LOCUS       sequence-237870-        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(L331D)
FEATURES             Location/Qualifiers
     source          1..7933
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     promoter        11..412
                     /gene="S. cerevisiae ADH1"
                     /label=ADH1 promoter
                     /note="promoter for alcohol dehydrogenase 1"
     CDS             458..478
                     /codon_start=1
                     /product="nuclear localization signal of SV40 (simian virus
                     40) large T antigen"
                     /label=SV40 NLS
                     /translation="PKKKRKV"
     CDS             494..835
                     /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             1906..1917
                     /note="strong bacterial ribosome binding site (Elowitz and 
                     Leibler, 2000)"
     terminator      2517..2704
                     /gene="S. cerevisiae ADH1"
                     /label=ADH1 terminator
                     /note="transcription terminator for the S. cerevisiae 
                     alcohol dehydrogenase 1 (ADH1) gene"
     primer_bind     complement(2572..2596)
                     /label=pHybLex-R
                     /note="ADH terminator, reverse primer"
     CDS             complement(2825..3919)
                     /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(3920..4325)
                     /gene="S. cerevisiae LEU2"
                     /label=LEU2 promoter
     primer_bind     complement(4367..4383)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(4367..4383)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(4380..4402)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    4391..4407
                     /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(4415..4421,4422..4439,4440..4445))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    4460..4481
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(4598..4615)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(4769..5357)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(4849..4868)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(5528..6319,6320..6388))
                     /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     6151..6170
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(6389..6493)
                     /gene="bla"
                     /label=AmpR promoter
     primer_bind     6561..6579
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     primer_bind     complement(6617..6639)
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     primer_bind     6739..6758
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     rep_origin      join(6775..7933,1..6)
                     /label=2u ori
                     /note="yeast 2u plasmid origin of replication"
ORIGIN
        1 ttcgttgctt gcatgcaact tcttttcttt ttttttcttt tctctctccc ccgttgttgt
       61 ctcaccatat ccgcaatgac aaaaaaatga tggaagacac taaaggaaaa aattaacgac
      121 aaagacagca ccaacagatg tcgttgttcc agagctgatg aggggtatct cgaagcacac
      181 gaaacttttt ccttccttca ttcacgcaca ctactctcta atgagcaacg gtatacggcc
      241 ttccttccag ttacttgaat ttgaaataaa aaaaagtttg ctgtcttgct atcaagtata
      301 aatagacctg caattattaa tcttttgttt cctcgtcatt gttctcgttc cctttcttcc
      361 ttgtttcttt ttctgcacaa tatttcaagc tataccaagc atacaatcaa ctccaagctt
      421 tgcaaagatg gataaagcgg aattaattcc cgagcctcca aaaaagaaga gaaaggtcga
      481 attgggtacc gccgccaatt ttaatcaaag tgggaatatt gctgatagct cattgtcctt
      541 cactttcact aacagtagca acggtccgaa cctcataaca actcaaacaa attctcaagc
      601 gctttcacaa ccaattgcct cctctaacgt tcatgataac ttcatgaata atgaaatcac
      661 ggctagtaaa attgatgatg gtaataattc aaaaccactg tcacctggtt ggacggacca
      721 aactgcgtat aacgcgtttg gaatcactac agggatgttt aataccacta caatggatga
      781 tgtatataac tatctattcg atgatgaaga taccccacca aacccaaaaa aagagatcga
      841 attccccggg ggatccatcg ataacgaagc agatgttaca aaatttgtta ataatgccag
      901 gaaaacgctg accgatgctc aacttttatg ttcaagtgct aatttaagga ttgtagatat
      961 taagaaaaaa ttgtcatctt ggcagttgag tatttcaaaa ctcaattttc taatagttgg
     1021 cttgagacag caaggaaagt ttctttacac tattttaaag gaaggcattg ggacaaagct
     1081 aatccagaaa caatggaatc aagctgtctt agtggtctta gttgatgaga tgaagtactg
     1141 gcaatatgaa atcacttcta aagtacaaag gttggatggt atagtaaatg aattgagcat
     1201 atcagaaaaa gatgataccg atccctctaa actaggagat tacatctcga gagacaacgt
     1261 taacttattg aatgacaaac tgaaagaagt gccagtaatt gagcgtcaaa ttgaaaacat
     1321 taagcttcaa tacgaaaata tggtcagaaa agttaacaaa gaactgattg ataccaagtt
     1381 gacggacgta actcaaaaat tccaaagtaa atttggtata gataacctga tggaaacaaa
     1441 tgttgcagag cagtttagca gggaactaac ggaccttgaa aaagatttag cagagataat
     1501 gaattcattg acacagcact ttgataagac attgctgtta caagataaaa aaattgataa
     1561 cgatgaacgt gaggagctgt ttaaggtggt acaaggcgac gacaaagaac tatacaacat
     1621 tttcaaaact ctgcatgagg taattgatga cgtggacaaa acaattctta acttgggtca
     1681 atttttgcag gcaaaaataa aggaaaagac agaactacac agcgaagttt ctgaaataat
     1741 aaacgatttc aatagaaatt tggaatatct attaatcttt aaagatattt ccaatctgat
     1801 tgatagcttc aaaaattcct gtacacaaga tattcaaaca actaaggaag actgtgaatt
     1861 ttatgataat tttgaagaaa gctacggtaa cttagttcta gaagcaaaga ggagaaagga
     1921 tgtggcaaac agaatgaaaa ctatattgaa agattgtgaa aagcagttgc agaatttaga
     1981 tgctcaggac caggaagaac gtcagaattt tatagcggaa aatggaactt atcttcctga
     2041 gacaatctgg cccggtaaaa ttgacgattt ttcttccctg tacactttaa attacaacgt
     2101 gaagaatcct tagaaaaaag gaaaaaaagg ctgcagagat ctatgaatcg tagatactga
     2161 aaaaccccgc aagttcactt caactgtgca tcgtgcacca tctcaatttc tttcatttat
     2221 acatcgtttt gccttctttt atgtaactat actcctctaa gtttcaatct tggccatgta
     2281 acctctgatc tatagaattt tttaaatgac tagaattaat gcccatcttt tttttggacc
     2341 taaattcttc atgaaaatat attacgaggg cttattcaga agctttggac ttcttcgcca
     2401 gaggtttggt caagtctcca atcaaggttg tcggcttgtc taccttgcca gaaatttacg
     2461 aaaagatgga aaagggtcaa atcgttggta gatacgttgt tgacacttct aaataagcga
     2521 atttcttatg atttatgatt tttattatta aataagttat aaaaaaaata agtgtataca
     2581 aattttaaag tgactcttag gttttaaaac gaaaattctt attcttgagt aactctttcc
     2641 tgtaggtcag gttgctttct caggtatagc atgaggtcgc tcttattgac cacacctcta
     2701 ccggcatgcc ggtcgaaatt cccctaccct atgaacatat tccattttgt aatttcgtgt
     2761 cgtttctatt atgaatttca tttataaagt ttatgtacaa atatcataaa aaaagagaat
     2821 ctttttaagc aaggattttc ttaacctctt cggcgacagc atcaccgact tcggtggtac
     2881 tgttggaacc acctaaatca ccagttctga tacctgcatc caaaaccttt ttaactgcat
     2941 cttcaatggc cttaccttct tcaggcaagt tcaatgacaa tttcaacatc attgcagcag
     3001 acaagatagt ggcgataggg ttgaccttat tctttggcaa atctggagca gaaccgtggc
     3061 atggttcgta caaaccaaat gcggtgttct tgtctggcaa agaggccaag gacgcagatg
     3121 gcaacaaacc caaggaacct gggataacgg aggcttcatc ggagacgata tcaccaaaca
     3181 tgttgctggt gattataata ccatttaggt gggttgggtt cctaactagg atcatggcgg
     3241 cagaatcaat caattgatgt tgaaccttca atgtaggaaa ttcgttcttg atggtttcct
     3301 ccacagtttt tctccataat cttgaagagg ccaaaacatt agctttatcc aaggaccaaa
     3361 taggcaatgg tggctcatgt tgtagggcca tgaaagcggc cattcttgtg attctttgca
     3421 cttctggaac ggtgtattgt tcactatccc aagcgacacc atcaccatcg tcttcctttc
     3481 tcttaccaaa gtaaatacct cccactaatt ctctgacaac aacgaagtca gtacctttag
     3541 caaattgtgg cttgattgga gataagtcta aaagagagtc ggatgcaaag ttacatggtc
     3601 ttaagttggc gtacaattga agttctttac ggatttttag taaaccttgt tcaggtctaa
     3661 cactacctgt accccattta ggaccaccca cagcacctaa caaaacggca tcagccttct
     3721 tggaggcttc cagcgcctca tctggaagtg ggacacctgt agcttcgata gcagcaccac
     3781 caattaaatg attttcgaaa tcgaacttga cattggaacg aacatcagaa atagctttaa
     3841 gaaccttaat ggcttcggct gtgatttctt gaccaacgtg gtcacctggc aaaacgacga
     3901 tcttcttagg ggcagacatt agaatggtat atccttgaaa tatatatata tattgctgaa
     3961 atgtaaaagg taagaaaagt tagaaagtaa gacgattgct aaccacctat tggaaaaaac
     4021 aataggtcct taaataatat tgtcaacttc aagtattgtg atgcaagcat ttagtcatga
     4081 acgcttctct attctatatg aaaagccggt tccggcgctc tcacctttcc tttttctccc
     4141 aatttttcag ttgaaaaagg tatatgcgtc aggcgacctc tgaaattaac aaaaaatttc
     4201 cagtcatcga atttgattct gtgcgatagc gcccctgtgt gttctcgtta tgttgaggaa
     4261 aaaaataatg gttgctaaga gattcgaact cttgcatctt acgatacctg agtattccca
     4321 cagttggggg atctcgactc tagctagagg atcaattcgt aatcatgtca tagctgtttc
     4381 ctgtgtgaaa ttgttatccg ctcacaattc cacacaacat acgagccgga agcataaagt
     4441 gtaaagcctg gggtgcctaa tgagtgagct aactcacatt aattgcgttg cgctcactgc
     4501 ccgctttcca gtcgggaaac ctgtcgtgcc agctgcatta atgaatcggc caacgcgcgg
     4561 ggagaggcgg tttgcgtatt gggcgctctt ccgcttcctc gctcactgac tcgctgcgct
     4621 cggtcgttcg gctgcggcga gcggtatcag ctcactcaaa ggcggtaata cggttatcca
     4681 cagaatcagg ggataacgca ggaaagaaca tgtgagcaaa aggccagcaa aaggccagga
     4741 accgtaaaaa ggccgcgttg ctggcgtttt tccataggct ccgcccccct gacgagcatc
     4801 acaaaaatcg acgctcaagt cagaggtggc gaaacccgac aggactataa agataccagg
     4861 cgtttccccc tggaagctcc ctcgtgcgct ctcctgttcc gaccctgccg cttaccggat
     4921 acctgtccgc ctttctccct tcgggaagcg tggcgctttc tcatagctca cgctgtaggt
     4981 atctcagttc ggtgtaggtc gttcgctcca agctgggctg tgtgcacgaa ccccccgttc
     5041 agcccgaccg ctgcgcctta tccggtaact atcgtcttga gtccaacccg gtaagacacg
     5101 acttatcgcc actggcagca gccactggta acaggattag cagagcgagg tatgtaggcg
     5161 gtgctacaga gttcttgaag tggtggccta actacggcta cactagaaga acagtatttg
     5221 gtatctgcgc tctgctgaag ccagttacct tcggaaaaag agttggtagc tcttgatccg
     5281 gcaaacaaac caccgctggt agcggtggtt tttttgtttg caagcagcag attacgcgca
     5341 gaaaaaaagg atctcaagaa gatcctttga tcttttctac ggggtctgac gctcagtgga
     5401 acgaaaactc acgttaaggg attttggtca tgagattatc aaaaaggatc ttcacctaga
     5461 tccttttaaa ttaaaaatga agttttaaat caatctaaag tatatatgag taaacttggt
     5521 ctgacagtta ccaatgctta atcagtgagg cacctatctc agcgatctgt ctatttcgtt
     5581 catccatagt tgcctgactc cccgtcgtgt agataactac gatacgggag ggcttaccat
     5641 ctggccccag tgctgcaatg ataccgcgag acccacgctc accggctcca gatttatcag
     5701 caataaacca gccagccgga agggccgagc gcagaagtgg tcctgcaact ttatccgcct
     5761 ccatccagtc tattaattgt tgccgggaag ctagagtaag tagttcgcca gttaatagtt
     5821 tgcgcaacgt tgttgccatt gctacaggca tcgtggtgtc acgctcgtcg tttggtatgg
     5881 cttcattcag ctccggttcc caacgatcaa ggcgagttac atgatccccc atgttgtgca
     5941 aaaaagcggt tagctccttc ggtcctccga tcgttgtcag aagtaagttg gccgcagtgt
     6001 tatcactcat ggttatggca gcactgcata attctcttac tgtcatgcca tccgtaagat
     6061 gcttttctgt gactggtgag tactcaacca agtcattctg agaatagtgt atgcggcgac
     6121 cgagttgctc ttgcccggcg tcaatacggg ataataccgc gccacatagc agaactttaa
     6181 aagtgctcat cattggaaaa cgttcttcgg ggcgaaaact ctcaaggatc ttaccgctgt
     6241 tgagatccag ttcgatgtaa cccactcgtg cacccaactg atcttcagca tcttttactt
     6301 tcaccagcgt ttctgggtga gcaaaaacag gaaggcaaaa tgccgcaaaa aagggaataa
     6361 gggcgacacg gaaatgttga atactcatac tcttcctttt tcaatattat tgaagcattt
     6421 atcagggtta ttgtctcatg agcggataca tatttgaatg tatttagaaa aataaacaaa
     6481 taggggttcc gcgcacattt ccccgaaaag tgccacctga cgtctaagaa accattatta
     6541 tcatgacatt aacctataaa aataggcgta tcacgaggcc ctttcgtctc gcgcgtttcg
     6601 gtgatgacgg tgaaaacctc tgacacatgc agctcccgga gacggtcaca gcttgtctgt
     6661 aagcggatgc cgggagcaga caagcccgtc agggcgcgtc agcgggtgtt ggcgggtgtc
     6721 ggggctggct taactatgcg gcatcagagc agattgtact gagagtgcac cataacgcat
     6781 ttaagcataa acacgcacta tgccgttctt ctcatgtata tatatataca ggcaacacgc
     6841 agatataggt gcgacgtgaa cagtgagctg tatgtgcgca gctcgcgttg cattttcgga
     6901 agcgctcgtt ttcggaaacg ctttgaagtt cctattccga agttcctatt ctctagctag
     6961 aaagtatagg aacttcagag cgcttttgaa aaccaaaagc gctctgaaga cgcactttca
     7021 aaaaaccaaa aacgcaccgg actgtaacga gctactaaaa tattgcgaat accgcttcca
     7081 caaacattgc tcaaaagtat ctctttgcta tatatctctg tgctatatcc ctatataacc
     7141 tacccatcca cctttcgctc cttgaacttg catctaaact cgacctctac attttttatg
     7201 tttatctcta gtattactct ttagacaaaa aaattgtagt aagaactatt catagagtga
     7261 atcgaaaaca atacgaaaat gtaaacattt cctatacgta gtatatagag acaaaataga
     7321 agaaaccgtt cataattttc tgaccaatga agaatcatca acgctatcac tttctgttca
     7381 caaagtatgc gcaatccaca tcggtataga atataatcgg ggatgccttt atcttgaaaa
     7441 aatgcacccg cagcttcgct agtaatcagt aaacgcggga agtggagtca ggcttttttt
     7501 atggaagaga aaatagacac caaagtagcc ttcttctaac cttaacggac ctacagtgca
     7561 aaaagttatc aagagactgc attatagagc gcacaaagga gaaaaaaagt aatctaagat
     7621 gctttgttag aaaaatagcg ctctcgggat gcatttttgt agaacaaaaa agaagtatag
     7681 attctttgtt ggtaaaatag cgctctcgcg ttgcatttct gttctgtaaa aatgcagctc
     7741 agattctttg tttgaaaaat tagcgctctc gcgttgcatt tttgttttac aaaaatgaag
     7801 cacagattct tcgttggtaa aatagcgctt tcgcgttgca tttctgttct gtaaaaatgc
     7861 agctcagatt ctttgtttga aaaattagcg ctctcgcgtt gcatttttgt tctacaaaat
     7921 gaagcacaga tgc
//
