LOCUS       sequence-239328-        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(F317Q)
FEATURES             Location/Qualifiers
     source          1..7933
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     promoter        114..515
                     /gene="S. cerevisiae ADH1"
                     /label=ADH1 promoter
                     /note="promoter for alcohol dehydrogenase 1"
     CDS             561..581
                     /codon_start=1
                     /product="nuclear localization signal of SV40 (simian virus
                     40) large T antigen"
                     /label=SV40 NLS
                     /translation="PKKKRKV"
     CDS             597..938
                     /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             2009..2020
                     /note="strong bacterial ribosome binding site (Elowitz and 
                     Leibler, 2000)"
     terminator      2620..2807
                     /gene="S. cerevisiae ADH1"
                     /label=ADH1 terminator
                     /note="transcription terminator for the S. cerevisiae 
                     alcohol dehydrogenase 1 (ADH1) gene"
     primer_bind     complement(2675..2699)
                     /label=pHybLex-R
                     /note="ADH terminator, reverse primer"
     CDS             complement(2928..4022)
                     /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(4023..4428)
                     /gene="S. cerevisiae LEU2"
                     /label=LEU2 promoter
     primer_bind     complement(4470..4486)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(4470..4486)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(4483..4505)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    4494..4510
                     /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(4518..4524,4525..4542,4543..4548))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    4563..4584
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(4701..4718)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(4872..5460)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(4952..4971)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(5631..6422,6423..6491))
                     /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     6254..6273
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(6492..6596)
                     /gene="bla"
                     /label=AmpR promoter
     primer_bind     6664..6682
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     primer_bind     complement(6720..6742)
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     primer_bind     6842..6861
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     rep_origin      join(6878..7933,1..109)
                     /label=2u ori
                     /note="yeast 2u plasmid origin of replication"
ORIGIN
        1 tcgcgttgca tttctgttct gtaaaaatgc agctcagatt ctttgtttga aaaattagcg
       61 ctctcgcgtt gcatttttgt tctacaaaat gaagcacaga tgcttcgttg cttgcatgca
      121 acttcttttc tttttttttc ttttctctct cccccgttgt tgtctcacca tatccgcaat
      181 gacaaaaaaa tgatggaaga cactaaagga aaaaattaac gacaaagaca gcaccaacag
      241 atgtcgttgt tccagagctg atgaggggta tctcgaagca cacgaaactt tttccttcct
      301 tcattcacgc acactactct ctaatgagca acggtatacg gccttccttc cagttacttg
      361 aatttgaaat aaaaaaaagt ttgctgtctt gctatcaagt ataaatagac ctgcaattat
      421 taatcttttg tttcctcgtc attgttctcg ttccctttct tccttgtttc tttttctgca
      481 caatatttca agctatacca agcatacaat caactccaag ctttgcaaag atggataaag
      541 cggaattaat tcccgagcct ccaaaaaaga agagaaaggt cgaattgggt accgccgcca
      601 attttaatca aagtgggaat attgctgata gctcattgtc cttcactttc actaacagta
      661 gcaacggtcc gaacctcata acaactcaaa caaattctca agcgctttca caaccaattg
      721 cctcctctaa cgttcatgat aacttcatga ataatgaaat cacggctagt aaaattgatg
      781 atggtaataa ttcaaaacca ctgtcacctg gttggacgga ccaaactgcg tataacgcgt
      841 ttggaatcac tacagggatg tttaatacca ctacaatgga tgatgtatat aactatctat
      901 tcgatgatga agatacccca ccaaacccaa aaaaagagat cgaattcccc gggggatcca
      961 tcgataacga agcagatgtt acaaaatttg ttaataatgc caggaaaacg ctgaccgatg
     1021 ctcaactttt atgttcaagt gctaatttaa ggattgtaga tattaagaaa aaattgtcat
     1081 cttggcagtt gagtatttca aaactcaatt ttctaatagt tggcttgaga cagcaaggaa
     1141 agtttcttta cactatttta aaggaaggca ttgggacaaa gctaatccag aaacaatgga
     1201 atcaagctgt cttagtggtc ttagttgatg agatgaagta ctggcaatat gaaatcactt
     1261 ctaaagtaca aaggttggat ggtatagtaa atgaattgag catatcagaa aaagatgata
     1321 ccgatccctc taaactagga gattacatct cgagagacaa cgttaactta ttgaatgaca
     1381 aactgaaaga agtgccagta attgagcgtc aaattgaaaa cattaagctt caatacgaaa
     1441 atatggtcag aaaagttaac aaagaactga ttgataccaa gttgacggac gtaactcaaa
     1501 aattccaaag taaatttggt atagataacc tgatggaaac aaatgttgca gagcagttta
     1561 gcagggaact aacggacctt gaaaaagatt tagcagagat aatgaattca ttgacacagc
     1621 actttgataa gacattgctg ttacaagata aaaaaattga taacgatgaa cgtgaggagc
     1681 tgtttaaggt ggtacaaggc gacgacaaag aactatacaa cattttcaaa actctgcatg
     1741 aggtaattga tgacgtggac aaaacaattc ttaacttggg tcaatttttg caggcaaaaa
     1801 taaaggaaaa gacagaacta cacagcgaag tttctgaaat aataaacgat ttcaatagaa
     1861 atttggaata tctattaatc tttaaagata tttccaatct gattgatagc caaaaaaatt
     1921 cctgtacaca agatattcaa acaactaagg aactttgtga attttatgat aattttgaag
     1981 aaagctacgg taacttagtt ctagaagcaa agaggagaaa ggatgtggca aacagaatga
     2041 aaactatatt gaaagattgt gaaaagcagt tgcagaattt agatgctcag gaccaggaag
     2101 aacgtcagaa ttttatagcg gaaaatggaa cttatcttcc tgagacaatc tggcccggta
     2161 aaattgacga tttttcttcc ctgtacactt taaattacaa cgtgaagaat ccttagaaaa
     2221 aaggaaaaaa aggctgcaga gatctatgaa tcgtagatac tgaaaaaccc cgcaagttca
     2281 cttcaactgt gcatcgtgca ccatctcaat ttctttcatt tatacatcgt tttgccttct
     2341 tttatgtaac tatactcctc taagtttcaa tcttggccat gtaacctctg atctatagaa
     2401 ttttttaaat gactagaatt aatgcccatc ttttttttgg acctaaattc ttcatgaaaa
     2461 tatattacga gggcttattc agaagctttg gacttcttcg ccagaggttt ggtcaagtct
     2521 ccaatcaagg ttgtcggctt gtctaccttg ccagaaattt acgaaaagat ggaaaagggt
     2581 caaatcgttg gtagatacgt tgttgacact tctaaataag cgaatttctt atgatttatg
     2641 atttttatta ttaaataagt tataaaaaaa ataagtgtat acaaatttta aagtgactct
     2701 taggttttaa aacgaaaatt cttattcttg agtaactctt tcctgtaggt caggttgctt
     2761 tctcaggtat agcatgaggt cgctcttatt gaccacacct ctaccggcat gccggtcgaa
     2821 attcccctac cctatgaaca tattccattt tgtaatttcg tgtcgtttct attatgaatt
     2881 tcatttataa agtttatgta caaatatcat aaaaaaagag aatcttttta agcaaggatt
     2941 ttcttaacct cttcggcgac agcatcaccg acttcggtgg tactgttgga accacctaaa
     3001 tcaccagttc tgatacctgc atccaaaacc tttttaactg catcttcaat ggccttacct
     3061 tcttcaggca agttcaatga caatttcaac atcattgcag cagacaagat agtggcgata
     3121 gggttgacct tattctttgg caaatctgga gcagaaccgt ggcatggttc gtacaaacca
     3181 aatgcggtgt tcttgtctgg caaagaggcc aaggacgcag atggcaacaa acccaaggaa
     3241 cctgggataa cggaggcttc atcggagacg atatcaccaa acatgttgct ggtgattata
     3301 ataccattta ggtgggttgg gttcctaact aggatcatgg cggcagaatc aatcaattga
     3361 tgttgaacct tcaatgtagg aaattcgttc ttgatggttt cctccacagt ttttctccat
     3421 aatcttgaag aggccaaaac attagcttta tccaaggacc aaataggcaa tggtggctca
     3481 tgttgtaggg ccatgaaagc ggccattctt gtgattcttt gcacttctgg aacggtgtat
     3541 tgttcactat cccaagcgac accatcacca tcgtcttcct ttctcttacc aaagtaaata
     3601 cctcccacta attctctgac aacaacgaag tcagtacctt tagcaaattg tggcttgatt
     3661 ggagataagt ctaaaagaga gtcggatgca aagttacatg gtcttaagtt ggcgtacaat
     3721 tgaagttctt tacggatttt tagtaaacct tgttcaggtc taacactacc tgtaccccat
     3781 ttaggaccac ccacagcacc taacaaaacg gcatcagcct tcttggaggc ttccagcgcc
     3841 tcatctggaa gtgggacacc tgtagcttcg atagcagcac caccaattaa atgattttcg
     3901 aaatcgaact tgacattgga acgaacatca gaaatagctt taagaacctt aatggcttcg
     3961 gctgtgattt cttgaccaac gtggtcacct ggcaaaacga cgatcttctt aggggcagac
     4021 attagaatgg tatatccttg aaatatatat atatattgct gaaatgtaaa aggtaagaaa
     4081 agttagaaag taagacgatt gctaaccacc tattggaaaa aacaataggt ccttaaataa
     4141 tattgtcaac ttcaagtatt gtgatgcaag catttagtca tgaacgcttc tctattctat
     4201 atgaaaagcc ggttccggcg ctctcacctt tcctttttct cccaattttt cagttgaaaa
     4261 aggtatatgc gtcaggcgac ctctgaaatt aacaaaaaat ttccagtcat cgaatttgat
     4321 tctgtgcgat agcgcccctg tgtgttctcg ttatgttgag gaaaaaaata atggttgcta
     4381 agagattcga actcttgcat cttacgatac ctgagtattc ccacagttgg gggatctcga
     4441 ctctagctag aggatcaatt cgtaatcatg tcatagctgt ttcctgtgtg aaattgttat
     4501 ccgctcacaa ttccacacaa catacgagcc ggaagcataa agtgtaaagc ctggggtgcc
     4561 taatgagtga gctaactcac attaattgcg ttgcgctcac tgcccgcttt ccagtcggga
     4621 aacctgtcgt gccagctgca ttaatgaatc ggccaacgcg cggggagagg cggtttgcgt
     4681 attgggcgct cttccgcttc ctcgctcact gactcgctgc gctcggtcgt tcggctgcgg
     4741 cgagcggtat cagctcactc aaaggcggta atacggttat ccacagaatc aggggataac
     4801 gcaggaaaga acatgtgagc aaaaggccag caaaaggcca ggaaccgtaa aaaggccgcg
     4861 ttgctggcgt ttttccatag gctccgcccc cctgacgagc atcacaaaaa tcgacgctca
     4921 agtcagaggt ggcgaaaccc gacaggacta taaagatacc aggcgtttcc ccctggaagc
     4981 tccctcgtgc gctctcctgt tccgaccctg ccgcttaccg gatacctgtc cgcctttctc
     5041 ccttcgggaa gcgtggcgct ttctcatagc tcacgctgta ggtatctcag ttcggtgtag
     5101 gtcgttcgct ccaagctggg ctgtgtgcac gaaccccccg ttcagcccga ccgctgcgcc
     5161 ttatccggta actatcgtct tgagtccaac ccggtaagac acgacttatc gccactggca
     5221 gcagccactg gtaacaggat tagcagagcg aggtatgtag gcggtgctac agagttcttg
     5281 aagtggtggc ctaactacgg ctacactaga agaacagtat ttggtatctg cgctctgctg
     5341 aagccagtta ccttcggaaa aagagttggt agctcttgat ccggcaaaca aaccaccgct
     5401 ggtagcggtg gtttttttgt ttgcaagcag cagattacgc gcagaaaaaa aggatctcaa
     5461 gaagatcctt tgatcttttc tacggggtct gacgctcagt ggaacgaaaa ctcacgttaa
     5521 gggattttgg tcatgagatt atcaaaaagg atcttcacct agatcctttt aaattaaaaa
     5581 tgaagtttta aatcaatcta aagtatatat gagtaaactt ggtctgacag ttaccaatgc
     5641 ttaatcagtg aggcacctat ctcagcgatc tgtctatttc gttcatccat agttgcctga
     5701 ctccccgtcg tgtagataac tacgatacgg gagggcttac catctggccc cagtgctgca
     5761 atgataccgc gagacccacg ctcaccggct ccagatttat cagcaataaa ccagccagcc
     5821 ggaagggccg agcgcagaag tggtcctgca actttatccg cctccatcca gtctattaat
     5881 tgttgccggg aagctagagt aagtagttcg ccagttaata gtttgcgcaa cgttgttgcc
     5941 attgctacag gcatcgtggt gtcacgctcg tcgtttggta tggcttcatt cagctccggt
     6001 tcccaacgat caaggcgagt tacatgatcc cccatgttgt gcaaaaaagc ggttagctcc
     6061 ttcggtcctc cgatcgttgt cagaagtaag ttggccgcag tgttatcact catggttatg
     6121 gcagcactgc ataattctct tactgtcatg ccatccgtaa gatgcttttc tgtgactggt
     6181 gagtactcaa ccaagtcatt ctgagaatag tgtatgcggc gaccgagttg ctcttgcccg
     6241 gcgtcaatac gggataatac cgcgccacat agcagaactt taaaagtgct catcattgga
     6301 aaacgttctt cggggcgaaa actctcaagg atcttaccgc tgttgagatc cagttcgatg
     6361 taacccactc gtgcacccaa ctgatcttca gcatctttta ctttcaccag cgtttctggg
     6421 tgagcaaaaa caggaaggca aaatgccgca aaaaagggaa taagggcgac acggaaatgt
     6481 tgaatactca tactcttcct ttttcaatat tattgaagca tttatcaggg ttattgtctc
     6541 atgagcggat acatatttga atgtatttag aaaaataaac aaataggggt tccgcgcaca
     6601 tttccccgaa aagtgccacc tgacgtctaa gaaaccatta ttatcatgac attaacctat
     6661 aaaaataggc gtatcacgag gccctttcgt ctcgcgcgtt tcggtgatga cggtgaaaac
     6721 ctctgacaca tgcagctccc ggagacggtc acagcttgtc tgtaagcgga tgccgggagc
     6781 agacaagccc gtcagggcgc gtcagcgggt gttggcgggt gtcggggctg gcttaactat
     6841 gcggcatcag agcagattgt actgagagtg caccataacg catttaagca taaacacgca
     6901 ctatgccgtt cttctcatgt atatatatat acaggcaaca cgcagatata ggtgcgacgt
     6961 gaacagtgag ctgtatgtgc gcagctcgcg ttgcattttc ggaagcgctc gttttcggaa
     7021 acgctttgaa gttcctattc cgaagttcct attctctagc tagaaagtat aggaacttca
     7081 gagcgctttt gaaaaccaaa agcgctctga agacgcactt tcaaaaaacc aaaaacgcac
     7141 cggactgtaa cgagctacta aaatattgcg aataccgctt ccacaaacat tgctcaaaag
     7201 tatctctttg ctatatatct ctgtgctata tccctatata acctacccat ccacctttcg
     7261 ctccttgaac ttgcatctaa actcgacctc tacatttttt atgtttatct ctagtattac
     7321 tctttagaca aaaaaattgt agtaagaact attcatagag tgaatcgaaa acaatacgaa
     7381 aatgtaaaca tttcctatac gtagtatata gagacaaaat agaagaaacc gttcataatt
     7441 ttctgaccaa tgaagaatca tcaacgctat cactttctgt tcacaaagta tgcgcaatcc
     7501 acatcggtat agaatataat cggggatgcc tttatcttga aaaaatgcac ccgcagcttc
     7561 gctagtaatc agtaaacgcg ggaagtggag tcaggctttt tttatggaag agaaaataga
     7621 caccaaagta gccttcttct aaccttaacg gacctacagt gcaaaaagtt atcaagagac
     7681 tgcattatag agcgcacaaa ggagaaaaaa agtaatctaa gatgctttgt tagaaaaata
     7741 gcgctctcgg gatgcatttt tgtagaacaa aaaagaagta tagattcttt gttggtaaaa
     7801 tagcgctctc gcgttgcatt tctgttctgt aaaaatgcag ctcagattct ttgtttgaaa
     7861 aattagcgct ctcgcgttgc atttttgttt tacaaaaatg aagcacagat tcttcgttgg
     7921 taaaatagcg ctt
//
