccat ctggccccag tgctgcaatg ataccgcgag
     5701 acccacgctc accggctcca gatttatcag caataaacca gccagccgga agggccgagc
     5761 gcagaagtgg tcctgcaact ttatccgcct ccatccagtc tattaattgt tgccgggaag
     5821 ctagagtaag tagttcgcca gttaatagtt tgcgcaacgt tgttgccatt gctacaggca
     5881 tcgtggtgtc acgctcgtcg tttggtatgg cttcattcag ctccggttcc caacgatcaa
     5941 ggcgagttac atgatccccc atgttgtgca aaaaagcggt tagctccttc ggtcctccga
     6001 tcgttgtcag aagtaagttg gccgcagtgt tatcactcat ggttatggca gcactgcata
     6061 attctcttac tgtcatgcca tccgtaagat gcttttctgt gactggtgag tactcaacca
     6121 agtcattctg agaatagtgt atgcggcgac cgagttgctc ttgcccggcg tcaatacggg
     6181 ataataccgc gccacatagc agaactttaa aagtgctcat cattggaaaa cgttcttcgg
     6241 ggcgaaaact ctcaaggatc ttaccgctgt tgagatccag ttcgatgtaa cccactcgtg
     6301 cacccaactg atcttcagca tcttttactt tcaccagcgt ttctgggtga gcaaaaacag
     6361 gaaggcaaaa tgccgcaaaa aagggaataa gggcgacacg gaaatgttga atactcatac
     6421 tcttcctttt tcaatattat tgaagcattt atcagggtta ttgtctcatg agcggataca
     6481 tatttgaatg tatttagaaa aataaacaaa taggggttcc gcgcacattt ccccgaaaag
     6541 tgccacctga cgtctaagaa accattatta tcatgacatt aacctataaa aataggcgta
     6601 tcacgaggcc ctttcgtctc gcgcgtttcg gtgatgacgg tgaaaacctc tgacacatgc
     6661 agctcccgga gacggtcaca gcttgtctgt aagcggatgc cgggagcaga caagcccgtc
     6721 agggcgcgtc agcgggtgtt ggcgggtgtc ggggctggct taactatgcg gcatcagagc
     6781 agattgtact gagagtgcac cataacgcat ttaagcataa acacgcacta tgccgttctt
     6841 ctcatgtata tatatataca ggcaacacgc agatataggt gcgacgtgaa cagtgagctg
     6901 tatgtgcgca gctcgcgttg cattttcgga agcgctcgtt ttcggaaacg ctttgaagtt
     6961 cctattccga agttcctatt ctctagctag aaagtatagg aacttcagag cgcttttgaa
     7021 aaccaaaagc gctctgaaga cgcactttca aaaaaccaaa aacgcaccgg actgtaacga
     7081 gctactaaaa tattgcgaat accgcttcca caaacattgc tcaaaagtat ctctttgcta
     7141 tatatctctg tgctatatcc ctatataacc tacccatcca cctttcgctc cttgaacttg
     7201 catctaaact cgacctctac attttttatg tttatctcta gtattactct ttagacaaaa
     7261 aaattgtagt aagaactatt catagagtga atcgaaaaca atacgaaaat gtaaacattt
     7321 cctatacgta gtatatagag acaaaataga agaaaccgtt cataattttc tgaccaatga
     7381 agaatcatca acgctatcac tttctgttca caaagtatgc gcaatccaca tcggtataga
     7441 atataatcgg ggatgccttt atcttgaaaa aatgcacccg cagcttcgct agtaatcagt
     7501 aaacgcggga agtggagtca ggcttttttt atggaagaga aaatagacac caaagtagcc
     7561 ttcttctaac cttaacggac ctacagtgca aaaagttatc aagagactgc attatagagc
     7621 gcacaaagga gaaaaaaagt aatctaagat gctttgttag aaaaatagcg ctctcgggat
     7681 gcatttttgt agaacaaaaa agaagtatag attctttgtt ggtaaaatag cgctctcgcg
     7741 ttgcatttct gttctgtaaa aatgcagctc agattctttg tttgaaaaat tagcgctctc
     7801 gcgttgcatt tttgttttac aaaaatgaag cacagattct tcgttggtaa aatagcgctt
     7861 tcgcgtt
//
