LOCUS       sequence-237710-        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(L313D I314K)
FEATURES             Location/Qualifiers
     source          1..7933
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     promoter        4..405
                     /gene="S. cerevisiae ADH1"
                     /label=ADH1 promoter
                     /note="promoter for alcohol dehydrogenase 1"
     CDS             451..471
                     /codon_start=1
                     /product="nuclear localization signal of SV40 (simian virus
                     40) large T antigen"
                     /label=SV40 NLS
                     /translation="PKKKRKV"
     CDS             487..828
                     /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             1899..1910
                     /note="strong bacterial ribosome binding site (Elowitz and 
                     Leibler, 2000)"
     terminator      2510..2697
                     /gene="S. cerevisiae ADH1"
                     /label=ADH1 terminator
                     /note="transcription terminator for the S. cerevisiae 
                     alcohol dehydrogenase 1 (ADH1) gene"
     primer_bind     complement(2565..2589)
                     /label=pHybLex-R
                     /note="ADH terminator, reverse primer"
     CDS             complement(2818..3912)
                     /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(3913..4318)
                     /gene="S. cerevisiae LEU2"
                     /label=LEU2 promoter
     primer_bind     complement(4360..4376)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(4360..4376)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(4373..4395)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    4384..4400
                     /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(4408..4414,4415..4432,4433..4438))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    4453..4474
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(4591..4608)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(4762..5350)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(4842..4861)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(5521..6312,6313..6381))
                     /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     6144..6163
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(6382..6486)
                     /gene="bla"
                     /label=AmpR promoter
     primer_bind     6554..6572
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     primer_bind     complement(6610..6632)
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     primer_bind     6732..6751
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     rep_origin      6768..7932
                     /label=2u ori
                     /note="yeast 2u plasmid origin of replication"
ORIGIN
        1 cttgcatgca acttcttttc tttttttttc ttttctctct cccccgttgt tgtctcacca
       61 tatccgcaat gacaaaaaaa tgatggaaga cactaaagga aaaaattaac gacaaagaca
      121 gcaccaacag atgtcgttgt tccagagctg atgaggggta tctcgaagca cacgaaactt
      181 tttccttcct tcattcacgc acactactct ctaatgagca acggtatacg gccttccttc
      241 cagttacttg aatttgaaat aaaaaaaagt ttgctgtctt gctatcaagt ataaatagac
      301 ctgcaattat taatcttttg tttcctcgtc attgttctcg ttccctttct tccttgtttc
      361 tttttctgca caatatttca agctatacca agcatacaat caactccaag ctttgcaaag
      421 atggataaag cggaattaat tcccgagcct ccaaaaaaga agagaaaggt cgaattgggt
      481 accgccgcca attttaatca aagtgggaat attgctgata gctcattgtc cttcactttc
      541 actaacagta gcaacggtcc gaacctcata acaactcaaa caaattctca agcgctttca
      601 caaccaattg cctcctctaa cgttcatgat aacttcatga ataatgaaat cacggctagt
      661 aaaattgatg atggtaataa ttcaaaacca ctgtcacctg gttggacgga ccaaactgcg
      721 tataacgcgt ttggaatcac tacagggatg tttaatacca ctacaatgga tgatgtatat
      781 aactatctat tcgatgatga agatacccca ccaaacccaa aaaaagagat cgaattcccc
      841 gggggatcca tcgataacga agcagatgtt acaaaatttg ttaataatgc caggaaaacg
      901 ctgaccgatg ctcaactttt atgttcaagt gctaatttaa ggattgtaga tattaagaaa
      961 aaattgtcat cttggcagtt gagtatttca aaactcaatt ttctaatagt tggcttgaga
     1021 cagcaaggaa agtttcttta cactatttta aaggaaggca ttgggacaaa gctaatccag
     1081 aaacaatgga atcaagctgt cttagtggtc ttagttgatg agatgaagta ctggcaatat
     1141 gaaatcactt ctaaagtaca aaggttggat ggtatagtaa atgaattgag catatcagaa
     1201 aaagatgata ccgatccctc taaactagga gattacatct cgagagacaa cgttaactta
     1261 ttgaatgaca aactgaaaga agtgccagta attgagcgtc aaattgaaaa cattaagctt
     1321 caatacgaaa atatggtcag aaaagttaac aaagaactga ttgataccaa gttgacggac
     1381 gtaactcaaa aattccaaag taaatttggt atagataacc tgatggaaac aaatgttgca
     1441 gagcagttta gcagggaact aacggacctt gaaaaagatt tagcagagat aatgaattca
     1501 ttgacacagc actttgataa gacattgctg ttacaagata aaaaaattga taacgatgaa
     1561 cgtgaggagc tgtttaaggt ggtacaaggc gacgacaaag aactatacaa cattttcaaa
     1621 actctgcatg aggtaattga tgacgtggac aaaacaattc ttaacttggg tcaatttttg
     1681 caggcaaaaa taaaggaaaa gacagaacta cacagcgaag tttctgaaat aataaacgat
     1741 ttcaatagaa atttggaata tctattaatc tttaaagata tttccaatga caaggatagc
     1801 ttcaaaaatt cctgtacaca agatattcaa acaactaagg aactttgtga attttatgat
     1861 aattttgaag aaagctacgg taacttagtt ctagaagcaa agaggagaaa ggatgtggca
     1921 aacagaatga aaactatatt gaaagattgt gaaaagcagt tgcagaattt agatgctcag
     1981 gaccaggaag aacgtcagaa ttttatagcg gaaaatggaa cttatcttcc tgagacaatc
     2041 tggcccggta aaattgacga tttttcttcc ctgtacactt taaattacaa cgtgaagaat
     2101 ccttagaaaa aaggaaaaaa aggctgcaga gatctatgaa tcgtagatac tgaaaaaccc
     2161 cgcaagttca cttcaactgt gcatcgtgca ccatctcaat ttctttcatt tatacatcgt
     2221 tttgccttct tttatgtaac tatactcctc taagtttcaa tcttggccat gtaacctctg
     2281 atctatagaa ttttttaaat gactagaatt aatgcccatc ttttttttgg acctaaattc
     2341 ttcatgaaaa tatattacga gggcttattc agaagctttg gacttcttcg ccagaggttt
     2401 ggtcaagtct ccaatcaagg ttgtcggctt gtctaccttg ccagaaattt acgaaaagat
     2461 ggaaaagggt caaatcgttg gtagatacgt tgttgacact tctaaataag cgaatttctt
     2521 atgatttatg atttttatta ttaaataagt tataaaaaaa ataagtgtat acaaatttta
     2581 aagtgactct taggttttaa aacgaaaatt cttattcttg agtaactctt tcctgtaggt
     2641 caggttgctt tctcaggtat agcatgaggt cgctcttatt gaccacacct ctaccggcat
     2701 gccggtcgaa attcccctac cctatgaaca tattccattt tgtaatttcg tgtcgtttct
     2761 attatgaatt tcatttataa agtttatgta caaatatcat aaaaaaagag aatcttttta
     2821 agcaaggatt ttcttaacct cttcggcgac agcatcaccg acttcggtgg tactgttgga
     2881 accacctaaa tcaccagttc tgatacctgc atccaaaacc tttttaactg catcttcaat
     2941 ggccttacct tcttcaggca agttcaatga caatttcaac atcattgcag cagacaagat
     3001 agtggcgata gggttgacct tattctttgg caaatctgga gcagaaccgt ggcatggttc
     3061 gtacaaacca aatgcggtgt tcttgtctgg caaagaggcc aaggacgcag atggcaacaa
     3121 acccaaggaa cctgggataa cggaggcttc atcggagacg atatcaccaa acatgttgct
     3181 ggtgattata ataccattta ggtgggttgg gttcctaact aggatcatgg cggcagaatc
     3241 aatcaattga tgttgaacct tcaatgtagg aaattcgttc ttgatggttt cctccacagt
     3301 ttttctccat aatcttgaag aggccaaaac attagcttta tccaaggacc aaataggcaa
     3361 tggtggctca tgttgtaggg ccatgaaagc ggccattctt gtgattcttt gcacttctgg
     3421 aacggtgtat tgttcactat cccaagcgac accatcacca tcgtcttcct ttctcttacc
     3481 aaagtaaata cctcccacta attctctgac aacaacgaag tcagtacctt tagcaaattg
     3541 tggcttgatt ggagataagt ctaaaagaga gtcggatgca aagttacatg gtcttaagtt
     3601 ggcgtacaat tgaagttctt tacggatttt tagtaaacct tgttcaggtc taacactacc
     3661 tgtaccccat ttaggaccac ccacagcacc taacaaaacg gcatcagcct tcttggaggc
     3721 ttccagcgcc tcatctggaa gtgggacacc tgtagcttcg atagcagcac caccaattaa
     3781 atgattttcg aaatcgaact tgacattgga acgaacatca gaaatagctt taagaacctt
     3841 aatggcttcg gctgtgattt cttgaccaac gtggtcacct ggcaaaacga cgatcttctt
     3901 aggggcagac attagaatgg tatatccttg aaatatatat atatattgct gaaatgtaaa
     3961 aggtaagaaa agttagaaag taagacgatt gctaaccacc tattggaaaa aacaataggt
     4021 ccttaaataa tattgtcaac ttcaagtatt gtgatgcaag catttagtca tgaacgcttc
     4081 tctattctat atgaaaagcc ggttccggcg ctctcacctt tcctttttct cccaattttt
     4141 cagttgaaaa aggtatatgc gtcaggcgac ctctgaaatt aacaaaaaat ttccagtcat
     4201 cgaatttgat tctgtgcgat agcgcccctg tgtgttctcg ttatgttgag gaaaaaaata
     4261 atggttgcta agagattcga actcttgcat cttacgatac ctgagtattc ccacagttgg
     4321 gggatctcga ctctagctag aggatcaatt cgtaatcatg tcatagctgt ttcctgtgtg
     4381 aaattgttat ccgctcacaa ttccacacaa catacgagcc ggaagcataa agtgtaaagc
     4441 ctggggtgcc taatgagtga gctaactcac attaattgcg ttgcgctcac tgcccgcttt
     4501 ccagtcggga aacctgtcgt gccagctgca ttaatgaatc ggccaacgcg cggggagagg
     4561 cggtttgcgt attgggcgct cttccgcttc ctcgctcact gactcgctgc gctcggtcgt
     4621 tcggctgcgg cgagcggtat cagctcactc aaaggcggta atacggttat ccacagaatc
     4681 aggggataac gcaggaaaga acatgtgagc aaaaggccag caaaaggcca ggaaccgtaa
     4741 aaaggccgcg ttgctggcgt ttttccatag gctccgcccc cctgacgagc atcacaaaaa
     4801 tcgacgctca agtcagaggt ggcgaaaccc gacaggacta taaagatacc aggcgtttcc
     4861 ccctggaagc tccctcgtgc gctctcctgt tccgaccctg ccgcttaccg gatacctgtc
     4921 cgcctttctc ccttcgggaa gcgtggcgct ttctcatagc tcacgctgta ggtatctcag
     4981 ttcggtgtag gtcgttcgct ccaagctggg ctgtgtgcac gaaccccccg ttcagcccga
     5041 ccgctgcgcc ttatccggta actatcgtct tgagtccaac ccggtaagac acgacttatc
     5101 gccactggca gcagccactg gtaacaggat tagcagagcg aggtatgtag gcggtgctac
     5161 agagttcttg aagtggtggc ctaactacgg ctacactaga agaacagtat ttggtatctg
     5221 cgctctgctg aagccagtta ccttcggaaa aagagttggt agctcttgat ccggcaaaca
     5281 aaccaccgct ggtagcggtg gtttttttgt ttgcaagcag cagattacgc gcagaaaaaa
     5341 aggatctcaa gaagatcctt tgatcttttc tacggggtct gacgctcagt ggaacgaaaa
     5401 ctcacgttaa gggattttgg tcatgagatt atcaaaaagg atcttcacct agatcctttt
     5461 aaattaaaaa tgaagtttta aatcaatcta aagtatatat gagtaaactt ggtctgacag
     5521 ttaccaatgc ttaatcagtg aggcacctat ctcagcgatc tgtctatttc gttcatccat
     5581 agttgcctga ctccccgtcg tgtagataac tacgatacgg gagggcttac catctggccc
     5641 cagtgctgca atgataccgc gagacccacg ctcaccggct ccagatttat cagcaataaa
     5701 ccagccagcc ggaagggccg agcgcagaag tggtcctgca actttatccg cctccatcca
     5761 gtctattaat tgttgccggg aagctagagt aagtagttcg ccagttaata gtttgcgcaa
     5821 cgttgttgcc attgctacag gcatcgtggt gtcacgctcg tcgtttggta tggcttcatt
     5881 cagctccggt tcccaacgat caaggcgagt tacatgatcc cccatgttgt gcaaaaaagc
     5941 ggttagctcc ttcggtcctc cgatcgttgt cagaagtaag ttggccgcag tgttatcact
     6001 catggttatg gcagcactgc ataattctct tactgtcatg ccatccgtaa gatgcttttc
     6061 tgtgactggt gagtactcaa ccaagtcatt ctgagaatag tgtatgcggc gaccgagttg
     6121 ctcttgcccg gcgtcaatac gggataatac cgcgccacat agcagaactt taaaagtgct
     6181 catcattgga aaacgttctt cggggcgaaa actctcaagg atcttaccgc tgttgagatc
     6241 cagttcgatg taacccactc gtgcacccaa ctgatcttca gcatctttta ctttcaccag
     6301 cgtttctggg tgagcaaaaa caggaaggca aaatgccgca aaaaagggaa taagggcgac
     6361 acggaaatgt tgaatactca tactcttcct ttttcaatat tattgaagca tttatcaggg
     6421 ttattgtctc atgagcggat acatatttga atgtatttag aaaaataaac aaataggggt
     6481 tccgcgcaca tttccccgaa aagtgccacc tgacgtctaa gaaaccatta ttatcatgac
     6541 attaacctat aaaaataggc gtatcacgag gccctttcgt ctcgcgcgtt tcggtgatga
     6601 cggtgaaaac ctctgacaca tgcagctccc ggagacggtc acagcttgtc tgtaagcgga
     6661 tgccgggagc agacaagccc gtcagggcgc gtcagcgggt gttggcgggt gtcggggctg
     6721 gcttaactat gcggcatcag agcagattgt actgagagtg caccataacg catttaagca
     6781 taaacacgca ctatgccgtt cttctcatgt atatatatat acaggcaaca cgcagatata
     6841 ggtgcgacgt gaacagtgag ctgtatgtgc gcagctcgcg ttgcattttc ggaagcgctc
     6901 gttttcggaa acgctttgaa gttcctattc cgaagttcct attctctagc tagaaagtat
     6961 aggaacttca gagcgctttt gaaaaccaaa agcgctctga agacgcactt tcaaaaaacc
     7021 aaaaacgcac cggactgtaa cgagctacta aaatattgcg aataccgctt ccacaaacat
     7081 tgctcaaaag tatctctttg ctatatatct ctgtgctata tccctatata acctacccat
     7141 ccacctttcg ctccttgaac ttgcatctaa actcgacctc tacatttttt atgtttatct
     7201 ctagtattac tctttagaca aaaaaattgt agtaagaact attcatagag tgaatcgaaa
     7261 acaatacgaa aatgtaaaca tttcctatac gtagtatata gagacaaaat agaagaaacc
     7321 gttcataatt ttctgaccaa tgaagaatca tcaacgctat cactttctgt tcacaaagta
     7381 tgcgcaatcc acatcggtat agaatataat cggggatgcc tttatcttga aaaaatgcac
     7441 ccgcagcttc gctagtaatc agtaaacgcg ggaagtggag tcaggctttt tttatggaag
     7501 agaaaataga caccaaagta gccttcttct aaccttaacg gacctacagt gcaaaaagtt
     7561 atcaagagac tgcattatag agcgcacaaa ggagaaaaaa agtaatctaa gatgctttgt
     7621 tagaaaaata gcgctctcgg gatgcatttt tgtagaacaa aaaagaagta tagattcttt
     7681 gttggtaaaa tagcgctctc gcgttgcatt tctgttctgt aaaaatgcag ctcagattct
     7741 ttgtttgaaa aattagcgct ctcgcgttgc atttttgttt tacaaaaatg aagcacagat
     7801 tcttcgttgg taaaatagcg ctttcgcgtt gcatttctgt tctgtaaaaa tgcagctcag
     7861 attctttgtt tgaaaaatta gcgctctcgc gttgcatttt tgttctacaa aatgaagcac
     7921 agatgcttcg ttg
//
