LOCUS       sequence-237708-        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(L105D)
FEATURES             Location/Qualifiers
     source          1..7933
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     CDS             445..465
                     /codon_start=1
                     /product="nuclear localization signal of SV40 (simian virus
                     40) large T antigen"
                     /label=SV40 NLS
                     /translation="PKKKRKV"
     CDS             481..822
                     /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             1893..1904
                     /note="strong bacterial ribosome binding site (Elowitz and 
                     Leibler, 2000)"
     terminator      2504..2691
                     /gene="S. cerevisiae ADH1"
                     /label=ADH1 terminator
                     /note="transcription terminator for the S. cerevisiae 
                     alcohol dehydrogenase 1 (ADH1) gene"
     primer_bind     complement(2559..2583)
                     /label=pHybLex-R
                     /note="ADH terminator, reverse primer"
     CDS             complement(2812..3906)
                     /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(3907..4312)
                     /gene="S. cerevisiae LEU2"
                     /label=LEU2 promoter
     primer_bind     complement(4354..4370)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(4354..4370)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(4367..4389)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    4378..4394
                     /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(4402..4408,4409..4426,4427..4432))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    4447..4468
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(4585..4602)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(4756..5344)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(4836..4855)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(5515..6306,6307..6375))
                     /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     6138..6157
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(6376..6480)
                     /gene="bla"
                     /label=AmpR promoter
     primer_bind     6548..6566
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     primer_bind     complement(6604..6626)
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     primer_bind     6726..6745
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     rep_origin      6762..7926
                     /label=2u ori
                     /note="yeast 2u plasmid origin of replication"
     promoter        join(7931..7933,1..399)
                     /gene="S. cerevisiae ADH1"
                     /label=ADH1 promoter
                     /note="promoter for alcohol dehydrogenase 1"
ORIGIN
        1 tgcaacttct tttctttttt tttcttttct ctctcccccg ttgttgtctc accatatccg
       61 caatgacaaa aaaatgatgg aagacactaa aggaaaaaat taacgacaaa gacagcacca
      121 acagatgtcg ttgttccaga gctgatgagg ggtatctcga agcacacgaa actttttcct
      181 tccttcattc acgcacacta ctctctaatg agcaacggta tacggccttc cttccagtta
      241 cttgaatttg aaataaaaaa aagtttgctg tcttgctatc aagtataaat agacctgcaa
      301 ttattaatct tttgtttcct cgtcattgtt ctcgttccct ttcttccttg tttctttttc
      361 tgcacaatat ttcaagctat accaagcata caatcaactc caagctttgc aaagatggat
      421 aaagcggaat taattcccga gcctccaaaa aagaagagaa aggtcgaatt gggtaccgcc
      481 gccaatttta atcaaagtgg gaatattgct gatagctcat tgtccttcac tttcactaac
      541 agtagcaacg gtccgaacct cataacaact caaacaaatt ctcaagcgct ttcacaacca
      601 attgcctcct ctaacgttca tgataacttc atgaataatg aaatcacggc tagtaaaatt
      661 gatgatggta ataattcaaa accactgtca cctggttgga cggaccaaac tgcgtataac
      721 gcgtttggaa tcactacagg gatgtttaat accactacaa tggatgatgt atataactat
      781 ctattcgatg atgaagatac cccaccaaac ccaaaaaaag agatcgaatt ccccggggga
      841 tccatcgata acgaagcaga tgttacaaaa tttgttaata atgccaggaa aacgctgacc
      901 gatgctcaac ttttatgttc aagtgctaat ttaaggattg tagatattaa gaaaaaattg
      961 tcatcttggc agttgagtat ttcaaaactc aattttctaa tagttggctt gagacagcaa
     1021 ggaaagtttc tttacactat tttaaaggaa ggcattggga caaagctaat ccagaaacaa
     1081 tggaatcaag ctgtcttagt ggtcttagtt gatgagatga agtactggca atatgaaatc
     1141 acttctaaag tacaaaggga cgatggtata gtaaatgaat tgagcatatc agaaaaagat
     1201 gataccgatc cctctaaact aggagattac atctcgagag acaacgttaa cttattgaat
     1261 gacaaactga aagaagtgcc agtaattgag cgtcaaattg aaaacattaa gcttcaatac
     1321 gaaaatatgg tcagaaaagt taacaaagaa ctgattgata ccaagttgac ggacgtaact
     1381 caaaaattcc aaagtaaatt tggtatagat aacctgatgg aaacaaatgt tgcagagcag
     1441 tttagcaggg aactaacgga ccttgaaaaa gatttagcag agataatgaa ttcattgaca
     1501 cagcactttg ataagacatt gctgttacaa gataaaaaaa ttgataacga tgaacgtgag
     1561 gagctgttta aggtggtaca aggcgacgac aaagaactat acaacatttt caaaactctg
     1621 catgaggtaa ttgatgacgt ggacaaaaca attcttaact tgggtcaatt tttgcaggca
     1681 aaaataaagg aaaagacaga actacacagc gaagtttctg aaataataaa cgatttcaat
     1741 agaaatttgg aatatctatt aatctttaaa gatatttcca atctgattga tagcttcaaa
     1801 aattcctgta cacaagatat tcaaacaact aaggaacttt gtgaatttta tgataatttt
     1861 gaagaaagct acggtaactt agttctagaa gcaaagagga gaaaggatgt ggcaaacaga
     1921 atgaaaacta tattgaaaga ttgtgaaaag cagttgcaga atttagatgc tcaggaccag
     1981 gaagaacgtc agaattttat agcggaaaat ggaacttatc ttcctgagac aatctggccc
     2041 ggtaaaattg acgatttttc ttccctgtac actttaaatt acaacgtgaa gaatccttag
     2101 aaaaaaggaa aaaaaggctg cagagatcta tgaatcgtag atactgaaaa accccgcaag
     2161 ttcacttcaa ctgtgcatcg tgcaccatct caatttcttt catttataca tcgttttgcc
     2221 ttcttttatg taactatact cctctaagtt tcaatcttgg ccatgtaacc tctgatctat
     2281 agaatttttt aaatgactag aattaatgcc catctttttt ttggacctaa attcttcatg
     2341 aaaatatatt acgagggctt attcagaagc tttggacttc ttcgccagag gtttggtcaa
     2401 gtctccaatc aaggttgtcg gcttgtctac cttgccagaa atttacgaaa agatggaaaa
     2461 gggtcaaatc gttggtagat acgttgttga cacttctaaa taagcgaatt tcttatgatt
     2521 tatgattttt attattaaat aagttataaa aaaaataagt gtatacaaat tttaaagtga
     2581 ctcttaggtt ttaaaacgaa aattcttatt cttgagtaac tctttcctgt aggtcaggtt
     2641 gctttctcag gtatagcatg aggtcgctct tattgaccac acctctaccg gcatgccggt
     2701 cgaaattccc ctaccctatg aacatattcc attttgtaat ttcgtgtcgt ttctattatg
     2761 aatttcattt ataaagttta tgtacaaata tcataaaaaa agagaatctt tttaagcaag
     2821 gattttctta acctcttcgg cgacagcatc accgacttcg gtggtactgt tggaaccacc
     2881 taaatcacca gttctgatac ctgcatccaa aaccttttta actgcatctt caatggcctt
     2941 accttcttca ggcaagttca atgacaattt caacatcatt gcagcagaca agatagtggc
     3001 gatagggttg accttattct ttggcaaatc tggagcagaa ccgtggcatg gttcgtacaa
     3061 accaaatgcg gtgttcttgt ctggcaaaga ggccaaggac gcagatggca acaaacccaa
     3121 ggaacctggg ataacggagg cttcatcgga gacgatatca ccaaacatgt tgctggtgat
     3181 tataatacca tttaggtggg ttgggttcct aactaggatc atggcggcag aatcaatcaa
     3241 ttgatgttga accttcaatg taggaaattc gttcttgatg gtttcctcca cagtttttct
     3301 ccataatctt gaagaggcca aaacattagc tttatccaag gaccaaatag gcaatggtgg
     3361 ctcatgttgt agggccatga aagcggccat tcttgtgatt ctttgcactt ctggaacggt
     3421 gtattgttca ctatcccaag cgacaccatc accatcgtct tcctttctct taccaaagta
     3481 aatacctccc actaattctc tgacaacaac gaagtcagta cctttagcaa attgtggctt
     3541 gattggagat aagtctaaaa gagagtcgga tgcaaagtta catggtctta agttggcgta
     3601 caattgaagt tctttacgga tttttagtaa accttgttca ggtctaacac tacctgtacc
     3661 ccatttagga ccacccacag cacctaacaa aacggcatca gccttcttgg aggcttccag
     3721 cgcctcatct ggaagtggga cacctgtagc ttcgatagca gcaccaccaa ttaaatgatt
     3781 ttcgaaatcg aacttgacat tggaacgaac atcagaaata gctttaagaa ccttaatggc
     3841 ttcggctgtg atttcttgac caacgtggtc acctggcaaa acgacgatct tcttaggggc
     3901 agacattaga atggtatatc cttgaaatat atatatatat tgctgaaatg taaaaggtaa
     3961 gaaaagttag aaagtaagac gattgctaac cacctattgg aaaaaacaat aggtccttaa
     4021 ataatattgt caacttcaag tattgtgatg caagcattta gtcatgaacg cttctctatt
     4081 ctatatgaaa agccggttcc ggcgctctca cctttccttt ttctcccaat ttttcagttg
     4141 aaaaaggtat atgcgtcagg cgacctctga aattaacaaa aaatttccag tcatcgaatt
     4201 tgattctgtg cgatagcgcc cctgtgtgtt ctcgttatgt tgaggaaaaa aataatggtt
     4261 gctaagagat tcgaactctt gcatcttacg atacctgagt attcccacag ttgggggatc
     4321 tcgactctag ctagaggatc aattcgtaat catgtcatag ctgtttcctg tgtgaaattg
     4381 ttatccgctc acaattccac acaacatacg agccggaagc ataaagtgta aagcctgggg
     4441 tgcctaatga gtgagctaac tcacattaat tgcgttgcgc tcactgcccg ctttccagtc
     4501 gggaaacctg tcgtgccagc tgcattaatg aatcggccaa cgcgcgggga gaggcggttt
     4561 gcgtattggg cgctcttccg cttcctcgct cactgactcg ctgcgctcgg tcgttcggct
     4621 gcggcgagcg gtatcagctc actcaaaggc ggtaatacgg ttatccacag aatcagggga
     4681 taacgcagga aagaacatgt gagcaaaagg ccagcaaaag gccaggaacc gtaaaaaggc
     4741 cgcgttgctg gcgtttttcc ataggctccg cccccctgac gagcatcaca aaaatcgacg
     4801 ctcaagtcag aggtggcgaa acccgacagg actataaaga taccaggcgt ttccccctgg
     4861 aagctccctc gtgcgctctc ctgttccgac cctgccgctt accggatacc tgtccgcctt
     4921 tctcccttcg ggaagcgtgg cgctttctca tagctcacgc tgtaggtatc tcagttcggt
     4981 gtaggtcgtt cgctccaagc tgggctgtgt gcacgaaccc cccgttcagc ccgaccgctg
     5041 cgccttatcc ggtaactatc gtcttgagtc caacccggta agacacgact tatcgccact
     5101 ggcagcagcc actggtaaca ggattagcag agcgaggtat gtaggcggtg ctacagagtt
     5161 cttgaagtgg tggcctaact acggctacac tagaagaaca gtatttggta tctgcgctct
     5221 gctgaagcca gttaccttcg gaaaaagagt tggtagctct tgatccggca aacaaaccac
     5281 cgctggtagc ggtggttttt ttgtttgcaa gcagcagatt acgcgcagaa aaaaaggatc
     5341 tcaagaagat cctttgatct tttctacggg gtctgacgct cagtggaacg aaaactcacg
     5401 ttaagggatt ttggtcatga gattatcaaa aaggatcttc acctagatcc ttttaaatta
     5461 aaaatgaagt tttaaatcaa tctaaagtat atatgagtaa acttggtctg acagttacca
     5521 atgcttaatc agtgaggcac ctatctcagc gatctgtcta tttcgttcat ccatagttgc
     5581 ctgactcccc gtcgtgtaga taactacgat acgggagggc ttaccatctg gccccagtgc
     5641 tgcaatgata ccgcgagacc cacgctcacc ggctccagat ttatcagcaa taaaccagcc
     5701 agccggaagg gccgagcgca gaagtggtcc tgcaacttta tccgcctcca tccagtctat
     5761 taattgttgc cgggaagcta gagtaagtag ttcgccagtt aatagtttgc gcaacgttgt
     5821 tgccattgct acaggcatcg tggtgtcacg ctcgtcgttt ggtatggctt cattcagctc
     5881 cggttcccaa cgatcaaggc gagttacatg atcccccatg ttgtgcaaaa aagcggttag
     5941 ctccttcggt cctccgatcg ttgtcagaag taagttggcc gcagtgttat cactcatggt
     6001 tatggcagca ctgcataatt ctcttactgt catgccatcc gtaagatgct tttctgtgac
     6061 tggtgagtac tcaaccaagt cattctgaga atagtgtatg cggcgaccga gttgctcttg
     6121 cccggcgtca atacgggata ataccgcgcc acatagcaga actttaaaag tgctcatcat
     6181 tggaaaacgt tcttcggggc gaaaactctc aaggatctta ccgctgttga gatccagttc
     6241 gatgtaaccc actcgtgcac ccaactgatc ttcagcatct tttactttca ccagcgtttc
     6301 tgggtgagca aaaacaggaa ggcaaaatgc cgcaaaaaag ggaataaggg cgacacggaa
     6361 atgttgaata ctcatactct tcctttttca atattattga agcatttatc agggttattg
     6421 tctcatgagc ggatacatat ttgaatgtat ttagaaaaat aaacaaatag gggttccgcg
     6481 cacatttccc cgaaaagtgc cacctgacgt ctaagaaacc attattatca tgacattaac
     6541 ctataaaaat aggcgtatca cgaggccctt tcgtctcgcg cgtttcggtg atgacggtga
     6601 aaacctctga cacatgcagc tcccggagac ggtcacagct tgtctgtaag cggatgccgg
     6661 gagcagacaa gcccgtcagg gcgcgtcagc gggtgttggc gggtgtcggg gctggcttaa
     6721 ctatgcggca tcagagcaga ttgtactgag agtgcaccat aacgcattta agcataaaca
     6781 cgcactatgc cgttcttctc atgtatatat atatacaggc aacacgcaga tataggtgcg
     6841 acgtgaacag tgagctgtat gtgcgcagct cgcgttgcat tttcggaagc gctcgttttc
     6901 ggaaacgctt tgaagttcct attccgaagt tcctattctc tagctagaaa gtataggaac
     6961 ttcagagcgc ttttgaaaac caaaagcgct ctgaagacgc actttcaaaa aaccaaaaac
     7021 gcaccggact gtaacgagct actaaaatat tgcgaatacc gcttccacaa acattgctca
     7081 aaagtatctc tttgctatat atctctgtgc tatatcccta tataacctac ccatccacct
     7141 ttcgctcctt gaacttgcat ctaaactcga cctctacatt ttttatgttt atctctagta
     7201 ttactcttta gacaaaaaaa ttgtagtaag aactattcat agagtgaatc gaaaacaata
     7261 cgaaaatgta aacatttcct atacgtagta tatagagaca aaatagaaga aaccgttcat
     7321 aattttctga ccaatgaaga atcatcaacg ctatcacttt ctgttcacaa agtatgcgca
     7381 atccacatcg gtatagaata taatcgggga tgcctttatc ttgaaaaaat gcacccgcag
     7441 cttcgctagt aatcagtaaa cgcgggaagt ggagtcaggc tttttttatg gaagagaaaa
     7501 tagacaccaa agtagccttc ttctaacctt aacggaccta cagtgcaaaa agttatcaag
     7561 agactgcatt atagagcgca caaaggagaa aaaaagtaat ctaagatgct ttgttagaaa
     7621 aatagcgctc tcgggatgca tttttgtaga acaaaaaaga agtatagatt ctttgttggt
     7681 aaaatagcgc tctcgcgttg catttctgtt ctgtaaaaat gcagctcaga ttctttgttt
     7741 gaaaaattag cgctctcgcg ttgcattttt gttttacaaa aatgaagcac agattcttcg
     7801 ttggtaaaat agcgctttcg cgttgcattt ctgttctgta aaaatgcagc tcagattctt
     7861 tgtttgaaaa attagcgctc tcgcgttgca tttttgttct acaaaatgaa gcacagatgc
     7921 ttcgttgctt gca
//
