LOCUS       sequence-239325-        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(F317D)
FEATURES             Location/Qualifiers
     source          1..7933
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     promoter        8..409
                     /gene="S. cerevisiae ADH1"
                     /label=ADH1 promoter
                     /note="promoter for alcohol dehydrogenase 1"
     CDS             455..475
                     /codon_start=1
                     /product="nuclear localization signal of SV40 (simian virus
                     40) large T antigen"
                     /label=SV40 NLS
                     /translation="PKKKRKV"
     CDS             491..832
                     /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             1903..1914
                     /note="strong bacterial ribosome binding site (Elowitz and 
                     Leibler, 2000)"
     terminator      2514..2701
                     /gene="S. cerevisiae ADH1"
                     /label=ADH1 terminator
                     /note="transcription terminator for the S. cerevisiae 
                     alcohol dehydrogenase 1 (ADH1) gene"
     primer_bind     complement(2569..2593)
                     /label=pHybLex-R
                     /note="ADH terminator, reverse primer"
     CDS             complement(2822..3916)
                     /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(3917..4322)
                     /gene="S. cerevisiae LEU2"
                     /label=LEU2 promoter
     primer_bind     complement(4364..4380)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(4364..4380)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(4377..4399)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    4388..4404
                     /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(4412..4418,4419..4436,4437..4442))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    4457..4478
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(4595..4612)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(4766..5354)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(4846..4865)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(5525..6316,6317..6385))
                     /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     6148..6167
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(6386..6490)
                     /gene="bla"
                     /label=AmpR promoter
     primer_bind     6558..6576
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     primer_bind     complement(6614..6636)
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     primer_bind     6736..6755
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     rep_origin      join(6772..7933,1..3)
                     /label=2u ori
                     /note="yeast 2u plasmid origin of replication"
ORIGIN
        1 gttgcttgca tgcaacttct tttctttttt tttcttttct ctctcccccg ttgttgtctc
       61 accatatccg caatgacaaa aaaatgatgg aagacactaa aggaaaaaat taacgacaaa
      121 gacagcacca acagatgtcg ttgttccaga gctgatgagg ggtatctcga agcacacgaa
      181 actttttcct tccttcattc acgcacacta ctctctaatg agcaacggta tacggccttc
      241 cttccagtta cttgaatttg aaataaaaaa aagtttgctg tcttgctatc aagtataaat
      301 agacctgcaa ttattaatct tttgtttcct cgtcattgtt ctcgttccct ttcttccttg
      361 tttctttttc tgcacaatat ttcaagctat accaagcata caatcaactc caagctttgc
      421 aaagatggat aaagcggaat taattcccga gcctccaaaa aagaagagaa aggtcgaatt
      481 gggtaccgcc gccaatttta atcaaagtgg gaatattgct gatagctcat tgtccttcac
      541 tttcactaac agtagcaacg gtccgaacct cataacaact caaacaaatt ctcaagcgct
      601 ttcacaacca attgcctcct ctaacgttca tgataacttc atgaataatg aaatcacggc
      661 tagtaaaatt gatgatggta ataattcaaa accactgtca cctggttgga cggaccaaac
      721 tgcgtataac gcgtttggaa tcactacagg gatgtttaat accactacaa tggatgatgt
      781 atataactat ctattcgatg atgaagatac cccaccaaac ccaaaaaaag agatcgaatt
      841 ccccggggga tccatcgata acgaagcaga tgttacaaaa tttgttaata atgccaggaa
      901 aacgctgacc gatgctcaac ttttatgttc aagtgctaat ttaaggattg tagatattaa
      961 gaaaaaattg tcatcttggc agttgagtat ttcaaaactc aattttctaa tagttggctt
     1021 gagacagcaa ggaaagtttc tttacactat tttaaaggaa ggcattggga caaagctaat
     1081 ccagaaacaa tggaatcaag ctgtcttagt ggtcttagtt gatgagatga agtactggca
     1141 atatgaaatc acttctaaag tacaaaggtt ggatggtata gtaaatgaat tgagcatatc
     1201 agaaaaagat gataccgatc cctctaaact aggagattac atctcgagag acaacgttaa
     1261 cttattgaat gacaaactga aagaagtgcc agtaattgag cgtcaaattg aaaacattaa
     1321 gcttcaatac gaaaatatgg tcagaaaagt taacaaagaa ctgattgata ccaagttgac
     1381 ggacgtaact caaaaattcc aaagtaaatt tggtatagat aacctgatgg aaacaaatgt
     1441 tgcagagcag tttagcaggg aactaacgga ccttgaaaaa gatttagcag agataatgaa
     1501 ttcattgaca cagcactttg ataagacatt gctgttacaa gataaaaaaa ttgataacga
     1561 tgaacgtgag gagctgttta aggtggtaca aggcgacgac aaagaactat acaacatttt
     1621 caaaactctg catgaggtaa ttgatgacgt ggacaaaaca attcttaact tgggtcaatt
     1681 tttgcaggca aaaataaagg aaaagacaga actacacagc gaagtttctg aaataataaa
     1741 cgatttcaat agaaatttgg aatatctatt aatctttaaa gatatttcca atctgattga
     1801 tagcgacaaa aattcctgta cacaagatat tcaaacaact aaggaacttt gtgaatttta
     1861 tgataatttt gaagaaagct acggtaactt agttctagaa gcaaagagga gaaaggatgt
     1921 ggcaaacaga atgaaaacta tattgaaaga ttgtgaaaag cagttgcaga atttagatgc
     1981 tcaggaccag gaagaacgtc agaattttat agcggaaaat ggaacttatc ttcctgagac
     2041 aatctggccc ggtaaaattg acgatttttc ttccctgtac actttaaatt acaacgtgaa
     2101 gaatccttag aaaaaaggaa aaaaaggctg cagagatcta tgaatcgtag atactgaaaa
     2161 accccgcaag ttcacttcaa ctgtgcatcg tgcaccatct caatttcttt catttataca
     2221 tcgttttgcc ttcttttatg taactatact cctctaagtt tcaatcttgg ccatgtaacc
     2281 tctgatctat agaatttttt aaatgactag aattaatgcc catctttttt ttggacctaa
     2341 attcttcatg aaaatatatt acgagggctt attcagaagc tttggacttc ttcgccagag
     2401 gtttggtcaa gtctccaatc aaggttgtcg gcttgtctac cttgccagaa atttacgaaa
     2461 agatggaaaa gggtcaaatc gttggtagat acgttgttga cacttctaaa taagcgaatt
     2521 tcttatgatt tatgattttt attattaaat aagttataaa aaaaataagt gtatacaaat
     2581 tttaaagtga ctcttaggtt ttaaaacgaa aattcttatt cttgagtaac tctttcctgt
     2641 aggtcaggtt gctttctcag gtatagcatg aggtcgctct tattgaccac acctctaccg
     2701 gcatgccggt cgaaattccc ctaccctatg aacatattcc attttgtaat ttcgtgtcgt
     2761 ttctattatg aatttcattt ataaagttta tgtacaaata tcataaaaaa agagaatctt
     2821 tttaagcaag gattttctta acctcttcgg cgacagcatc accgacttcg gtggtactgt
     2881 tggaaccacc taaatcacca gttctgatac ctgcatccaa aaccttttta actgcatctt
     2941 caatggcctt accttcttca ggcaagttca atgacaattt caacatcatt gcagcagaca
     3001 agatagtggc gatagggttg accttattct ttggcaaatc tggagcagaa ccgtggcatg
     3061 gttcgtacaa accaaatgcg gtgttcttgt ctggcaaaga ggccaaggac gcagatggca
     3121 acaaacccaa ggaacctggg ataacggagg cttcatcgga gacgatatca ccaaacatgt
     3181 tgctggtgat tataatacca tttaggtggg ttgggttcct aactaggatc atggcggcag
     3241 aatcaatcaa ttgatgttga accttcaatg taggaaattc gttcttgatg gtttcctcca
     3301 cagtttttct ccataatctt gaagaggcca aaacattagc tttatccaag gaccaaatag
     3361 gcaatggtgg ctcatgttgt agggccatga aagcggccat tcttgtgatt ctttgcactt
     3421 ctggaacggt gtattgttca ctatcccaag cgacaccatc accatcgtct tcctttctct
     3481 taccaaagta aatacctccc actaattctc tgacaacaac gaagtcagta cctttagcaa
     3541 attgtggctt gattggagat aagtctaaaa gagagtcgga tgcaaagtta catggtctta
     3601 agttggcgta caattgaagt tctttacgga tttttagtaa accttgttca ggtctaacac
     3661 tacctgtacc ccatttagga ccacccacag cacctaacaa aacggcatca gccttcttgg
     3721 aggcttccag cgcctcatct ggaagtggga cacctgtagc ttcgatagca gcaccaccaa
     3781 ttaaatgatt ttcgaaatcg aacttgacat tggaacgaac atcagaaata gctttaagaa
     3841 ccttaatggc ttcggctgtg atttcttgac caacgtggtc acctggcaaa acgacgatct
     3901 tcttaggggc agacattaga atggtatatc cttgaaatat atatatatat tgctgaaatg
     3961 taaaaggtaa gaaaagttag aaagtaagac gattgctaac cacctattgg aaaaaacaat
     4021 aggtccttaa ataatattgt caacttcaag tattgtgatg caagcattta gtcatgaacg
     4081 cttctctatt ctatatgaaa agccggttcc ggcgctctca cctttccttt ttctcccaat
     4141 ttttcagttg aaaaaggtat atgcgtcagg cgacctctga aattaacaaa aaatttccag
     4201 tcatcgaatt tgattctgtg cgatagcgcc cctgtgtgtt ctcgttatgt tgaggaaaaa
     4261 aataatggtt gctaagagat tcgaactctt gcatcttacg atacctgagt attcccacag
     4321 ttgggggatc tcgactctag ctagaggatc aattcgtaat catgtcatag ctgtttcctg
     4381 tgtgaaattg ttatccgctc acaattccac acaacatacg agccggaagc ataaagtgta
     4441 aagcctgggg tgcctaatga gtgagctaac tcacattaat tgcgttgcgc tcactgcccg
     4501 ctttccagtc gggaaacctg tcgtgccagc tgcattaatg aatcggccaa cgcgcgggga
     4561 gaggcggttt gcgtattggg cgctcttccg cttcctcgct cactgactcg ctgcgctcgg
     4621 tcgttcggct gcggcgagcg gtatcagctc actcaaaggc ggtaatacgg ttatccacag
     4681 aatcagggga taacgcagga aagaacatgt gagcaaaagg ccagcaaaag gccaggaacc
     4741 gtaaaaaggc cgcgttgctg gcgtttttcc ataggctccg cccccctgac gagcatcaca
     4801 aaaatcgacg ctcaagtcag aggtggcgaa acccgacagg actataaaga taccaggcgt
     4861 ttccccctgg aagctccctc gtgcgctctc ctgttccgac cctgccgctt accggatacc
     4921 tgtccgcctt tctcccttcg ggaagcgtgg cgctttctca tagctcacgc tgtaggtatc
     4981 tcagttcggt gtaggtcgtt cgctccaagc tgggctgtgt gcacgaaccc cccgttcagc
     5041 ccgaccgctg cgccttatcc ggtaactatc gtcttgagtc caacccggta agacacgact
     5101 tatcgccact ggcagcagcc actggtaaca ggattagcag agcgaggtat gtaggcggtg
     5161 ctacagagtt cttgaagtgg tggcctaact acggctacac tagaagaaca gtatttggta
     5221 tctgcgctct gctgaagcca gttaccttcg gaaaaagagt tggtagctct tgatccggca
     5281 aacaaaccac cgctggtagc ggtggttttt ttgtttgcaa gcagcagatt acgcgcagaa
     5341 aaaaaggatc tcaagaagat cctttaatct tttctacggg gtctgacgct cagtggaacg
     5401 aaaactcacg ttaagggatt ttggtcatga gattatcaaa aaggatcttc acctagatcc
     5461 ttttaaatta aaaatgaagt tttaaatcaa tctaaagtat atatgagtaa acttggtctg
     5521 acagttacca atgcttaatc agtgaggcac ctatctcagc gatctgtcta tttcgttcat
     5581 ccatagttgc ctgactcccc gtcgtgtaga taactacgat acgggagggc ttaccatctg
     5641 gccccagtgc tgcaatgata ccgcgagacc cacgctcacc ggctccagat ttatcagcaa
     5701 taaaccagcc agccggaagg gccgagcgca gaagtggtcc tgcaacttta tccgcctcca
     5761 tccagtctat taattgttgc cgggaagcta gagtaagtag ttcgccagtt aatagtttgc
     5821 gcaacgttgt tgccattgct acaggcatcg tggtgtcacg ctcgtcgttt ggtatggctt
     5881 cattcagctc cggttcccaa cgatcaaggc gagttacatg atcccccatg ttgtgcaaaa
     5941 aagcggttag ctccttcggt cctccgatcg ttgtcagaag taagttggcc gcagtgttat
     6001 cactcatggt tatggcagca ctgcataatt ctcttactgt catgccatcc gtaagatgct
     6061 tttctgtgac tggtgagtac tcaaccaagt cattctgaga atagtgtatg cggcgaccga
     6121 gttgctcttg cccggcgtca atacgggata ataccgcgcc acatagcaga actttaaaag
     6181 tgctcatcat tggaaaacgt tcttcggggc gaaaactctc aaggatctta ccgctgttga
     6241 gatccagttc gatgtaaccc actcgtgcac ccaactgatc ttcagcatct tttactttca
     6301 ccagcgtttc tgggtgagca aaaacaggaa ggcaaaatgc cgcaaaaaag ggaataaggg
     6361 cgacacggaa atgttgaata ctcatactct tcctttttca atattattga agcatttatc
     6421 agggttattg tctcatgagc ggatacatat ttgaatgtat ttagaaaaat aaacaaatag
     6481 gggttccgcg cacatttccc cgaaaagtgc cacctgacgt ctaagaaacc attattatca
     6541 tgacattaac ctataaaaat aggcgtatca cgaggccctt tcgtctcgcg cgtttcggtg
     6601 atgacggtga aaacctctga cacatgcagc tcccggagac ggtcacagct tgtctgtaag
     6661 cggatgccgg gagcagacaa gcccgtcagg gcgcgtcagc gggtgttggc gggtgtcggg
     6721 gctggcttaa ctatgcggca tcagagcaga ttgtactgag agtgcaccat aacgcattta
     6781 agcataaaca cgcactatgc cgttcttctc atgtatatat atatacaggc aacacgcaga
     6841 tataggtgcg acgtgaacag tgagctgtat gtgcgcagct cgcgttgcat tttcggaagc
     6901 gctcgttttc ggaaacgctt tgaagttcct attccgaagt tcctattctc tagctagaaa
     6961 gtataggaac ttcagagcgc ttttgaaaac caaaagcgct ctgaagacgc actttcaaaa
     7021 aaccaaaaac gcaccggact gtaacgagct actaaaatat tgcgaatacc gcttccacaa
     7081 acattgctca aaagtatctc tttgctatat atctctgtgc tatatcccta tataacctac
     7141 ccatccacct ttcgctcctt gaacttgcat ctaaactcga cctctacatt ttttatgttt
     7201 atctctagta ttactcttta gacaaaaaaa ttgtagtaag aactattcat agagtgaatc
     7261 gaaaacaata cgaaaatgta aacatttcct atacgtagta tatagagaca aaatagaaga
     7321 aaccgttcat aattttctga ccaatgaaga atcatcaacg ctatcacttt ctgttcacaa
     7381 agtatgcgca atccacatcg gtatagaata taatcgggga tgcctttatc ttgaaaaaat
     7441 gcacccgcag cttcgctagt aatcagtaaa cgcgggaagt ggagtcaggc tttttttatg
     7501 gaagagaaaa tagacaccaa agtagccttc ttctaacctt aacggaccta cagtgcaaaa
     7561 agttatcaag agactgcatt atagagcgca caaaggagaa aaaaagtaat ctaagatgct
     7621 ttgttagaaa aatagcgctc tcgggatgca tttttgtaga acaaaaaaga agtatagatt
     7681 ctttgttggt aaaatagcgc tctcgcgttg catttctgtt ctgtaaaaat gcagctcaga
     7741 ttctttgttt gaaaaattag cgctctcgcg ttgcattttt gttttacaaa aatgaagcac
     7801 agattcttcg ttggtaaaat agcgctttcg cgttgcattt ctgttctgta aaaatgcagc
     7861 tcagattctt tgtttgaaaa attagcgctc tcgcgttgca tttttgttct acaaaatgaa
     7921 gcacagatgc ttc
//
