LOCUS       sequence-236914-        7888 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 7888)
  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 7888)
  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(∆92-106)
FEATURES             Location/Qualifiers
     source          1..7888
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     promoter        16..417
                     /gene="S. cerevisiae ADH1"
                     /label=ADH1 promoter
                     /note="promoter for alcohol dehydrogenase 1"
     CDS             463..483
                     /codon_start=1
                     /product="nuclear localization signal of SV40 (simian virus
                     40) large T antigen"
                     /label=SV40 NLS
                     /translation="PKKKRKV"
     CDS             499..840
                     /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             1866..1877
                     /note="strong bacterial ribosome binding site (Elowitz and 
                     Leibler, 2000)"
     terminator      2477..2664
                     /gene="S. cerevisiae ADH1"
                     /label=ADH1 terminator
                     /note="transcription terminator for the S. cerevisiae 
                     alcohol dehydrogenase 1 (ADH1) gene"
     primer_bind     complement(2532..2556)
                     /label=pHybLex-R
                     /note="ADH terminator, reverse primer"
     CDS             complement(2785..3879)
                     /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(3880..4285)
                     /gene="S. cerevisiae LEU2"
                     /label=LEU2 promoter
     primer_bind     complement(4327..4343)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(4327..4343)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(4340..4362)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    4351..4367
                     /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(4375..4381,4382..4399,4400..4405))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    4420..4441
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(4558..4575)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(4729..5317)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(4809..4828)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(5488..6279,6280..6348))
                     /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     6111..6130
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(6349..6453)
                     /gene="bla"
                     /label=AmpR promoter
     primer_bind     6521..6539
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     primer_bind     complement(6577..6599)
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     primer_bind     6699..6718
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     rep_origin      join(6735..7888,1..11)
                     /label=2u ori
                     /note="yeast 2u plasmid origin of replication"
ORIGIN
        1 gatgcttcgt tgcttgcatg caacttcttt tctttttttt tcttttctct ctcccccgtt
       61 gttgtctcac catatccgca atgacaaaaa aatgatggaa gacactaaag gaaaaaatta
      121 acgacaaaga cagcaccaac agatgtcgtt gttccagagc tgatgagggg tatctcgaag
      181 cacacgaaac tttttccttc cttcattcac gcacactact ctctaatgag caacggtata
      241 cggccttcct tccagttact tgaatttgaa ataaaaaaaa gtttgctgtc ttgctatcaa
      301 gtataaatag acctgcaatt attaatcttt tgtttcctcg tcattgttct cgttcccttt
      361 cttccttgtt tctttttctg cacaatattt caagctatac caagcataca atcaactcca
      421 agctttgcaa agatggataa agcggaatta attcccgagc ctccaaaaaa gaagagaaag
      481 gtcgaattgg gtaccgccgc caattttaat caaagtggga atattgctga tagctcattg
      541 tccttcactt tcactaacag tagcaacggt ccgaacctca taacaactca aacaaattct
      601 caagcgcttt cacaaccaat tgcctcctct aacgttcatg ataacttcat gaataatgaa
      661 atcacggcta gtaaaattga tgatggtaat aattcaaaac cactgtcacc tggttggacg
      721 gaccaaactg cgtataacgc gtttggaatc actacaggga tgtttaatac cactacaatg
      781 gatgatgtat ataactatct attcgatgat gaagataccc caccaaaccc aaaaaaagag
      841 atcgaattcc ccgggggatc catcgataac gaagcagatg ttacaaaatt tgttaataat
      901 gccaggaaaa cgctgaccga tgctcaactt ttatgttcaa gtgctaattt aaggattgta
      961 gatattaaga aaaaattgtc atcttggcag ttgagtattt caaaactcaa ttttctaata
     1021 gttggcttga gacagcaagg aaagtttctt tacactattt taaaggaagg cattgggaca
     1081 aagctaatcc agaaacaatg gaatcaagct gtcttagtgg tcttagttga tgagatgggt
     1141 atagtaaatg aattgagcat atcagaaaaa gatgataccg atccctctaa actaggagat
     1201 tacatctcga gagacaacgt taacttattg aatgacaaac tgaaagaagt gccagtaatt
     1261 gagcgtcaaa ttgaaaacat taagcttcaa tacgaaaata tggtcagaaa agttaacaaa
     1321 gaactgattg ataccaagtt gacggacgta actcaaaaat tccaaagtaa atttggtata
     1381 gataacctga tggaaacaaa tgttgcagag cagtttagca gggaactaac ggaccttgaa
     1441 aaagatttag cagagataat gaattcattg acacagcact ttgataagac attgctgtta
     1501 caagataaaa aaattgataa cgatgaacgt gaggagctgt ttaaggtggt acaaggcgac
     1561 gacaaagaac tatacaacat tttcaaaact ctgcatgagg taattgatga cgtggacaaa
     1621 acaattctta acttgggtca atttttgcag gcaaaaataa aggaaaagac agaactacac
     1681 agcgaagttt ctgaaataat aaacgatttc aatagaaatt tggaatatct attaatcttt
     1741 aaagatattt ccaatctgat tgatagcttc aaaaattcct gtacacaaga tattcaaaca
     1801 actaaggaac tttgtgaatt ttatgataat tttgaagaaa gctacggtaa cttagttcta
     1861 gaagcaaaga ggagaaagga tgtggcaaac agaatgaaaa ctatattgaa agattgtgaa
     1921 aagcagttgc agaatttaga tgctcaggac caggaagaac gtcagaattt tatagcggaa
     1981 aatggaactt atcttcctga gacaatctgg cccggtaaaa ttgacgattt ttcttccctg
     2041 tacactttaa attacaacgt gaagaatcct tagaaaaaag gaaaaaaagg ctgcagagat
     2101 ctatgaatcg tagatactga aaaaccccgc aagttcactt caactgtgca tcgtgcacca
     2161 tctcaatttc tttcatttat acatcgtttt gccttctttt atgtaactat actcctctaa
     2221 gtttcaatct tggccatgta acctctgatc tatagaattt tttaaatgac tagaattaat
     2281 gcccatcttt tttttggacc taaattcttc atgaaaatat attacgaggg cttattcaga
     2341 agctttggac ttcttcgcca gaggtttggt caagtctcca atcaaggttg tcggcttgtc
     2401 taccttgcca gaaatttacg aaaagatgga aaagggtcaa atcgttggta gatacgttgt
     2461 tgacacttct aaataagcga atttcttatg atttatgatt tttattatta aataagttat
     2521 aaaaaaaata agtgtataca aattttaaag tgactcttag gttttaaaac gaaaattctt
     2581 attcttgagt aactctttcc tgtaggtcag gttgctttct caggtatagc atgaggtcgc
     2641 tcttattgac cacacctcta ccggcatgcc ggtcgaaatt cccctaccct atgaacatat
     2701 tccattttgt aatttcgtgt cgtttctatt atgaatttca tttataaagt ttatgtacaa
     2761 atatcataaa aaaagagaat ctttttaagc aaggattttc ttaacctctt cggcgacagc
     2821 atcaccgact tcggtggtac tgttggaacc acctaaatca ccagttctga tacctgcatc
     2881 caaaaccttt ttaactgcat cttcaatggc cttaccttct tcaggcaagt tcaatgacaa
     2941 tttcaacatc attgcagcag acaagatagt ggcgataggg ttgaccttat tctttggcaa
     3001 atctggagca gaaccgtggc atggttcgta caaaccaaat gcggtgttct tgtctggcaa
     3061 agaggccaag gacgcagatg gcaacaaacc caaggaacct gggataacgg aggcttcatc
     3121 ggagacgata tcaccaaaca tgttgctggt gattataata ccatttaggt gggttgggtt
     3181 cctaactagg atcatggcgg cagaatcaat caattgatgt tgaaccttca atgtaggaaa
     3241 ttcgttcttg atggtttcct ccacagtttt tctccataat cttgaagagg ccaaaacatt
     3301 agctttatcc aaggaccaaa taggcaatgg tggctcatgt tgtagggcca tgaaagcggc
     3361 cattcttgtg attctttgca cttctggaac ggtgtattgt tcactatccc aagcgacacc
     3421 atcaccatcg tcttcctttc tcttaccaaa gtaaatacct cccactaatt ctctgacaac
     3481 aacgaagtca gtacctttag caaattgtgg cttgattgga gataagtcta aaagagagtc
     3541 ggatgcaaag ttacatggtc ttaagttggc gtacaattga agttctttac ggatttttag
     3601 taaaccttgt tcaggtctaa cactacctgt accccattta ggaccaccca cagcacctaa
     3661 caaaacggca tcagccttct tggaggcttc cagcgcctca tctggaagtg ggacacctgt
     3721 agcttcgata gcagcaccac caattaaatg attttcgaaa tcgaacttga cattggaacg
     3781 aacatcagaa atagctttaa gaaccttaat ggcttcggct gtgatttctt gaccaacgtg
     3841 gtcacctggc aaaacgacga tcttcttagg ggcagacatt agaatggtat atccttgaaa
     3901 tatatatata tattgctgaa atgtaaaagg taagaaaagt tagaaagtaa gacgattgct
     3961 aaccacctat tggaaaaaac aataggtcct taaataatat tgtcaacttc aagtattgtg
     4021 atgcaagcat ttagtcatga acgcttctct attctatatg aaaagccggt tccggcgctc
     4081 tcacctttcc tttttctccc aatttttcag ttgaaaaagg tatatgcgtc aggcgacctc
     4141 tgaaattaac aaaaaatttc cagtcatcga atttgattct gtgcgatagc gcccctgtgt
     4201 gttctcgtta tgttgaggaa aaaaataatg gttgctaaga gattcgaact cttgcatctt
     4261 acgatacctg agtattccca cagttggggg atctcgactc tagctagagg atcaattcgt
     4321 aatcatgtca tagctgtttc ctgtgtgaaa ttgttatccg ctcacaattc cacacaacat
     4381 acgagccgga agcataaagt gtaaagcctg gggtgcctaa tgagtgagct aactcacatt
     4441 aattgcgttg cgctcactgc ccgctttcca gtcgggaaac ctgtcgtgcc agctgcatta
     4501 atgaatcggc caacgcgcgg ggagaggcgg tttgcgtatt gggcgctctt ccgcttcctc
     4561 gctcactgac tcgctgcgct cggtcgttcg gctgcggcga gcggtatcag ctcactcaaa
     4621 ggcggtaata cggttatcca cagaatcagg ggataacgca ggaaagaaca tgtgagcaaa
     4681 aggccagcaa aaggccagga accgtaaaaa ggccgcgttg ctggcgtttt tccataggct
     4741 ccgcccccct gacgagcatc acaaaaatcg acgctcaagt cagaggtggc gaaacccgac
     4801 aggactataa agataccagg cgtttccccc tggaagctcc ctcgtgcgct ctcctgttcc
     4861 gaccctgccg cttaccggat acctgtccgc ctttctccct tcgggaagcg tggcgctttc
     4921 tcatagctca cgctgtaggt atctcagttc ggtgtaggtc gttcgctcca agctgggctg
     4981 tgtgcacgaa ccccccgttc agcccgaccg ctgcgcctta tccggtaact atcgtcttga
     5041 gtccaacccg gtaagacacg acttatcgcc actggcagca gccactggta acaggattag
     5101 cagagcgagg tatgtaggcg gtgctacaga gttcttgaag tggtggccta actacggcta
     5161 cactagaaga acagtatttg gtatctgcgc tctgctgaag ccagttacct tcggaaaaag
     5221 agttggtagc tcttgatccg gcaaacaaac caccgctggt agcggtggtt tttttgtttg
     5281 caagcagcag attacgcgca gaaaaaaagg atctcaagaa gatcctttga tcttttctac
     5341 ggggtctgac gctcagtgga acgaaaactc acgttaaggg attttggtca tgagattatc
     5401 aaaaaggatc ttcacctaga tccttttaaa ttaaaaatga agttttaaat caatctaaag
     5461 tatatatgag taaacttggt ctgacagtta ccaatgctta atcagtgagg cacctatctc
     5521 agcgatctgt ctatttcgtt catccatagt tgcctgactc cccgtcgtgt agataactac
     5581 gatacgggag ggcttaccat ctggccccag tgctgcaatg ataccgcgag acccacgctc
     5641 accggctcca gatttatcag caataaacca gccagccgga agggccgagc gcagaagtgg
     5701 tcctgcaact ttatccgcct ccatccagtc tattaattgt tgccgggaag ctagagtaag
     5761 tagttcgcca gttaatagtt tgcgcaacgt tgttgccatt gctacaggca tcgtggtgtc
     5821 acgctcgtcg tttggtatgg cttcattcag ctccggttcc caacgatcaa ggcgagttac
     5881 atgatccccc atgttgtgca aaaaagcggt tagctccttc ggtcctccga tcgttgtcag
     5941 aagtaagttg gccgcagtgt tatcactcat ggttatggca gcactgcata attctcttac
     6001 tgtcatgcca tccgtaagat gcttttctgt gactggtgag tactcaacca agtcattctg
     6061 agaatagtgt atgcggcgac cgagttgctc ttgcccggcg tcaatacggg ataataccgc
     6121 gccacatagc agaactttaa aagtgctcat cattggaaaa cgttcttcgg ggcgaaaact
     6181 ctcaaggatc ttaccgctgt tgagatccag ttcgatgtaa cccactcgtg cacccaactg
     6241 atcttcagca tcttttactt tcaccagcgt ttctgggtga gcaaaaacag gaaggcaaaa
     6301 tgccgcaaaa aagggaataa gggcgacacg gaaatgttga atactcatac tcttcctttt
     6361 tcaatattat tgaagcattt atcagggtta ttgtctcatg agcggataca tatttgaatg
     6421 tatttagaaa aataaacaaa taggggttcc gcgcacattt ccccgaaaag tgccacctga
     6481 cgtctaagaa accattatta tcatgacatt aacctataaa aataggcgta tcacgaggcc
     6541 ctttcgtctc gcgcgtttcg gtgatgacgg tgaaaacctc tgacacatgc agctcccgga
     6601 gacggtcaca gcttgtctgt aagcggatgc cgggagcaga caagcccgtc agggcgcgtc
     6661 agcgggtgtt ggcgggtgtc ggggctggct taactatgcg gcatcagagc agattgtact
     6721 gagagtgcac cataacgcat ttaagcataa acacgcacta tgccgttctt ctcatgtata
     6781 tatatataca ggcaacacgc agatataggt gcgacgtgaa cagtgagctg tatgtgcgca
     6841 gctcgcgttg cattttcgga agcgctcgtt ttcggaaacg ctttgaagtt cctattccga
     6901 agttcctatt ctctagctag aaagtatagg aacttcagag cgcttttgaa aaccaaaagc
     6961 gctctgaaga cgcactttca aaaaaccaaa aacgcaccgg actgtaacga gctactaaaa
     7021 tattgcgaat accgcttcca caaacattgc tcaaaagtat ctctttgcta tatatctctg
     7081 tgctatatcc ctatataacc tacccatcca cctttcgctc cttgaacttg catctaaact
     7141 cgacctctac attttttatg tttatctcta gtattactct ttagacaaaa aaattgtagt
     7201 aagaactatt catagagtga atcgaaaaca atacgaaaat gtaaacattt cctatacgta
     7261 gtatatagag acaaaataga agaaaccgtt cataattttc tgaccaatga agaatcatca
     7321 acgctatcac tttctgttca caaagtatgc gcaatccaca tcggtataga atataatcgg
     7381 ggatgccttt atcttgaaaa aatgcacccg cagcttcgct agtaatcagt aaacgcggga
     7441 agtggagtca ggcttttttt atggaagaga aaatagacac caaagtagcc ttcttctaac
     7501 cttaacggac ctacagtgca aaaagttatc aagagactgc attatagagc gcacaaagga
     7561 gaaaaaaagt aatctaagat gctttgttag aaaaatagcg ctctcgggat gcatttttgt
     7621 agaacaaaaa agaagtatag attctttgtt ggtaaaatag cgctctcgcg ttgcatttct
     7681 gttctgtaaa aatgcagctc agattctttg tttgaaaaat tagcgctctc gcgttgcatt
     7741 tttgttttac aaaaatgaag cacagattct tcgttggtaa aatagcgctt tcgcgttgca
     7801 tttctgttct gtaaaaatgc agctcagatt ctttgtttga aaaattagcg ctctcgcgtt
     7861 gcatttttgt tctacaaaat gaagcaca
//
