LOCUS       sequence-239261-        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(F317K)
FEATURES             Location/Qualifiers
     source          1..7933
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     CDS             345..365
                     /codon_start=1
                     /product="nuclear localization signal of SV40 (simian virus
                     40) large T antigen"
                     /label=SV40 NLS
                     /translation="PKKKRKV"
     CDS             381..722
                     /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             1793..1804
                     /note="strong bacterial ribosome binding site (Elowitz and 
                     Leibler, 2000)"
     terminator      2404..2591
                     /gene="S. cerevisiae ADH1"
                     /label=ADH1 terminator
                     /note="transcription terminator for the S. cerevisiae 
                     alcohol dehydrogenase 1 (ADH1) gene"
     primer_bind     complement(2459..2483)
                     /label=pHybLex-R
                     /note="ADH terminator, reverse primer"
     CDS             complement(2712..3806)
                     /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(3807..4212)
                     /gene="S. cerevisiae LEU2"
                     /label=LEU2 promoter
     primer_bind     complement(4254..4270)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(4254..4270)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(4267..4289)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    4278..4294
                     /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(4302..4308,4309..4326,4327..4332))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    4347..4368
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(4485..4502)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(4656..5244)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(4736..4755)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(5415..6206,6207..6275))
                     /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     6038..6057
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(6276..6380)
                     /gene="bla"
                     /label=AmpR promoter
     primer_bind     6448..6466
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     primer_bind     complement(6504..6526)
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     primer_bind     6626..6645
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     rep_origin      6662..7826
                     /label=2u ori
                     /note="yeast 2u plasmid origin of replication"
     promoter        join(7831..7933,1..299)
                     /gene="S. cerevisiae ADH1"
                     /label=ADH1 promoter
                     /note="promoter for alcohol dehydrogenase 1"
ORIGIN
        1 taacgacaaa gacagcacca acagatgtcg ttgttccaga gctgatgagg ggtatctcga
       61 agcacacgaa actttttcct tccttcattc acgcacacta ctctctaatg agcaacggta
      121 tacggccttc cttccagtta cttgaatttg aaataaaaaa aagtttgctg tcttgctatc
      181 aagtataaat agacctgcaa ttattaatct tttgtttcct cgtcattgtt ctcgttccct
      241 ttcttccttg tttctttttc tgcacaatat ttcaagctat accaagcata caatcaactc
      301 caagctttgc aaagatggat aaagcggaat taattcccga gcctccaaaa aagaagagaa
      361 aggtcgaatt gggtaccgcc gccaatttta atcaaagtgg gaatattgct gatagctcat
      421 tgtccttcac tttcactaac agtagcaacg gtccgaacct cataacaact caaacaaatt
      481 ctcaagcgct ttcacaacca attgcctcct ctaacgttca tgataacttc atgaataatg
      541 aaatcacggc tagtaaaatt gatgatggta ataattcaaa accactgtca cctggttgga
      601 cggaccaaac tgcgtataac gcgtttggaa tcactacagg gatgtttaat accactacaa
      661 tggatgatgt atataactat ctattcgatg atgaagatac cccaccaaac ccaaaaaaag
      721 agatcgaatt ccccggggga tccatcgata acgaagcaga tgttacaaaa tttgttaata
      781 atgccaggaa aacgctgacc gatgctcaac ttttatgttc aagtgctaat ttaaggattg
      841 tagatattaa gaaaaaattg tcatcttggc agttgagtat ttcaaaactc aattttctaa
      901 tagttggctt gagacagcaa ggaaagtttc tttacactat tttaaaggaa ggcattggga
      961 caaagctaat ccagaaacaa tggaatcaag ctgtcttagt ggtcttagtt gatgagatga
     1021 agtactggca atatgaaatc acttctaaag tacaaaggtt ggatggtata gtaaatgaat
     1081 tgagcatatc agaaaaagat gataccgatc cctctaaact aggagattac atctcgagag
     1141 acaacgttaa cttattgaat gacaaactga aagaagtgcc agtaattgag cgtcaaattg
     1201 aaaacattaa gcttcaatac gaaaatatgg tcagaaaagt taacaaagaa ctgattgata
     1261 ccaagttgac ggacgtaact caaaaattcc aaagtaaatt tggtatagat aacctgatgg
     1321 aaacaaatgt tgcagagcag tttagcaggg aactaacgga ccttgaaaaa gatttagcag
     1381 agataatgaa ttcattgaca cagcactttg ataagacatt gctgttacaa gataaaaaaa
     1441 ttgataacga tgaacgtgag gagctgttta aggtggtaca aggcgacgac aaagaactat
     1501 acaacatttt caaaactctg catgaggtaa ttgatgacgt ggacaaaaca attcttaact
     1561 tgggtcaatt tttgcaggca aaaataaagg aaaagacaga actacacagc gaagtttctg
     1621 aaataataaa cgatttcaat agaaatttgg aatatctatt aatctttaaa gatatttcca
     1681 atctgattga tagcaagaaa aattcctgta cacaagatat tcaaacaact aaggaacttt
     1741 gtgaatttta tgataatttt gaagaaagct acggtaactt agttctagaa gcaaagagga
     1801 gaaaggatgt ggcaaacaga atgaaaacta tattgaaaga ttgtgaaaag cagttgcaga
     1861 atttagatgc tcaggaccag gaagaacgtc agaattttat agcggaaaat ggaacttatc
     1921 ttcctgagac aatctggccc ggtaaaattg acgatttttc ttccctgtac actttaaatt
     1981 acaacgtgaa gaatccttag aaaaaaggaa aaaaaggctg cagagatcta tgaatcgtag
     2041 atactgaaaa accccgcaag ttcacttcaa ctgtgcatcg tgcaccatct caatttcttt
     2101 catttataca tcgttttgcc ttcttttatg taactatact cctctaagtt tcaatcttgg
     2161 ccatgtaacc tctgatctat agaatttttt aaatgactag aattaatgcc catctttttt
     2221 ttggacctaa attcttcatg aaaatatatt acgagggctt attcagaagc tttggacttc
     2281 ttcgccagag gtttggtcaa gtctccaatc aaggttgtcg gcttgtctac cttgccagaa
     2341 atttacgaaa agatggaaaa gggtcaaatc gttggtagat acgttgttga cacttctaaa
     2401 taagcgaatt tcttatgatt tatgattttt attattaaat aagttataaa aaaaataagt
     2461 gtatacaaat tttaaagtga ctcttaggtt ttaaaacgaa aattcttatt cttgagtaac
     2521 tctttcctgt aggtcaggtt gctttctcag gtatagcatg aggtcgctct tattgaccac
     2581 acctctaccg gcatgccggt cgaaattccc ctaccctatg aacatattcc attttgtaat
     2641 ttcgtgtcgt ttctattatg aatttcattt ataaagttta tgtacaaata tcataaaaaa
     2701 agagaatctt tttaagcaag gattttctta acctcttcgg cgacagcatc accgacttcg
     2761 gtggtactgt tggaaccacc taaatcacca gttctgatac ctgcatccaa aaccttttta
     2821 actgcatctt caatggcctt accttcttca ggcaagttca atgacaattt caacatcatt
     2881 gcagcagaca agatagtggc gatagggttg accttattct ttggcaaatc tggagcagaa
     2941 ccgtggcatg gttcgtacaa accaaatgcg gtgttcttgt ctggcaaaga ggccaaggac
     3001 gcagatggca acaaacccaa ggaacctggg ataacggagg cttcatcgga gacgatatca
     3061 ccaaacatgt tgctggtgat tataatacca tttaggtggg ttgggttcct aactaggatc
     3121 atggcggcag aatcaatcaa ttgatgttga accttcaatg taggaaattc gttcttgatg
     3181 gtttcctcca cagtttttct ccataatctt gaagaggcca aaacattagc tttatccaag
     3241 gaccaaatag gcaatggtgg ctcatgttgt agggccatga aagcggccat tcttgtgatt
     3301 ctttgcactt ctggaacggt gtattgttca ctatcccaag cgacaccatc accatcgtct
     3361 tcctttctct taccaaagta aatacctccc actaattctc tgacaacaac gaagtcagta
     3421 cctttagcaa attgtggctt gattggagat aagtctaaaa gagagtcgga tgcaaagtta
     3481 catggtctta agttggcgta caattgaagt tctttacgga tttttagtaa accttgttca
     3541 ggtctaacac tacctgtacc ccatttagga ccacccacag cacctaacaa aacggcatca
     3601 gccttcttgg aggcttccag cgcctcatct ggaagtggga cacctgtagc ttcgatagca
     3661 gcaccaccaa ttaaatgatt ttcgaaatcg aacttgacat tggaacgaac atcagaaata
     3721 gctttaagaa ccttaatggc ttcggctgtg atttcttgac caacgtggtc acctggcaaa
     3781 acgacgatct tcttaggggc agacattaga atggtatatc cttgaaatat atatatatat
     3841 tgctgaaatg taaaaggtaa gaaaagttag aaagtaagac gattgctaac cacctattgg
     3901 aaaaaacaat aggtccttaa ataatattgt caacttcaag tattgtgatg caagcattta
     3961 gtcatgaacg cttctctatt ctatatgaaa agccggttcc ggcgctctca cctttccttt
     4021 ttctcccaat ttttcagttg aaaaaggtat atgcgtcagg cgacctctga aattaacaaa
     4081 aaatttccag tcatcgaatt tgattctgtg cgatagcgcc cctgtgtgtt ctcgttatgt
     4141 tgaggaaaaa aataatggtt gctaagagat tcgaactctt gcatcttacg atacctgagt
     4201 attcccacag ttgggggatc tcgactctag ctagaggatc aattcgtaat catgtcatag
     4261 ctgtttcctg tgtgaaattg ttatccgctc acaattccac acaacatacg agccggaagc
     4321 ataaagtgta aagcctgggg tgcctaatga gtgagctaac tcacattaat tgcgttgcgc
     4381 tcactgcccg ctttccagtc gggaaacctg tcgtgccagc tgcattaatg aatcggccaa
     4441 cgcgcgggga gaggcggttt gcgtattggg cgctcttccg cttcctcgct cactgactcg
     4501 ctgcgctcgg tcgttcggct gcggcgagcg gtatcagctc actcaaaggc ggtaatacgg
     4561 ttatccacag aatcagggga taacgcagga aagaacatgt gagcaaaagg ccagcaaaag
     4621 gccaggaacc gtaaaaaggc cgcgttgctg gcgtttttcc ataggctccg cccccctgac
     4681 gagcatcaca aaaatcgacg ctcaagtcag aggtggcgaa acccgacagg actataaaga
     4741 taccaggcgt ttccccctgg aagctccctc gtgcgctctc ctgttccgac cctgccgctt
     4801 accggatacc tgtccgcctt tctcccttcg ggaagcgtgg cgctttctca tagctcacgc
     4861 tgtaggtatc tcagttcggt gtaggtcgtt cgctccaagc tgggctgtgt gcacgaaccc
     4921 cccgttcagc ccgaccgctg cgccttatcc ggtaactatc gtcttgagtc caacccggta
     4981 agacacgact tatcgccact ggcagcagcc actggtaaca ggattagcag agcgaggtat
     5041 gtaggcggtg ctacagagtt cttgaagtgg tggcctaact acggctacac tagaagaaca
     5101 gtatttggta tctgcgctct gctgaagcca gttaccttcg gaaaaagagt tggtagctct
     5161 tgatccggca aacaaaccac cgctggtagc ggtggttttt ttgtttgcaa gcagcagatt
     5221 acgcgcagaa aaaaaggatc tcaagaagat cctttgatct tttctacggg gtctgacgct
     5281 cagtggaacg aaaactcacg ttaagggatt ttggtcatga gattatcaaa aaggatcttc
     5341 acctagatcc ttttaaatta aaaatgaagt tttaaatcaa tctaaagtat atatgagtaa
     5401 acttggtctg acagttacca atgcttaatc agtgaggcac ctatctcagc gatctgtcta
     5461 tttcgttcat ccatagttgc ctgactcccc gtcgtgtaga taactacgat acgggagggc
     5521 ttaccatctg gccccagtgc tgcaatgata ccgcgagacc cacgctcacc ggctccagat
     5581 ttatcagcaa taaaccagcc agccggaagg gccgagcgca gaagtggtcc tgcaacttta
     5641 tccgcctcca tccagtctat taattgttgc cgggaagcta gagtaagtag ttcgccagtt
     5701 aatagtttgc gcaacgttgt tgccattgct acaggcatcg tggtgtcacg ctcgtcgttt
     5761 ggtatggctt cattcagctc cggttcccaa cgatcaaggc gagttacatg atcccccatg
     5821 ttgtgcaaaa aagcggttag ctccttcggt cctccgatcg ttgtcagaag taagttggcc
     5881 gcagtgttat cactcatggt tatggcagca ctgcataatt ctcttactgt catgccatcc
     5941 gtaagatgct tttctgtgac tggtgagtac tcaaccaagt cattctgaga atagtgtatg
     6001 cggcgaccga gttgctcttg cccggcgtca atacgggata ataccgcgcc acatagcaga
     6061 actttaaaag tgctcatcat tggaaaacgt tcttcggggc gaaaactctc aaggatctta
     6121 ccgctgttga gatccagttc gatgtaaccc actcgtgcac ccaactgatc ttcagcatct
     6181 tttactttca ccagcgtttc tgggtgagca aaaacaggaa ggcaaaatgc cgcaaaaaag
     6241 ggaataaggg cgacacggaa atgttgaata ctcatactct tcctttttca atattattga
     6301 agcatttatc agggttattg tctcatgagc ggatacatat ttgaatgtat ttagaaaaat
     6361 aaacaaatag gggttccgcg cacatttccc cgaaaagtgc cacctgacgt ctaagaaacc
     6421 attattatca tgacattaac ctataaaaat aggcgtatca cgaggccctt tcgtctcgcg
     6481 cgtttcggtg atgacggtga aaacctctga cacatgcagc tcccggagac ggtcacagct
     6541 tgtctgtaag cggatgccgg gagcagacaa gcccgtcagg gcgcgtcagc gggtgttggc
     6601 gggtgtcggg gctggcttaa ctatgcggca tcagagcaga ttgtactgag agtgcaccat
     6661 aacgcattta agcataaaca cgcactatgc cgttcttctc atgtatatat atatacaggc
     6721 aacacgcaga tataggtgcg acgtgaacag tgagctgtat gtgcgcagct cgcgttgcat
     6781 tttcggaagc gctcgttttc ggaaacgctt tgaagttcct attccgaagt tcctattctc
     6841 tagctagaaa gtataggaac ttcagagcgc ttttgaaaac caaaagcgct ctgaagacgc
     6901 actttcaaaa aaccaaaaac gcaccggact gtaacgagct actaaaatat tgcgaatacc
     6961 gcttccacaa acattgctca aaagtatctc tttgctatat atctctgtgc tatatcccta
     7021 tataacctac ccatccacct ttcgctcctt gaacttgcat ctaaactcga cctctacatt
     7081 ttttatgttt atctctagta ttactcttta gacaaaaaaa ttgtagtaag aactattcat
     7141 agagtgaatc gaaaacaata cgaaaatgta aacatttcct atacgtagta tatagagaca
     7201 aaatagaaga aaccgttcat aattttctga ccaatgaaga atcatcaacg ctatcacttt
     7261 ctgttcacaa agtatgcgca atccacatcg gtatagaata taatcgggga tgcctttatc
     7321 ttgaaaaaat gcacccgcag cttcgctagt aatcagtaaa cgcgggaagt ggagtcaggc
     7381 tttttttatg gaagagaaaa tagacaccaa agtagccttc ttctaacctt aacggaccta
     7441 cagtgcaaaa agttatcaag agactgcatt atagagcgca caaaggagaa aaaaagtaat
     7501 ctaagatgct ttgttagaaa aatagcgctc tcgggatgca tttttgtaga acaaaaaaga
     7561 agtatagatt ctttgttggt aaaatagcgc tctcgcgttg catttctgtt ctgtaaaaat
     7621 gcagctcaga ttctttgttt gaaaaattag cgctctcgcg ttgcattttt gttttacaaa
     7681 aatgaagcac agattcttcg ttggtaaaat agcgctttcg cgttgcattt ctgttctgta
     7741 aaaatgcagc tcagattctt tgtttgaaaa attagcgctc tcgcgttgca tttttgttct
     7801 acaaaatgaa gcacagatgc ttcgttgctt gcatgcaact tcttttcttt ttttttcttt
     7861 tctctctccc ccgttgttgt ctcaccatat ccgcaatgac aaaaaaatga tggaagacac
     7921 taaaggaaaa aat
//
