LOCUS       sequence-239281-        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(F317G)
FEATURES             Location/Qualifiers
     source          1..7933
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     promoter        3..404
                     /gene="S. cerevisiae ADH1"
                     /label=ADH1 promoter
                     /note="promoter for alcohol dehydrogenase 1"
     CDS             450..470
                     /codon_start=1
                     /product="nuclear localization signal of SV40 (simian virus
                     40) large T antigen"
                     /label=SV40 NLS
                     /translation="PKKKRKV"
     CDS             486..827
                     /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             1898..1909
                     /note="strong bacterial ribosome binding site (Elowitz and 
                     Leibler, 2000)"
     terminator      2509..2696
                     /gene="S. cerevisiae ADH1"
                     /label=ADH1 terminator
                     /note="transcription terminator for the S. cerevisiae 
                     alcohol dehydrogenase 1 (ADH1) gene"
     primer_bind     complement(2564..2588)
                     /label=pHybLex-R
                     /note="ADH terminator, reverse primer"
     CDS             complement(2817..3911)
                     /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(3912..4317)
                     /gene="S. cerevisiae LEU2"
                     /label=LEU2 promoter
     primer_bind     complement(4359..4375)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(4359..4375)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(4372..4394)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    4383..4399
                     /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(4407..4413,4414..4431,4432..4437))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    4452..4473
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(4590..4607)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(4761..5349)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(4841..4860)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(5520..6311,6312..6380))
                     /codon_start=1
                     /gene="bla"
                     /product="beta-lactamase"
                     /label=AmpR
                     /note="confers resistance to ampicillin, carbenicillin, and
                     related antibiotics"
                     /translation="MSIQHFRVALIPFFAAFCLPVFAHPETLVKVKDAEDQLGARVGYI
                     ELDLNSGKILESFRPEERFPMMSTFKVLLCGAVLSRIDAGQEQLGRRIHYSQNDLVEYS
                     PVTEKHLTDGMTVRELCSAAITMSDNTAANLLLTTIGGPKELTAFLHNIGDHVTRLDRW
                     EPELNEAIPNDERDTTMPVAMATTLRKLLTGELLTLASRQQLIDWMEADKVAGPLLRSA
                     LPAGWFIADKSGAGERGSRGIIAALGPDGKPSRIVVIYTTGSQATMDERNRQIAEIGAS
                     LIKHW"
     primer_bind     6143..6162
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(6381..6485)
                     /gene="bla"
                     /label=AmpR promoter
     primer_bind     6553..6571
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     primer_bind     complement(6609..6631)
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     primer_bind     6731..6750
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     rep_origin      6767..7931
                     /label=2u ori
                     /note="yeast 2u plasmid origin of replication"
ORIGIN
        1 ttgcatgcaa cttcttttct ttttttttct tttctctctc ccccgttgtt gtctcaccat
       61 atccgcaatg acaaaaaaat gatggaagac actaaaggaa aaaattaacg acaaagacag
      121 caccaacaga tgtcgttgtt ccagagctga tgaggggtat ctcgaagcac acgaaacttt
      181 ttccttcctt cattcacgca cactactctc taatgagcaa cggtatacgg ccttccttcc
      241 agttacttga atttgaaata aaaaaaagtt tgctgtcttg ctatcaagta taaatagacc
      301 tgcaattatt aatcttttgt ttcctcgtca ttgttctcgt tccctttctt ccttgtttct
      361 ttttctgcac aatatttcaa gctataccaa gcatacaatc aactccaagc tttgcaaaga
      421 tggataaagc ggaattaatt cccgagcctc caaaaaagaa gagaaaggtc gaattgggta
      481 ccgccgccaa ttttaatcaa agtgggaata ttgctgatag ctcattgtcc ttcactttca
      541 ctaacagtag caacggtccg aacctcataa caactcaaac aaattctcaa gcgctttcac
      601 aaccaattgc ctcctctaac gttcatgata acttcatgaa taatgaaatc acggctagta
      661 aaattgatga tggtaataat tcaaaaccac tgtcacctgg ttggacggac caaactgcgt
      721 ataacgcgtt tggaatcact acagggatgt ttaataccac tacaatggat gatgtatata
      781 actatctatt cgatgatgaa gataccccac caaacccaaa aaaagagatc gaattccccg
      841 ggggatccat cgataacgaa gcagatgtta caaaatttgt taataatgcc aggaaaacgc
      901 tgaccgatgc tcaactttta tgttcaagtg ctaatttaag gattgtagat attaagaaaa
      961 aattgtcatc ttggcagttg agtatttcaa aactcaattt tctaatagtt ggcttgagac
     1021 agcaaggaaa gtttctttac actattttaa aggaaggcat tgggacaaag ctaatccaga
     1081 aacaatggaa tcaagctgtc ttagtggtct tagttgatga gatgaagtac tggcaatatg
     1141 aaatcacttc taaagtacaa aggttggatg gtatagtaaa tgaattgagc atatcagaaa
     1201 aagatgatac cgatccctct aaactaggag attacatctc gagagacaac gttaacttat
     1261 tgaatgacaa actgaaagaa gtgccagtaa ttgagcgtca aattgaaaac attaagcttc
     1321 aatacgaaaa tatggtcaga aaagttaaca aagaactgat tgataccaag ttgacggacg
     1381 taactcaaaa attccaaagt aaatttggta tagataacct gatggaaaca aatgttgcag
     1441 agcagtttag cagggaacta acggaccttg aaaaagattt agcagagata atgaattcat
     1501 tgacacagca ctttgataag acattgctgt tacaagataa aaaaattgat aacgatgaac
     1561 gtgaggagct gtttaaggtg gtacaaggcg acgacaaaga actatacaac attttcaaaa
     1621 ctctgcatga ggtaattgat gacgtggaca aaacaattct taacttgggt caatttttgc
     1681 aggcaaaaat aaaggaaaag acagaactac acagcgaagt ttctgaaata ataaacgatt
     1741 tcaatagaaa tttggaatat ctattaatct ttaaagatat ttccaatctg attgatagcg
     1801 gtaaaaattc ctgtacacaa gatattcaaa caactaagga actttgtgaa ttttatgata
     1861 attttgaaga aagctacggt aacttagttc tagaagcaaa gaggagaaag gatgtggcaa
     1921 acagaatgaa aactatattg aaagattgtg aaaagcagtt gcagaattta gatgctcagg
     1981 accaggaaga acgtcagaat tttatagcgg aaaatggaac ttatcttcct gagacaatct
     2041 ggcccggtaa aattgacgat ttttcttccc tgtacacttt aaattacaac gtgaagaatc
     2101 cttagaaaaa aggaaaaaaa ggctgcagag atctatgaat cgtagatact gaaaaacccc
     2161 gcaagttcac ttcaactgtg catcgtgcac catctcaatt tctttcattt atacatcgtt
     2221 ttgccttctt ttatgtaact atactcctct aagtttcaat cttggccatg taacctctga
     2281 tctatagaat tttttaaatg actagaatta atgcccatct tttttttgga cctaaattct
     2341 tcatgaaaat atattacgag ggcttattca gaagctttgg acttcttcgc cagaggtttg
     2401 gtcaagtctc caatcaaggt tgtcggcttg tctaccttgc cagaaattta cgaaaagatg
     2461 gaaaagggtc aaatcgttgg tagatacgtt gttgacactt ctaaataagc gaatttctta
     2521 tgatttatga tttttattat taaataagtt ataaaaaaaa taagtgtata caaattttaa
     2581 agtgactctt aggttttaaa acgaaaattc ttattcttga gtaactcttt cctgtaggtc
     2641 aggttgcttt ctcaggtata gcatgaggtc gctcttattg accacacctc taccggcatg
     2701 ccggtcgaaa ttcccctacc ctatgaacat attccatttt gtaatttcgt gtcgtttcta
     2761 ttatgaattt catttataaa gtttatgtac aaatatcata aaaaaagaga atctttttaa
     2821 gcaaggattt tcttaacctc ttcggcgaca gcatcaccga cttcggtggt actgttggaa
     2881 ccacctaaat caccagttct gatacctgca tccaaaacct ttttaactgc atcttcaatg
     2941 gccttacctt cttcaggcaa gttcaatgac aatttcaaca tcattgcagc agacaagata
     3001 gtggcgatag ggttgacctt attctttggc aaatctggag cagaaccgtg gcatggttcg
     3061 tacaaaccaa atgcggtgtt cttgtctggc aaagaggcca aggacgcaga tggcaacaaa
     3121 cccaaggaac ctgggataac ggaggcttca tcggagacga tatcaccaaa catgttgctg
     3181 gtgattataa taccatttag gtgggttggg ttcctaacta ggatcatggc ggcagaatca
     3241 atcaattgat gttgaacctt caatgtagga aattcgttct tgatggtttc ctccacagtt
     3301 tttctccata atcttgaaga ggccaaaaca ttagctttat ccaaggacca aataggcaat
     3361 ggtggctcat gttgtagggc catgaaagcg gccattcttg tgattctttg cacttctgga
     3421 acggtgtatt gttcactatc ccaagcgaca ccatcaccat cgtcttcctt tctcttacca
     3481 aagtaaatac ctcccactaa ttctctgaca acaacgaagt cagtaccttt agcaaattgt
     3541 ggcttgattg gagataagtc taaaagagag tcggatgcaa agttacatgg tcttaagttg
     3601 gcgtacaatt gaagttcttt acggattttt agtaaacctt gttcaggtct aacactacct
     3661 gtaccccatt taggaccacc cacagcacct aacaaaacgg catcagcctt cttggaggct
     3721 tccagcgcct catctggaag tgggacacct gtagcttcga tagcagcacc accaattaaa
     3781 tgattttcga aatcgaactt gacattggaa cgaacatcag aaatagcttt aagaacctta
     3841 atggcttcgg ctgtgatttc ttgaccaacg tggtcacctg gcaaaacgac gatcttctta
     3901 ggggcagaca ttagaatggt atatccttga aatatatata tatattgctg aaatgtaaaa
     3961 ggtaagaaaa gttagaaagt aagacgattg ctaaccacct attggaaaaa acaataggtc
     4021 cttaaataat attgtcaact tcaagtattg tgatgcaagc atttagtcat gaacgcttct
     4081 ctattctata tgaaaagccg gttccggcgc tctcaccttt cctttttctc ccaatttttc
     4141 agttgaaaaa ggtatatgcg tcaggcgacc tctgaaatta acaaaaaatt tccagtcatc
     4201 gaatttgatt ctgtgcgata gcgcccctgt gtgttctcgt tatgttgagg aaaaaaataa
     4261 tggttgctaa gagattcgaa ctcttgcatc ttacgatacc tgagtattcc cacagttggg
     4321 ggatctcgac tctagctaga ggatcaattc gtaatcatgt catagctgtt tcctgtgtga
     4381 aattgttatc cgctcacaat tccacacaac atacgagccg gaagcataaa gtgtaaagcc
     4441 tggggtgcct aatgagtgag ctaactcaca ttaattgcgt tgcgctcact gcccgctttc
     4501 cagtcgggaa acctgtcgtg ccagctgcat taatgaatcg gccaacgcgc ggggagaggc
     4561 ggtttgcgta ttgggcgctc ttccgcttcc tcgctcactg actcgctgcg ctcggtcgtt
     4621 cggctgcggc gagcggtatc agctcactca aaggcggtaa tacggttatc cacagaatca
     4681 ggggataacg caggaaagaa catgtgagca aaaggccagc aaaaggccag gaaccgtaaa
     4741 aaggccgcgt tgctggcgtt tttccatagg ctccgccccc ctgacgagca tcacaaaaat
     4801 cgacgctcaa gtcagaggtg gcgaaacccg acaggactat aaagatacca ggcgtttccc
     4861 cctggaagct ccctcgtgcg ctctcctgtt ccgaccctgc cgcttaccgg atacctgtcc
     4921 gcctttctcc cttcgggaag cgtggcgctt tctcatagct cacgctgtag gtatctcagt
     4981 tcggtgtagg tcgttcgctc caagctgggc tgtgtgcacg aaccccccgt tcagcccgac
     5041 cgctgcgcct tatccggtaa ctatcgtctt gagtccaacc cggtaagaca cgacttatcg
     5101 ccactggcag cagccactgg taacaggatt agcagagcga ggtatgtagg cggtgctaca
     5161 gagttcttga agtggtggcc taactacggc tacactagaa gaacagtatt tggtatctgc
     5221 gctctgctga agccagttac cttcggaaaa agagttggta gctcttgatc cggcaaacaa
     5281 accaccgctg gtagcggtgg tttttttgtt tgcaagcagc agattacgcg cagaaaaaaa
     5341 ggatctcaag aagatccttt gatcttttct acggggtctg acgctcagtg gaacgaaaac
     5401 tcacgttaag ggattttggt catgagatta tcaaaaagga tcttcaccta gatcctttta
     5461 aattaaaaat gaagttttaa atcaatctaa agtatatatg agtaaacttg gtctgacagt
     5521 taccaatgct taatcagtga ggcacctatc tcagcgatct gtctatttcg ttcatccata
     5581 gttgcctgac tccccgtcgt gtagataact acgatacggg agggcttacc atctggcccc
     5641 agtgctgcaa tgataccgcg agacccacgc tcaccggctc cagatttatc agcaataaac
     5701 cagccagccg gaagggccga gcgcagaagt ggtcctgcaa ctttatccgc ctccatccag
     5761 tctattaatt gttgccggga agctagagta agtagttcgc cagttaatag tttgcgcaac
     5821 gttgttgcca ttgctacagg catcgtggtg tcacgctcgt cgtttggtat ggcttcattc
     5881 agctccggtt cccaacgatc aaggcgagtt acatgatccc ctatgttgtg caaaaaagcg
     5941 gttagctcct tcggtcctcc gatcgttgtc agaagtaagt tggccgcagt gttatcactc
     6001 atggttatgg cagcactgca taattctctt actgtcatgc catccgtaag atgcttttct
     6061 gtgactggtg agtactcaac caagtcattc tgagaatagt gtatgcggcg accgagttgc
     6121 tcttgcccgg cgtcaatacg ggataatacc gcgccacata gcagaacttt aaaagtgctc
     6181 atcattggaa aacgttcttc ggggcgaaaa ctctcaagga tcttaccgct gttgagatcc
     6241 agttcgatgt aacccactcg tgcacccaac tgatcttcag catcttttac tttcaccagc
     6301 gtttctgggt gagcaaaaac aggaaggcaa aatgccgcaa aaaagggaat aagggcgaca
     6361 cggaaatgtt gaatactcat actcttcctt tttcaatatt attgaagcat ttatcagggt
     6421 tattgtctca tgagcggata catatttgaa tgtatttaga aaaataaaca aataggggtt
     6481 ccgcgcacat ttccccgaaa agtgccacct gacgtctaag aaaccattat tatcatgaca
     6541 ttaacctata aaaataggcg tatcacgagg ccctttcgtc tcgcgcgttt cggtgatgac
     6601 ggtgaaaacc tctgacacat gcagctcccg gagacggtca cagcttgtct gtaagcggat
     6661 gccgggagca gacaagcccg tcagggcgcg tcagcgggtg ttggcgggtg tcggggctgg
     6721 cttaactatg cggcatcaga gcagattgta ctgagagtgc accataacgc atttaagcat
     6781 aaacacgcac tatgccgttc ttctcatgta tatatatata caggcaacac gcagatatag
     6841 gtgcgacgtg aacagtgagc tgtatgtgcg cagctcgcgt tgcattttcg gaagcgctcg
     6901 ttttcggaaa cgctttgaag ttcctattcc gaagttccta ttctctagct agaaagtata
     6961 ggaacttcag agcgcttttg aaaaccaaaa gcgctctgaa gacgcacttt caaaaaacca
     7021 aaaacgcacc ggactgtaac gagctactaa aatattgcga ataccgcttc cacaaacatt
     7081 gctcaaaagt atctctttgc tatatatctc tgtgctatat ccctatataa cctacccatc
     7141 cacctttcgc tccttgaact tgcatctaaa ctcgacctct acatttttta tgtttatctc
     7201 tagtattact ctttagacaa aaaaattgta gtaagaacta ttcatagagt gaatcgaaaa
     7261 caatacgaaa atgtaaacat ttcctatacg tagtatatag agacaaaata gaagaaaccg
     7321 ttcataattt tctgaccaat gaagaatcat caacgctatc actttctgtt cacaaagtat
     7381 gcgcaatcca catcggtata gaatataatc ggggatgcct ttatcttgaa aaaatgcacc
     7441 cgcagcttcg ctagtaatca gtaaacgcgg gaagtggagt caggcttttt ttatggaaga
     7501 gaaaatagac accaaagtag ccttcttcta accttaacgg acctacagtg caaaaagtta
     7561 tcaagagact gcattataga gcgcacaaag gagaaaaaaa gtaatctaag atgctttgtt
     7621 agaaaaatag cgctctcggg atgcattttt gtagaacaaa aaagaagtat agattctttg
     7681 ttggtaaaat agcgctctcg cgttgcattt ctgttctgta aaaatgcagc tcagattctt
     7741 tgtttgaaaa attagcgctc tcgcgttgca tttttgtttt acaaaaatga agcacagatt
     7801 cttcgttggt aaaatagcgc tttcgcgttg catttctgtt ctgtaaaaat gcagctcaga
     7861 ttctttgttt gaaaaattag cgctctcgcg ttgcattttt gttctacaaa atgaagcaca
     7921 gatgcttcgt tgc
//
