LOCUS       sequence-240231-        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(F317H)
FEATURES             Location/Qualifiers
     source          1..7933
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     promoter        10..411
                     /gene="S. cerevisiae ADH1"
                     /label=ADH1 promoter
                     /note="promoter for alcohol dehydrogenase 1"
     CDS             457..477
                     /codon_start=1
                     /product="nuclear localization signal of SV40 (simian virus
                     40) large T antigen"
                     /label=SV40 NLS
                     /translation="PKKKRKV"
     CDS             493..834
                     /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             1905..1916
                     /note="strong bacterial ribosome binding site (Elowitz and 
                     Leibler, 2000)"
     terminator      2516..2703
                     /gene="S. cerevisiae ADH1"
                     /label=ADH1 terminator
                     /note="transcription terminator for the S. cerevisiae 
                     alcohol dehydrogenase 1 (ADH1) gene"
     primer_bind     complement(2571..2595)
                     /label=pHybLex-R
                     /note="ADH terminator, reverse primer"
     CDS             complement(2824..3918)
                     /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(3919..4324)
                     /gene="S. cerevisiae LEU2"
                     /label=LEU2 promoter
     primer_bind     complement(4366..4382)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(4366..4382)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(4379..4401)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    4390..4406
                     /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(4414..4420,4421..4438,4439..4444))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    4459..4480
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(4597..4614)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(4768..5356)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(4848..4867)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(5527..6318,6319..6387))
                     /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     6150..6169
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(6388..6492)
                     /gene="bla"
                     /label=AmpR promoter
     primer_bind     6560..6578
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     primer_bind     complement(6616..6638)
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     primer_bind     6738..6757
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     rep_origin      join(6774..7933,1..5)
                     /label=2u ori
                     /note="yeast 2u plasmid origin of replication"
ORIGIN
        1 tcgttgcttg catgcaactt cttttctttt tttttctttt ctctctcccc cgttgttgtc
       61 tcaccatatc cgcaatgaca aaaaaatgat ggaagacact aaaggaaaaa attaacgaca
      121 aagacagcac caacagatgt cgttgttcca gagctgatga ggggtatctc gaagcacacg
      181 aaactttttc cttccttcat tcacgcacac tactctctaa tgagcaacgg tatacggcct
      241 tccttccagt tacttgaatt tgaaataaaa aaaagtttgc tgtcttgcta tcaagtataa
      301 atagacctgc aattattaat cttttgtttc ctcgtcattg ttctcgttcc ctttcttcct
      361 tgtttctttt tctgcacaat atttcaagct ataccaagca tacaatcaac tccaagcttt
      421 gcaaagatgg ataaagcgga attaattccc gagcctccaa aaaagaagag aaaggtcgaa
      481 ttgggtaccg ccgccaattt taatcaaagt gggaatattg ctgatagctc attgtccttc
      541 actttcacta acagtagcaa cggtccgaac ctcataacaa ctcaaacaaa ttctcaagcg
      601 ctttcacaac caattgcctc ctctaacgtt catgataact tcatgaataa tgaaatcacg
      661 gctagtaaaa ttgatgatgg taataattca aaaccactgt cacctggttg gacggaccaa
      721 actgcgtata acgcgtttgg aatcactaca gggatgttta ataccactac aatggatgat
      781 gtatataact atctattcga tgatgaagat accccaccaa acccaaaaaa agagatcgaa
      841 ttccccgggg gatccatcga taacgaagca gatgttacaa aatttgttaa taatgccagg
      901 aaaacgctga ccgatgctca acttttatgt tcaagtgcta atttaaggat tgtagatatt
      961 aagaaaaaat tgtcatcttg gcagttgagt atttcaaaac tcaattttct aatagttggc
     1021 ttgagacagc aaggaaagtt tctttacact attttaaagg aaggcattgg gacaaagcta
     1081 atccagaaac aatggaatca agctgtctta gtggtcttag ttgatgagat gaagtactgg
     1141 caatatgaaa tcacttctaa agtacaaagg ttggatggta tagtaaatga attgagcata
     1201 tcagaaaaag atgataccga tccctctaaa ctaggagatt acatctcgag agacaacgtt
     1261 aacttattga atgacaaact gaaagaagtg ccagtaattg agcgtcaaat tgaaaacatt
     1321 aagcttcaat acgaaaatat ggtcagaaaa gttaacaaag aactgattga taccaagttg
     1381 acggacgtaa ctcaaaaatt ccaaagtaaa tttggtatag ataacctgat ggaaacaaat
     1441 gttgcagagc agtttagcag ggaactaacg gaccttgaaa aagatttagc agagataatg
     1501 aattcattga cacagcactt tgataagaca ttgctgttac aagataaaaa aattgataac
     1561 gatgaacgtg aggagctgtt taaggtggta caaggcgacg acaaagaact atacaacatt
     1621 ttcaaaactc tgcatgaggt aattgatgac gtggacaaaa caattcttaa cttgggtcaa
     1681 tttttgcagg caaaaataaa ggaaaagaca gaactacaca gcgaagtttc tgaaataata
     1741 aacgatttca atagaaattt ggaatatcta ttaatcttta aagatatttc caatctgatt
     1801 gatagccata aaaattcctg tacacaagat attcaaacaa ctaaggaact ttgtgaattt
     1861 tatgataatt ttgaagaaag ctacggtaac ttagttctag aagcaaagag gagaaaggat
     1921 gtggcaaaca gaatgaaaac tatattgaaa gattgtgaaa agcagttgca gaatttagat
     1981 gctcaggacc aggaagaacg tcagaatttt atagcggaaa atggaactta tcttcctgag
     2041 acaatctggc ccggtaaaat tgacgatttt tcttccctgt acactttaaa ttacaacgtg
     2101 aagaatcctt agaaaaaagg aaaaaaaggc tgcagagatc tatgaatcgt agatactgaa
     2161 aaaccccgca agttcacttc aactgtgcat cgtgcaccat ctcaatttct ttcatttata
     2221 catcgttttg ccttctttta tgtaactata ctcctctaag tttcaatctt ggccatgtaa
     2281 cctctgatct atagaatttt ttaaatgact agaattaatg cccatctttt ttttggacct
     2341 aaattcttca tgaaaatata ttacgagggc ttattcagaa gctttggact tcttcgccag
     2401 aggtttggtc aagtctccaa tcaaggttgt cggcttgtct accttgccag aaatttacga
     2461 aaagatggaa aagggtcaaa tcgttggtag atacgttgtt gacacttcta aataagcgaa
     2521 tttcttatga tttatgattt ttattattaa ataagttata aaaaaaataa gtgtatacaa
     2581 attttaaagt gactcttagg ttttaaaacg aaaattctta ttcttgagta actctttcct
     2641 gtaggtcagg ttgctttctc aggtatagca tgaggtcgct cttattgacc acacctctac
     2701 cggcatgccg gtcgaaattc ccctacccta tgaacatatt ccattttgta atttcgtgtc
     2761 gtttctatta tgaatttcat ttataaagtt tatgtacaaa tatcataaaa aaagagaatc
     2821 tttttaagca aggattttct taacctcttc ggcgacagca tcaccgactt cggtggtact
     2881 gttggaacca cctaaatcac cagttctgat acctgcatcc aaaacctttt taactgcatc
     2941 ttcaatggcc ttaccttctt caggcaagtt caatgacaat ttcaacatca ttgcagcaga
     3001 caagatagtg gcgatagggt tgaccttatt ctttggcaaa tctggagcag aaccgtggca
     3061 tggttcgtac aaaccaaatg cggtgttctt gtctggcaaa gaggccaagg acgcagatgg
     3121 caacaaaccc aaggaacctg ggataacgga ggcttcatcg gagacgatat caccaaacat
     3181 gttgctggtg attataatac catttaggtg ggttgggttc ctaactagga tcatggcggc
     3241 agaatcaatc aattgatgtt gaaccttcaa tgtaggaaat tcgttcttga tggtttcctc
     3301 cacagttttt ctccataatc ttgaagaggc caaaacatta gctttatcca aggaccaaat
     3361 aggcaatggt ggctcatgtt gtagggccat gaaagcggcc attcttgtga ttctttgcac
     3421 ttctggaacg gtgtattgtt cactatccca agcgacacca tcaccatcgt cttcctttct
     3481 cttaccaaag taaatacctc ccactaattc tctgacaaca acgaagtcag tacctttagc
     3541 aaattgtggc ttgattggag ataagtctaa aagagagtcg gatgcaaagt tacatggtct
     3601 taagttggcg tacaattgaa gttctttacg gatttttagt aaaccttgtt caggtctaac
     3661 actacctgta ccccatttag gaccacccac agcacctaac aaaacggcat cagccttctt
     3721 ggaggcttcc agcgcctcat ctggaagtgg gacacctgta gcttcgatag cagcaccacc
     3781 aattaaatga ttttcgaaat cgaacttgac attggaacga acatcagaaa tagctttaag
     3841 aaccttaatg gcttcggctg tgatttcttg accaacgtgg tcacctggca aaacgacgat
     3901 cttcttaggg gcagacatta gaatggtata tccttgaaat atatatatat attgctgaaa
     3961 tgtaaaaggt aagaaaagtt agaaagtaag acgattgcta accacctatt ggaaaaaaca
     4021 ataggtcctt aaataatatt gtcaacttca agtattgtga tgcaagcatt tagtcatgaa
     4081 cgcttctcta ttctatatga aaagccggtt ccggcgctct cacctttcct ttttctccca
     4141 atttttcagt tgaaaaaggt atatgcgtca ggcgacctct gaaattaaca aaaaatttcc
     4201 agtcatcgaa tttgattctg tgcgatagcg cccctgtgtg ttctcgttat gttgaggaaa
     4261 aaaataatgg ttgctaagag attcgaactc ttgcatctta cgatacctga gtattcccac
     4321 agttggggga tctcgactct agctagagga tcaattcgta atcatgtcat agctgtttcc
     4381 tgtgtgaaat tgttatccgc tcacaattcc acacaacata cgagccggaa gcataaagtg
     4441 taaagcctgg ggtgcctaat gagtgagcta actcacatta attgcgttgc gctcactgcc
     4501 cgctttccag tcgggaaacc tgtcgtgcca gctgcattaa tgaatcggcc aacgcgcggg
     4561 gagaggcggt ttgcgtattg ggcgctcttc cgcttcctcg ctcactgact cgctgcgctc
     4621 ggtcgttcgg ctgcggcgag cggtatcagc tcactcaaag gcggtaatac ggttatccac
     4681 agaatcaggg gataacgcag gaaagaacat gtgagcaaaa ggccagcaaa aggccaggaa
     4741 ccgtaaaaag gccgcgttgc tggcgttttt ccataggctc cgcccccctg acgagcatca
     4801 caaaaatcga cgctcaagtc agaggtggcg aaacccgaca ggactataaa gataccaggc
     4861 gtttccccct ggaagctccc tcgtgcgctc tcctgttccg accctgccgc ttaccggata
     4921 cctgtccgcc tttctccctt cgggaagcgt ggcgctttct catagctcac gctgtaggta
     4981 tctcagttcg gtgtaggtcg ttcgctccaa gctgggctgt gtgcacgaac cccccgttca
     5041 gcccgaccgc tgcgccttat ccggtaacta tcgtcttgag tccaacccgg taagacacga
     5101 cttatcgcca ctggcagcag ccactggtaa caggattagc agagcgaggt atgtaggcgg
     5161 tgctacagag ttcttgaagt ggtggcctaa ctacggctac actagaagaa cagtatttgg
     5221 tatctgcgct ctgctgaagc cagttacctt cggaaaaaga gttggtagct cttgatccgg
     5281 caaacaaacc accgctggta gcggtggttt ttttgtttgc aagcagcaga ttacgcgcag
     5341 aaaaaaagga tctcaagaag atcctttgat cttttctacg gggtctgacg ctcagtggaa
     5401 cgaaaactca cgttaaggga ttttggtcat gagattatca aaaaggatct tcacctagat
     5461 ccttttaaat taaaaatgaa gttttaaatc aatctaaagt atatatgagt aaacttggtc
     5521 tgacagttac caatgcttaa tcagtgaggc acctatctca gcgatctgtc tatttcgttc
     5581 atccatagtt gcctgactcc ccgtcgtgta gataactacg atacgggagg gcttaccatc
     5641 tggccccagt gctgcaatga taccgcgaga cccacgctca ccggctccag atttatcagc
     5701 aataaaccag ccagccggaa gggccgagcg cagaagtggt cctgcaactt tatccgcctc
     5761 catccagtct attaattgtt gccgggaagc tagagtaagt agttcgccag ttaatagttt
     5821 gcgcaacgtt gttgccattg ctacaggcat cgtggtgtca cgctcgtcgt ttggtatggc
     5881 ttcattcagc tccggttccc aacgatcaag gcgagttaca tgatccccca tgttgtgcaa
     5941 aaaagcggtt agctccttcg gtcctccgat cgttgtcaga agtaagttgg ccgcagtgtt
     6001 atcactcatg gttatggcag cactgcataa ttctcttact gtcatgccat ccgtaagatg
     6061 cttttctgtg actggtgagt actcaaccaa gtcattctga gaatagtgta tgcggcgacc
     6121 gagttgctct tgcccggcgt caatacggga taataccgcg ccacatagca gaactttaaa
     6181 agtgctcatc attggaaaac gttcttcggg gcgaaaactc tcaaggatct taccgctgtt
     6241 gagatccagt tcgatgtaac ccactcgtgc acccaactga tcttcagcat cttttacttt
     6301 caccagcgtt tctgggtgag caaaaacagg aaggcaaaat gccgcaaaaa agggaataag
     6361 ggcgacacgg aaatgttgaa tactcatact cttccttttt caatattatt gaagcattta
     6421 tcagggttat tgtctcatga gcggatacat atttgaatgt atttagaaaa ataaacaaat
     6481 aggggttccg cgcacatttc cccgaaaagt gccacctgac gtctaagaaa ccattattat
     6541 catgacatta acctataaaa ataggcgtat cacgaggccc tttcgtctcg cgcgtttcgg
     6601 tgatgacggt gaaaacctct gacacatgca gctcccggag acggtcacag cttgtctgta
     6661 agcggatgcc gggagcagac aagcccgtca gggcgcgtca gcgggtgttg gcgggtgtcg
     6721 gggctggctt aactatgcgg catcagagca gattgtactg agagtgcacc ataacgcatt
     6781 taagcataaa cacgcactat gccgttcttc tcatgtatat atatatacag gcaacacgca
     6841 gatataggtg cgacgtgaac agtgagctgt atgtgcgcag ctcgcgttgc attttcggaa
     6901 gcgctcgttt tcggaaacgc tttgaagttc ctattccgaa gttcctattc tctagctaga
     6961 aagtatagga acttcagagc gcttttgaaa accaaaagcg ctctgaagac gcactttcaa
     7021 aaaaccaaaa acgcaccgga ctgtaacgag ctactaaaat attgcgaata ccgcttccac
     7081 aaacattgct caaaagtatc tctttgctat atatctctgt gctatatccc tatataacct
     7141 acccatccac ctttcgctcc ttgaacttgc atctaaactc gacctctaca ttttttatgt
     7201 ttatctctag tattactctt tagacaaaaa aattgtagta agaactattc atagagtgaa
     7261 tcgaaaacaa tacgaaaatg taaacatttc ctatacgtag tatatagaga caaaatagaa
     7321 gaaaccgttc ataattttct gaccaatgaa gaatcatcaa cgctatcact ttctgttcac
     7381 aaagtatgcg caatccacat cggtatagaa tataatcggg gatgccttta tcttgaaaaa
     7441 atgcacccgc agcttcgcta gtaatcagta aacgcgggaa gtggagtcag gcttttttta
     7501 tggaagagaa aatagacacc aaagtagcct tcttctaacc ttaacggacc tacagtgcaa
     7561 aaagttatca agagactgca ttatagagcg cacaaaggag aaaaaaagta atctaagatg
     7621 ctttgttaga aaaatagcgc tctcgggatg catttttgta gaacaaaaaa gaagtataga
     7681 ttctttgttg gtaaaatagc gctctcgcgt tgcatttctg ttctgtaaaa atgcagctca
     7741 gattctttgt ttgaaaaatt agcgctctcg cgttgcattt ttgttttaca aaaatgaagc
     7801 acagattctt cgttggtaaa atagcgcttt cgcgttgcat ttctgttctg taaaaatgca
     7861 gctcagattc tttgtttgaa aaattagcgc tctcgcgttg catttttgtt ctacaaaatg
     7921 aagcacagat gct
//
