LOCUS       sequence-239335-        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(I325A)
FEATURES             Location/Qualifiers
     source          1..7933
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     promoter        804..1205
                     /gene="S. cerevisiae ADH1"
                     /label=ADH1 promoter
                     /note="promoter for alcohol dehydrogenase 1"
     CDS             1251..1271
                     /codon_start=1
                     /product="nuclear localization signal of SV40 (simian virus
                     40) large T antigen"
                     /label=SV40 NLS
                     /translation="PKKKRKV"
     CDS             1287..1628
                     /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             2699..2710
                     /note="strong bacterial ribosome binding site (Elowitz and 
                     Leibler, 2000)"
     terminator      3310..3497
                     /gene="S. cerevisiae ADH1"
                     /label=ADH1 terminator
                     /note="transcription terminator for the S. cerevisiae 
                     alcohol dehydrogenase 1 (ADH1) gene"
     primer_bind     complement(3365..3389)
                     /label=pHybLex-R
                     /note="ADH terminator, reverse primer"
     CDS             complement(3618..4712)
                     /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(4713..5118)
                     /gene="S. cerevisiae LEU2"
                     /label=LEU2 promoter
     primer_bind     complement(5160..5176)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(5160..5176)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(5173..5195)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    5184..5200
                     /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(5208..5214,5215..5232,5233..5238))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    5253..5274
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(5391..5408)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(5562..6150)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(5642..5661)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(6321..7112,7113..7181))
                     /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     6944..6963
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(7182..7286)
                     /gene="bla"
                     /label=AmpR promoter
     primer_bind     7354..7372
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     primer_bind     complement(7410..7432)
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     primer_bind     7532..7551
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     rep_origin      join(7568..7933,1..799)
                     /label=2u ori
                     /note="yeast 2u plasmid origin of replication"
ORIGIN
        1 tacccatcca cctttcgctc cttgaacttg catctaaact cgacctctac attttttatg
       61 tttatctcta gtattactct ttagacaaaa aaattgtagt aagaactatt catagagtga
      121 atcgaaaaca atacgaaaat gtaaacattt cctatacgta gtatatagag acaaaataga
      181 agaaaccgtt cataattttc tgaccaatga agaatcatca acgctatcac tttctgttca
      241 caaagtatgc gcaatccaca tcggtataga atataatcgg ggatgccttt atcttgaaaa
      301 aatgcacccg cagcttcgct agtaatcagt aaacgcggga agtggagtca ggcttttttt
      361 atggaagaga aaatagacac caaagtagcc ttcttctaac cttaacggac ctacagtgca
      421 aaaagttatc aagagactgc attatagagc gcacaaagga gaaaaaaagt aatctaagat
      481 gctttgttag aaaaatagcg ctctcgggat gcatttttgt agaacaaaaa agaagtatag
      541 attctttgtt ggtaaaatag cgctctcgcg ttgcatttct gttctgtaaa aatgcagctc
      601 agattctttg tttgaaaaat tagcgctctc gcgttgcatt tttgttttac aaaaatgaag
      661 cacagattct tcgttggtaa aatagcgctt tcgcgttgca tttctgttct gtaaaaatgc
      721 agctcagatt ctttgtttga aaaattagcg ctctcgcgtt gcatttttgt tctacaaaat
      781 gaagcacaga tgcttcgttg cttgcatgca acttcttttc tttttttttc ttttctctct
      841 cccccgttgt tgtctcacca tatccgcaat gacaaaaaaa tgatggaaga cactaaagga
      901 aaaaattaac gacaaagaca gcaccaacag atgtcgttgt tccagagctg atgaggggta
      961 tctcgaagca cacgaaactt tttccttcct tcattcacgc acactactct ctaatgagca
     1021 acggtatacg gccttccttc cagttacttg aatttgaaat aaaaaaaagt ttgctgtctt
     1081 gctatcaagt ataaatagac ctgcaattat taatcttttg tttcctcgtc attgttctcg
     1141 ttccctttct tccttgtttc tttttctgca caatatttca agctatacca agcatacaat
     1201 caactccaag ctttgcaaag atggataaag cggaattaat tcccgagcct ccaaaaaaga
     1261 agagaaaggt cgaattgggt accgccgcca attttaatca aagtgggaat attgctgata
     1321 gctcattgtc cttcactttc actaacagta gcaacggtcc gaacctcata acaactcaaa
     1381 caaattctca agcgctttca caaccaattg cctcctctaa cgttcatgat aacttcatga
     1441 ataatgaaat cacggctagt aaaattgatg atggtaataa ttcaaaacca ctgtcacctg
     1501 gttggacgga ccaaactgcg tataacgcgt ttggaatcac tacagggatg tttaatacca
     1561 ctacaatgga tgatgtatat aactatctat tcgatgatga agatacccca ccaaacccaa
     1621 aaaaagagat cgaattcccc gggggatcca tcgataacga agcagatgtt acaaaatttg
     1681 ttaataatgc caggaaaacg ctgaccgatg ctcaactttt atgttcaagt gctaatttaa
     1741 ggattgtaga tattaagaaa aaattgtcat cttggcagtt gagtatttca aaactcaatt
     1801 ttctaatagt tggcttgaga cagcaaggaa agtttcttta cactatttta aaggaaggca
     1861 ttgggacaaa gctaatccag aaacaatgga atcaagctgt cttagtggtc ttagttgatg
     1921 agatgaagta ctggcaatat gaaatcactt ctaaagtaca aaggttggat ggtatagtaa
     1981 atgaattgag catatcagaa aaagatgata ccgatccctc taaactagga gattacatct
     2041 cgagagacaa cgttaactta ttgaatgaca aactgaaaga agtgccagta attgagcgtc
     2101 aaattgaaaa cattaagctt caatacgaaa atatggtcag aaaagttaac aaagaactga
     2161 ttgataccaa gttgacggac gtaactcaaa aattccaaag taaatttggt atagataacc
     2221 tgatggaaac aaatgttgca gagcagttta gcagggaact aacggacctt gaaaaagatt
     2281 tagcagagat aatgaattca ttgacacagc actttgataa gacattgctg ttacaagata
     2341 aaaaaattga taacgatgaa cgtgaggagc tgtttaaggt ggtacaaggc gacgacaaag
     2401 aactatacaa cattttcaaa actctgcatg aggtaattga tgacgtggac aaaacaattc
     2461 ttaacttggg tcaatttttg caggcaaaaa taaaggaaaa gacagaacta cacagcgaag
     2521 tttctgaaat aataaacgat ttcaatagaa atttggaata tctattaatc tttaaagata
     2581 tttccaatct gattgatagc ttcaaaaatt cctgtacaca agatgctcaa acaactaagg
     2641 aactttgtga attttatgat aattttgaag aaagctacgg taacttagtt ctagaagcaa
     2701 agaggagaaa ggatgtggca aacagaatga aaactatatt gaaagattgt gaaaagcagt
     2761 tgcagaattt agatgctcag gaccaggaag aacgtcagaa ttttatagcg gaaaatggaa
     2821 cttatcttcc tgagacaatc tggcccggta aaattgacga tttttcttcc ctgtacactt
     2881 taaattacaa cgtgaagaat ccttagaaaa aaggaaaaaa aggctgcaga gatctatgaa
     2941 tcgtagatac tgaaaaaccc cgcaagttca cttcaactgt gcatcgtgca ccatctcaat
     3001 ttctttcatt tatacatcgt tttgccttct tttatgtaac tatactcctc taagtttcaa
     3061 tcttggccat gtaacctctg atctatagaa ttttttaaat gactagaatt aatgcccatc
     3121 ttttttttgg acctaaattc ttcatgaaaa tatattacga gggcttattc agaagctttg
     3181 gacttcttcg ccagaggttt ggtcaagtct ccaatcaagg ttgtcggctt gtctaccttg
     3241 ccagaaattt acgaaaagat ggaaaagggt caaatcgttg gtagatacgt tgttgacact
     3301 tctaaataag cgaatttctt atgatttatg atttttatta ttaaataagt tataaaaaaa
     3361 ataagtgtat acaaatttta aagtgactct taggttttaa aacgaaaatt cttattcttg
     3421 agtaactctt tcctgtaggt caggttgctt tctcaggtat agcatgaggt cgctcttatt
     3481 gaccacacct ctaccggcat gccggtcgaa attcccctac cctatgaaca tattccattt
     3541 tgtaatttcg tgtcgtttct attatgaatt tcatttataa agtttatgta caaatatcat
     3601 aaaaaaagag aatcttttta agcaaggatt ttcttaacct cttcggcgac agcatcaccg
     3661 acttcggtgg tactgttgga accacctaaa tcaccagttc tgatacctgc atccaaaacc
     3721 tttttaactg catcttcaat ggccttacct tcttcaggca agttcaatga caatttcaac
     3781 atcattgcag cagacaagat agtggcgata gggttgacct tattctttgg caaatctgga
     3841 gcagaaccgt ggcatggttc gtacaaacca aatgcggtgt tcttgtctgg caaagaggcc
     3901 aaggacgcag atggcaacaa acccaaggaa cctgggataa cggaggcttc atcggagacg
     3961 atatcaccaa acatgttgct ggtgattata ataccattta ggtgggttgg gttcctaact
     4021 aggatcatgg cggcagaatc aatcaattga tgttgaacct tcaatgtagg aaattcgttc
     4081 ttgatggttt cctccacagt ttttctccat aatcttgaag aggccaaaac attagcttta
     4141 tccaaggacc aaataggcaa tggtggctca tgttgtaggg ccatgaaagc ggccattctt
     4201 gtgattcttt gcacttctgg aacggtgtat tgttcactat cccaagcgac accatcacca
     4261 tcgtcttcct ttctcttacc aaagtaaata cctcccacta attctctgac aacaacgaag
     4321 tcagtacctt tagcaaattg tggcttgatt ggagataagt ctaaaagaga gtcggatgca
     4381 aagttacatg gtcttaagtt ggcgtacaat tgaagttctt tacggatttt tagtaaacct
     4441 tgttcaggtc taacactacc tgtaccccat ttaggaccac ccacagcacc taacaaaacg
     4501 gcatcagcct tcttggaggc ttccagcgcc tcatctggaa gtgggacacc tgtagcttcg
     4561 atagcagcac caccaattaa atgattttcg aaatcgaact tgacattgga acgaacatca
     4621 gaaatagctt taagaacctt aatggcttcg gctgtgattt cttgaccaac gtggtcacct
     4681 ggcaaaacga cgatcttctt aggggcagac attagaatgg tatatccttg aaatatatat
     4741 atatattgct gaaatgtaaa aggtaagaaa agttagaaag taagacgatt gctaaccacc
     4801 tattggaaaa aacaataggt ccttaaataa tattgtcaac ttcaagtatt gtgatgcaag
     4861 catttagtca tgaacgcttc tctattctat atgaaaagcc ggttccggcg ctctcacctt
     4921 tcctttttct cccaattttt cagttgaaaa aggtatatgc gtcaggcgac ctctgaaatt
     4981 aacaaaaaat ttccagtcat cgaatttgat tctgtgcgat agcgcccctg tgtgttctcg
     5041 ttatgttgag gaaaaaaata atggttgcta agagattcga actcttgcat cttacgatac
     5101 ctgagtattc ccacagttgg gggatctcga ctctagctag aggatcaatt cgtaatcatg
     5161 tcatagctgt ttcctgtgtg aaattgttat ccgctcacaa ttccacacaa catacgagcc
     5221 ggaagcataa agtgtaaagc ctggggtgcc taatgagtga gctaactcac attaattgcg
     5281 ttgcgctcac tgcccgcttt ccagtcggga aacctgtcgt gccagctgca ttaatgaatc
     5341 ggccaacgcg cggggagagg cggtttgcgt attgggcgct cttccgcttc ctcgctcact
     5401 gactcgctgc gctcggtcgt tcggctgcgg cgagcggtat cagctcactc aaaggcggta
     5461 atacggttat ccacagaatc aggggataac gcaggaaaga acatgtgagc aaaaggccag
     5521 caaaaggcca ggaaccgtaa aaaggccgcg ttgctggcgt ttttccatag gctccgcccc
     5581 cctgacgagc atcacaaaaa tcgacgctca agtcagaggt ggcgaaaccc gacaggacta
     5641 taaagatacc aggcgtttcc ccctggaagc tccctcgtgc gctctcctgt tccgaccctg
     5701 ccgcttaccg gatacctgtc cgcctttctc ccttcgggaa gcgtggcgct ttctcatagc
     5761 tcacgctgta ggtatctcag ttcggtgtag gtcgttcgct ccaagctggg ctgtgtgcac
     5821 gaaccccccg ttcagcccga ccgctgcgcc ttatccggta actatcgtct tgagtccaac
     5881 ccggtaagac acgacttatc gccactggca gcagccactg gtaacaggat tagcagagcg
     5941 aggtatgtag gcggtgctac agagttcttg aagtggtggc ctaactacgg ctacactaga
     6001 agaacagtat ttggtatctg cgctctgctg aagccagtta ccttcggaaa aagagttggt
     6061 agctcttgat ccggcaaaca aaccaccgct ggtagcggtg gtttttttgt ttgcaagcag
     6121 cagattacgc gcagaaaaaa aggatctcaa gaagatcctt tgatcttttc tacggggtct
     6181 gacgctcagt ggaacgaaaa ctcacgttaa gggattttgg tcatgagatt atcaaaaagg
     6241 atcttcacct agatcctttt aaattaaaaa tgaagtttta aatcaatcta aagtatatat
     6301 gagtaaactt ggtctgacag ttaccaatgc ttaatcagtg aggcacctat ctcagcgatc
     6361 tgtctatttc gttcatccat agttgcctga ctccccgtcg tgtagataac tacgatacgg
     6421 gagggcttac catctggccc cagtgctgca atgataccgc gagacccacg ctcaccggct
     6481 ccagatttat cagcaataaa ccagccagcc ggaagggccg agcgcagaag tggtcctgca
     6541 actttatccg cctccatcca gtctattaat tgttgccggg aagctagagt aagtagttcg
     6601 ccagttaata gtttgcgcaa cgttgttgcc attgctacag gcatcgtggt gtcacgctcg
     6661 tcgtttggta tggcttcatt cagctccggt tcccaacgat caaggcgagt tacatgatcc
     6721 cccatgttgt gcaaaaaagc ggttagctcc ttcggtcctc cgatcgttgt cagaagtaag
     6781 ttggccgcag tgttatcact catggttatg gcagcactgc ataattctct tactgtcatg
     6841 ccatccgtaa gatgcttttc tgtgactggt gagtactcaa ccaagtcatt ctgagaatag
     6901 tgtatgcggc gaccgagttg ctcttgcccg gcgtcaatac gggataatac cgcgccacat
     6961 agcagaactt taaaagtgct catcattgga aaacgttctt cggggcgaaa actctcaagg
     7021 atcttaccgc tgttgagatc cagttcgatg taacccactc gtgcacccaa ctgatcttca
     7081 gcatctttta ctttcaccag cgtttctggg tgagcaaaaa caggaaggca aaatgccgca
     7141 aaaaagggaa taagggcgac acggaaatgt tgaatactca tactcttcct ttttcaatat
     7201 tattgaagca tttatcaggg ttattgtctc atgagcggat acatatttga atgtatttag
     7261 aaaaataaac aaataggggt tccgcgcaca tttccccgaa aagtgccacc tgacgtctaa
     7321 gaaaccatta ttatcatgac attaacctat aaaaataggc gtatcacgag gccctttcgt
     7381 ctcgcgcgtt tcggtgatga cggtgaaaac ctctgacaca tgcagctccc ggagacggtc
     7441 acagcttgtc tgtaagcgga tgccgggagc agacaagccc gtcagggcgc gtcagcgggt
     7501 gttggcgggt gtcggggctg gcttaactat gcggcatcag agcagattgt actgagagtg
     7561 caccataacg catttaagca taaacacgca ctatgccgtt cttctcatgt atatatatat
     7621 acaggcaaca cgcagatata ggtgcgacgt gaacagtgag ctgtatgtgc gcagctcgcg
     7681 ttgcattttc ggaagcgctc gttttcggaa acgctttgaa gttcctattc cgaagttcct
     7741 attctctagc tagaaagtat aggaacttca gagcgctttt gaaaaccaaa agcgctctga
     7801 agacgcactt tcaaaaaacc aaaaacgcac cggactgtaa cgagctacta aaatattgcg
     7861 aataccgctt ccacaaacat tgctcaaaag tatctctttg ctatatatct ctgtgctata
     7921 tccctatata acc
//
