LOCUS       sequence-236317-        7882 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 7882)
  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 7882)
  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(∆279-295)
FEATURES             Location/Qualifiers
     source          1..7882
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     promoter        35..436
                     /gene="S. cerevisiae ADH1"
                     /label=ADH1 promoter
                     /note="promoter for alcohol dehydrogenase 1"
     CDS             482..502
                     /codon_start=1
                     /product="nuclear localization signal of SV40 (simian virus
                     40) large T antigen"
                     /label=SV40 NLS
                     /translation="PKKKRKV"
     CDS             518..859
                     /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             1879..1890
                     /note="strong bacterial ribosome binding site (Elowitz and 
                     Leibler, 2000)"
     terminator      2490..2677
                     /gene="S. cerevisiae ADH1"
                     /label=ADH1 terminator
                     /note="transcription terminator for the S. cerevisiae 
                     alcohol dehydrogenase 1 (ADH1) gene"
     primer_bind     complement(2545..2569)
                     /label=pHybLex-R
                     /note="ADH terminator, reverse primer"
     CDS             complement(2798..3892)
                     /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(3893..4298)
                     /gene="S. cerevisiae LEU2"
                     /label=LEU2 promoter
     primer_bind     complement(4340..4356)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(4340..4356)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(4353..4375)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    4364..4380
                     /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(4388..4394,4395..4412,4413..4418))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    4433..4454
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(4571..4588)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(4742..5330)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(4822..4841)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(5501..6292,6293..6361))
                     /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     6124..6143
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(6362..6466)
                     /gene="bla"
                     /label=AmpR promoter
     primer_bind     6534..6552
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     primer_bind     complement(6590..6612)
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     primer_bind     6712..6731
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     rep_origin      join(6748..7882,1..30)
                     /label=2u ori
                     /note="yeast 2u plasmid origin of replication"
ORIGIN
        1 ttctacaaaa tgaagcacag atgcttcgtt gcttgcatgc aacttctttt cttttttttt
       61 cttttctctc tcccccgttg ttgtctcacc atatccgcaa tgacaaaaaa atgatggaag
      121 acactaaagg aaaaaattaa cgacaaagac agcaccaaca gatgtcgttg ttccagagct
      181 gatgaggggt atctcgaagc acacgaaact ttttccttcc ttcattcacg cacactactc
      241 tctaatgagc aacggtatac ggccttcctt ccagttactt gaatttgaaa taaaaaaaag
      301 tttgctgtct tgctatcaag tataaataga cctgcaatta ttaatctttt gtttcctcgt
      361 cattgttctc gttccctttc ttccttgttt ctttttctgc acaatatttc aagctatacc
      421 aagcatacaa tcaactccaa gctttgcaaa gatggataaa gcggaattaa ttcccgagcc
      481 tccaaaaaag aagagaaagg tcgaattggg taccgccgcc aattttaatc aaagtgggaa
      541 tattgctgat agctcattgt ccttcacttt cactaacagt agcaacggtc cgaacctcat
      601 aacaactcaa acaaattctc aagcgctttc acaaccaatt gcctcctcta acgttcatga
      661 taacttcatg aataatgaaa tcacggctag taaaattgat gatggtaata attcaaaacc
      721 actgtcacct ggttggacgg accaaactgc gtataacgcg tttggaatca ctacagggat
      781 gtttaatacc actacaatgg atgatgtata taactatcta ttcgatgatg aagatacccc
      841 accaaaccca aaaaaagaga tcgaattccc cgggggatcc atcgataacg aagcagatgt
      901 tacaaaattt gttaataatg ccaggaaaac gctgaccgat gctcaacttt tatgttcaag
      961 tgctaattta aggattgtag atattaagaa aaaattgtca tcttggcagt tgagtatttc
     1021 aaaactcaat tttctaatag ttggcttgag acagcaagga aagtttcttt acactatttt
     1081 aaaggaaggc attgggacaa agctaatcca gaaacaatgg aatcaagctg tcttagtggt
     1141 cttagttgat gagatgaagt actggcaata tgaaatcact tctaaagtac aaaggttgga
     1201 tggtatagta aatgaattga gcatatcaga aaaagatgat accgatccct ctaaactagg
     1261 agattacatc tcgagagaca acgttaactt attgaatgac aaactgaaag aagtgccagt
     1321 aattgagcgt caaattgaaa acattaagct tcaatacgaa aatatggtca gaaaagttaa
     1381 caaagaactg attgatacca agttgacgga cgtaactcaa aaattccaaa gtaaatttgg
     1441 tatagataac ctgatggaaa caaatgttgc agagcagttt agcagggaac taacggacct
     1501 tgaaaaagat ttagcagaga taatgaattc attgacacag cactttgata agacattgct
     1561 gttacaagat aaaaaaattg ataacgatga acgtgaggag ctgtttaagg tggtacaagg
     1621 cgacgacaaa gaactataca acattttcaa aactctgcat gaggtaattg atgacgtgga
     1681 caaaacaatt cttaacttgg gtcaattttt gcaggcagat ttcaatagaa atttggaata
     1741 tctattaatc tttaaagata tttccaatct gattgatagc ttcaaaaatt cctgtacaca
     1801 agatattcaa acaactaagg aactttgtga attttatgat aattttgaag aaagctacgg
     1861 taacttagtt ctagaagcaa agaggagaaa ggatgtggca aacagaatga aaactatatt
     1921 gaaagattgt gaaaagcagt tgcagaattt agatgctcag gaccaggaag aacgtcagaa
     1981 ttttatagcg gaaaatggaa cttatcttcc tgagacaatc tggcccggta aaattgacga
     2041 tttttcttcc ctgtacactt taaattacaa cgtgaagaat ccttagaaaa aaggaaaaaa
     2101 aggctgcaga gatctatgaa tcgtagatac tgaaaaaccc cgcaagttca cttcaactgt
     2161 gcatcgtgca ccatctcaat ttctttcatt tatacatcgt tttgccttct tttatgtaac
     2221 tatactcctc taagtttcaa tcttggccat gtaacctctg atctatagaa ttttttaaat
     2281 gactagaatt aatgcccatc ttttttttgg acctaaattc ttcatgaaaa tatattacga
     2341 gggcttattc agaagctttg gacttcttcg ccagaggttt ggtcaagtct ccaatcaagg
     2401 ttgtcggctt gtctaccttg ccagaaattt acgaaaagat ggaaaagggt caaatcgttg
     2461 gtagatacgt tgttgacact tctaaataag cgaatttctt atgatttatg atttttatta
     2521 ttaaataagt tataaaaaaa ataagtgtat acaaatttta aagtgactct taggttttaa
     2581 aacgaaaatt cttattcttg agtaactctt tcctgtaggt caggttgctt tctcaggtat
     2641 agcatgaggt cgctcttatt gaccacacct ctaccggcat gccggtcgaa attcccctac
     2701 cctatgaaca tattccattt tgtaatttcg tgtcgtttct attatgaatt tcatttataa
     2761 agtttatgta caaatatcat aaaaaaagag aatcttttta agcaaggatt ttcttaacct
     2821 cttcggcgac agcatcaccg acttcggtgg tactgttgga accacctaaa tcaccagttc
     2881 tgatacctgc atccaaaacc tttttaactg catcttcaat ggccttacct tcttcaggca
     2941 agttcaatga caatttcaac atcattgcag cagacaagat agtggcgata gggttgacct
     3001 tattctttgg caaatctgga gcagaaccgt ggcatggttc gtacaaacca aatgcggtgt
     3061 tcttgtctgg caaagaggcc aaggacgcag atggcaacaa acccaaggaa cctgggataa
     3121 cggaggcttc atcggagacg atatcaccaa acatgttgct ggtgattata ataccattta
     3181 ggtgggttgg gttcctaact aggatcatgg cggcagaatc aatcaattga tgttgaacct
     3241 tcaatgtagg aaattcgttc ttgatggttt cctccacagt ttttctccat aatcttgaag
     3301 aggccaaaac attagcttta tccaaggacc aaataggcaa tggtggctca tgttgtaggg
     3361 ccatgaaagc ggccattctt gtgattcttt gcacttctgg aacggtgtat tgttcactat
     3421 cccaagcgac accatcacca tcgtcttcct ttctcttacc aaagtaaata cctcccacta
     3481 attctctgac aacaacgaag tcagtacctt tagcaaattg tggcttgatt ggagataagt
     3541 ctaaaagaga gtcggatgca aagttacatg gtcttaagtt ggcgtacaat tgaagttctt
     3601 tacggatttt tagtaaacct tgttcaggtc taacactacc tgtaccccat ttaggaccac
     3661 ccacagcacc taacaaaacg gcatcagcct tcttggaggc ttccagcgcc tcatctggaa
     3721 gtgggacacc tgtagcttcg atagcagcac caccaattaa atgattttcg aaatcgaact
     3781 tgacattgga acgaacatca gaaatagctt taagaacctt aatggcttcg gctgtgattt
     3841 cttgaccaac gtggtcacct ggcaaaacga cgatcttctt aggggcagac attagaatgg
     3901 tatatccttg aaatatatat atatattgct gaaatgtaaa aggtaagaaa agttagaaag
     3961 taagacgatt gctaaccacc tattggaaaa aacaataggt ccttaaataa tattgtcaac
     4021 ttcaagtatt gtgatgcaag catttagtca tgaacgcttc tctattctat atgaaaagcc
     4081 ggttccggcg ctctcacctt tcctttttct cccaattttt cagttgaaaa aggtatatgc
     4141 gtcaggcgac ctctgaaatt aacaaaaaat ttccagtcat cgaatttgat tctgtgcgat
     4201 agcgcccctg tgtgttctcg ttatgttgag gaaaaaaata atggttgcta agagattcga
     4261 actcttgcat cttacgatac ctgagtattc ccacagttgg gggatctcga ctctagctag
     4321 aggatcaatt cgtaatcatg tcatagctgt ttcctgtgtg aaattgttat ccgctcacaa
     4381 ttccacacaa catacgagcc ggaagcataa agtgtaaagc ctggggtgcc taatgagtga
     4441 gctaactcac attaattgcg ttgcgctcac tgcccgcttt ccagtcggga aacctgtcgt
     4501 gccagctgca ttaatgaatc ggccaacgcg cggggagagg cggtttgcgt attgggcgct
     4561 cttccgcttc ctcgctcact gactcgctgc gctcggtcgt tcggctgcgg cgagcggtat
     4621 cagctcactc aaaggcggta atacggttat ccacagaatc aggggataac gcaggaaaga
     4681 acatgtgagc aaaaggccag caaaaggcca ggaaccgtaa aaaggccgcg ttgctggcgt
     4741 ttttccatag gctccgcccc cctgacgagc atcacaaaaa tcgacgctca agtcagaggt
     4801 ggcgaaaccc gacaggacta taaagatacc aggcgtttcc ccctggaagc tccctcgtgc
     4861 gctctcctgt tccgaccctg ccgcttaccg gatacctgtc cgcctttctc ccttcgggaa
     4921 gcgtggcgct ttctcatagc tcacgctgta ggtatctcag ttcggtgtag gtcgttcgct
     4981 ccaagctggg ctgtgtgcac gaaccccccg ttcagcccga ccgctgcgcc ttatccggta
     5041 actatcgtct tgagtccaac ccggtaagac acgacttatc gccactggca gcagccactg
     5101 gtaacaggat tagcagagcg aggtatgtag gcggtgctac agagttcttg aagtggtggc
     5161 ctaactacgg ctacactaga agaacagtat ttggtatctg cgctctgctg aagccagtta
     5221 ccttcggaaa aagagttggt agctcttgat ccggcaaaca aaccaccgct ggtagcggtg
     5281 gtttttttgt ttgcaagcag cagattacgc gcagaaaaaa aggatctcaa gaagatcctt
     5341 tgatcttttc tacggggtct gacgctcagt ggaacgaaaa ctcacgttaa gggattttgg
     5401 tcatgagatt atcaaaaagg atcttcacct agatcctttt aaattaaaaa tgaagtttta
     5461 aatcaatcta aagtatatat gagtaaactt ggtctgacag ttaccaatgc ttaatcagtg
     5521 aggcacctat ctcagcgatc tgtctatttc gttcatccat agttgcctga ctccccgtcg
     5581 tgtagataac tacgatacgg gagggcttac catctggccc cagtgctgca atgataccgc
     5641 gagacccacg ctcaccggct ccagatttat cagcaataaa ccagccagcc ggaagggccg
     5701 agcgcagaag tggtcctgca actttatccg cctccatcca gtctattaat tgttgccggg
     5761 aagctagagt aagtagttcg ccagttaata gtttgcgcaa cgttgttgcc attgctacag
     5821 gcatcgtggt gtcacgctcg tcgtttggta tggcttcatt cagctccggt tcccaacgat
     5881 caaggcgagt tacatgatcc cccatgttgt gcaaaaaagc ggttagctcc ttcggtcctc
     5941 cgatcgttgt cagaagtaag ttggccgcag tgttatcact catggttatg gcagcactgc
     6001 ataattctct tactgtcatg ccatccgtaa gatgcttttc tgtgactggt gagtactcaa
     6061 ccaagtcatt ctgagaatag tgtatgcggc gaccgagttg ctcttgcccg gcgtcaatac
     6121 gggataatac cgcgccacat agcagaactt taaaagtgct catcattgga aaacgttctt
     6181 cggggcgaaa actctcaagg atcttaccgc tgttgagatc cagttcgatg taacccactc
     6241 gtgcacccaa ctgatcttca gcatctttta ctttcaccag cgtttctggg tgagcaaaaa
     6301 caggaaggca aaatgccgca aaaaagggaa taagggcgac acggaaatgt tgaatactca
     6361 tactcttcct ttttcaatat tattgaagca tttatcaggg ttattgtctc atgagcggat
     6421 acatatttga atgtatttag aaaaataaac aaataggggt tccgcgcaca tttccccgaa
     6481 aagtgccacc tgacgtctaa gaaaccatta ttatcatgac attaacctat aaaaataggc
     6541 gtatcacgag gccctttcgt ctcgcgcgtt tcggtgatga cggtgaaaac ctctgacaca
     6601 tgcagctccc ggagacggtc acagcttgtc tgtaagcgga tgccgggagc agacaagccc
     6661 gtcagggcgc gtcagcgggt gttggcgggt gtcggggctg gcttaactat gcggcatcag
     6721 agcagattgt actgagagtg caccataacg catttaagca taaacacgca ctatgccgtt
     6781 cttctcatgt atatatatat acaggcaaca cgcagatata ggtgcgacgt gaacagtgag
     6841 ctgtatgtgc gcagctcgcg ttgcattttc ggaagcgctc gttttcggaa acgctttgaa
     6901 gttcctattc cgaagttcct attctctagc tagaaagtat aggaacttca gagcgctttt
     6961 gaaaaccaaa agcgctctga agacgcactt tcaaaaaacc aaaaacgcac cggactgtaa
     7021 cgagctacta aaatattgcg aataccgctt ccacaaacat tgctcaaaag tatctctttg
     7081 ctatatatct ctgtgctata tccctatata acctacccat ccacctttcg ctccttgaac
     7141 ttgcatctaa actcgacctc tacatttttt atgtttatct ctagtattac tctttagaca
     7201 aaaaaattgt agtaagaact attcatagag tgaatcgaaa acaatacgaa aatgtaaaca
     7261 tttcctatac gtagtatata gagacaaaat agaagaaacc gttcataatt ttctgaccaa
     7321 tgaagaatca tcaacgctat cactttctgt tcacaaagta tgcgcaatcc acatcggtat
     7381 agaatataat cggggatgcc tttatcttga aaaaatgcac ccgcagcttc gctagtaatc
     7441 agtaaacgcg ggaagtggag tcaggctttt tttatggaag agaaaataga caccaaagta
     7501 gccttcttct aaccttaacg gacctacagt gcaaaaagtt atcaagagac tgcattatag
     7561 agcgcacaaa ggagaaaaaa agtaatctaa gatgctttgt tagaaaaata gcgctctcgg
     7621 gatgcatttt tgtagaacaa aaaagaagta tagattcttt gttggtaaaa tagcgctctc
     7681 gcgttgcatt tctgttctgt aaaaatgcag ctcagattct ttgtttgaaa aattagcgct
     7741 ctcgcgttgc atttttgttt tacaaaaatg aagcacagat tcttcgttgg taaaatagcg
     7801 ctttcgcgtt gcatttctgt tctgtaaaaa tgcagctcag attctttgtt tgaaaaatta
     7861 gcgctctcgc gttgcatttt tg
//
