LOCUS       sequence-237704-        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(I325D)
FEATURES             Location/Qualifiers
     source          1..7933
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     CDS             447..467
                     /codon_start=1
                     /product="nuclear localization signal of SV40 (simian virus
                     40) large T antigen"
                     /label=SV40 NLS
                     /translation="PKKKRKV"
     CDS             483..824
                     /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             1895..1906
                     /note="strong bacterial ribosome binding site (Elowitz and 
                     Leibler, 2000)"
     terminator      2506..2693
                     /gene="S. cerevisiae ADH1"
                     /label=ADH1 terminator
                     /note="transcription terminator for the S. cerevisiae 
                     alcohol dehydrogenase 1 (ADH1) gene"
     primer_bind     complement(2561..2585)
                     /label=pHybLex-R
                     /note="ADH terminator, reverse primer"
     CDS             complement(2814..3908)
                     /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(3909..4314)
                     /gene="S. cerevisiae LEU2"
                     /label=LEU2 promoter
     primer_bind     complement(4356..4372)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(4356..4372)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(4369..4391)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    4380..4396
                     /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(4404..4410,4411..4428,4429..4434))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    4449..4470
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(4587..4604)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(4758..5346)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(4838..4857)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(5517..6308,6309..6377))
                     /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     6140..6159
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(6378..6482)
                     /gene="bla"
                     /label=AmpR promoter
     primer_bind     6550..6568
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     primer_bind     complement(6606..6628)
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     primer_bind     6728..6747
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     rep_origin      6764..7928
                     /label=2u ori
                     /note="yeast 2u plasmid origin of replication"
     promoter        join(7933,1..401)
                     /gene="S. cerevisiae ADH1"
                     /label=ADH1 promoter
                     /note="promoter for alcohol dehydrogenase 1"
ORIGIN
        1 catgcaactt cttttctttt tttttctttt ctctctcccc cgttgttgtc tcaccatatc
       61 cgcaatgaca aaaaaatgat ggaagacact aaaggaaaaa attaacgaca aagacagcac
      121 caacagatgt cgttgttcca gagctgatga ggggtatctc gaagcacacg aaactttttc
      181 cttccttcat tcacgcacac tactctctaa tgagcaacgg tatacggcct tccttccagt
      241 tacttgaatt tgaaataaaa aaaagtttgc tgtcttgcta tcaagtataa atagacctgc
      301 aattattaat cttttgtttc ctcgtcattg ttctcgttcc ctttcttcct tgtttctttt
      361 tctgcacaat atttcaagct ataccaagca tacaatcaac tccaagcttt gcaaagatgg
      421 ataaagcgga attaattccc gagcctccaa aaaagaagag aaaggtcgaa ttgggtaccg
      481 ccgccaattt taatcaaagt gggaatattg ctgatagctc attgtccttc actttcacta
      541 acagtagcaa cggtccgaac ctcataacaa ctcaaacaaa ttctcaagcg ctttcacaac
      601 caattgcctc ctctaacgtt catgataact tcatgaataa tgaaatcacg gctagtaaaa
      661 ttgatgatgg taataattca aaaccactgt cacctggttg gacggaccaa actgcgtata
      721 acgcgtttgg aatcactaca gggatgttta ataccactac aatggatgat gtatataact
      781 atctattcga tgatgaagat accccaccaa acccaaaaaa agagatcgaa ttccccgggg
      841 gatccatcga taacgaagca gatgttacaa aatttgttaa taatgccagg aaaacgctga
      901 ccgatgctca acttttatgt tcaagtgcta atttaaggat tgtagatatt aagaaaaaat
      961 tgtcatcttg gcagttgagt atttcaaaac tcaattttct aatagttggc ttgagacagc
     1021 aaggaaagtt tctttacact attttaaagg aaggcattgg gacaaagcta atccagaaac
     1081 aatggaatca agctgtctta gtggtcttag ttgatgagat gaagtactgg caatatgaaa
     1141 tcacttctaa agtacaaagg ttggatggta tagtaaatga attgagcata tcagaaaaag
     1201 atgataccga tccctctaaa ctaggagatt acatctcgag agacaacgtt aacttattga
     1261 atgacaaact gaaagaagtg ccagtaattg agcgtcaaat tgaaaacatt aagcttcaat
     1321 acgaaaatat ggtcagaaaa gttaacaaag aactgattga taccaagttg acggacgtaa
     1381 ctcaaaaatt ccaaagtaaa tttggtatag ataacctgat ggaaacaaat gttgcagagc
     1441 agtttagcag ggaactaacg gaccttgaaa aagatttagc agagataatg aattcattga
     1501 cacagcactt tgataagaca ttgctgttac aagataaaaa aattgataac gatgaacgtg
     1561 aggagctgtt taaggtggta caaggcgacg acaaagaact atacaacatt ttcaaaactc
     1621 tgcatgaggt aattgatgac gtggacaaaa caattcttaa cttgggtcaa tttttgcagg
     1681 caaaaataaa ggaaaagaca gaactacaca gcgaagtttc tgaaataata aacgatttca
     1741 atagaaattt ggaatatcta ttaatcttta aagatatttc caatctgatt gatagcttca
     1801 aaaattcctg tacacaagat gatcaaacaa ctaaggaact ttgtgaattt tatgataatt
     1861 ttgaagaaag ctacggtaac ttagttctag aagcaaagag gagaaaggat gtggcaaaca
     1921 gaatgaaaac tatattgaaa gattgtgaaa agcagttgca gaatttagat gctcaggacc
     1981 aggaagaacg tcagaatttt atagcggaaa atggaactta tcttcctgag acaatctggc
     2041 ccggtaaaat tgacgatttt tcttccctgt acactttaaa ttacaacgtg aagaatcctt
     2101 agaaaaaagg aaaaaaaggc tgcagagatc tatgaatcgt agatactgaa aaaccccgca
     2161 agttcacttc aactgtgcat cgtgcaccat ctcaatttct ttcatttata catcgttttg
     2221 ccttctttta tgtaactata ctcctctaag tttcaatctt ggccatgtaa cctctgatct
     2281 atagaatttt ttaaatgact agaattaatg cccatctttt ttttggacct aaattcttca
     2341 tgaaaatata ttacgagggc ttattcagaa gctttggact tcttcgccag aggtttggtc
     2401 aagtctccaa tcaaggttgt cggcttgtct accttgccag aaatttacga aaagatggaa
     2461 aagggtcaaa tcgttggtag atacgttgtt gacacttcta aataagcgaa tttcttatga
     2521 tttatgattt ttattattaa ataagttata aaaaaaataa gtgtatacaa attttaaagt
     2581 gactcttagg ttttaaaacg aaaattctta ttcttgagta actctttcct gtaggtcagg
     2641 ttgctttctc aggtatagca tgaggtcgct cttattgacc acacctctac cggcatgccg
     2701 gtcgaaattc ccctacccta tgaacatatt ccattttgta atttcgtgtc gtttctatta
     2761 tgaatttcat ttataaagtt tatgtacaaa tatcataaaa aaagagaatc tttttaagca
     2821 aggattttct taacctcttc ggcgacagca tcaccgactt cggtggtact gttggaacca
     2881 cctaaatcac cagttctgat acctgcatcc aaaacctttt taactgcatc ttcaatggcc
     2941 ttaccttctt caggcaagtt caatgacaat ttcaacatca ttgcagcaga caagatagtg
     3001 gcgatagggt tgaccttatt ctttggcaaa tctggagcag aaccgtggca tggttcgtac
     3061 aaaccaaatg cggtgttctt gtctggcaaa gaggccaagg acgcagatgg caacaaaccc
     3121 aaggaacctg ggataacgga ggcttcatcg gagacgatat caccaaacat gttgctggtg
     3181 attataatac catttaggtg ggttgggttc ctaactagga tcatggcggc agaatcaatc
     3241 aattgatgtt gaaccttcaa tgtaggaaat tcgttcttga tggtttcctc cacagttttt
     3301 ctccataatc ttgaagaggc caaaacatta gctttatcca aggaccaaat aggcaatggt
     3361 ggctcatgtt gtagggccat gaaagcggcc attcttgtga ttctttgcac ttctggaacg
     3421 gtgtattgtt cactatccca agcgacacca tcaccatcgt cttcctttct cttaccaaag
     3481 taaatacctc ccactaattc tctgacaaca acgaagtcag tacctttagc aaattgtggc
     3541 ttgattggag ataagtctaa aagagagtcg gatgcaaagt tacatggtct taagttggcg
     3601 tacaattgaa gttctttacg gatttttagt aaaccttgtt caggtctaac actacctgta
     3661 ccccatttag gaccacccac agcacctaac aaaacggcat cagccttctt ggaggcttcc
     3721 agcgcctcat ctggaagtgg gacacctgta gcttcgatag cagcaccacc aattaaatga
     3781 ttttcgaaat cgaacttgac attggaacga acatcagaaa tagctttaag aaccttaatg
     3841 gcttcggctg tgatttcttg accaacgtgg tcacctggca aaacgacgat cttcttaggg
     3901 gcagacatta gaatggtata tccttgaaat atatatatat attgctgaaa tgtaaaaggt
     3961 aagaaaagtt agaaagtaag acgattgcta accacctatt ggaaaaaaca ataggtcctt
     4021 aaataatatt gtcaacttca agtattgtga tgcaagcatt tagtcatgaa cgcttctcta
     4081 ttctatatga aaagccggtt ccggcgctct cacctttcct ttttctccca atttttcagt
     4141 tgaaaaaggt atatgcgtca ggcgacctct gaaattaaca aaaaatttcc agtcatcgaa
     4201 tttgattctg tgcgatagcg cccctgtgtg ttctcgttat gttgaggaaa aaaataatgg
     4261 ttgctaagag attcgaactc ttgcatctta cgatacctga gtattcccac agttggggga
     4321 tctcgactct agctagagga tcaattcgta atcatgtcat agctgtttcc tgtgtgaaat
     4381 tgttatccgc tcacaattcc acacaacata cgagccggaa gcataaagtg taaagcctgg
     4441 ggtgcctaat gagtgagcta actcacatta attgcgttgc gctcactgcc cgctttccag
     4501 tcgggaaacc tgtcgtgcca gctgcattaa tgaatcggcc aacgcgcggg gagaggcggt
     4561 ttgcgtattg ggcgctcttc cgcttcctcg ctcactgact cgctgcgctc ggtcgttcgg
     4621 ctgcggcgag cggtatcagc tcactcaaag gcggtaatac ggttatccac agaatcaggg
     4681 gataacgcag gaaagaacat gtgagcaaaa ggccagcaaa aggccaggaa ccgtaaaaag
     4741 gccgcgttgc tggcgttttt ccataggctc cgcccccctg acgagcatca caaaaatcga
     4801 cgctcaagtc agaggtggcg aaacccgaca ggactataaa gataccaggc gtttccccct
     4861 ggaagctccc tcgtgcgctc tcctgttccg accctgccgc ttaccggata cctgtccgcc
     4921 tttctccctt cgggaagcgt ggcgctttct catagctcac gctgtaggta tctcagttcg
     4981 gtgtaggtcg ttcgctccaa gctgggctgt gtgcacgaac cccccgttca gcccgaccgc
     5041 tgcgccttat ccggtaacta tcgtcttgag tccaacccgg taagacacga cttatcgcca
     5101 ctggcagcag ccactggtaa caggattagc agagcgaggt atgtaggcgg tgctacagag
     5161 ttcttgaagt ggtggcctaa ctacggctac actagaagaa cagtatttgg tatctgcgct
     5221 ctgctgaagc cagttacctt cggaaaaaga gttggtagct cttgatccgg caaacaaacc
     5281 accgctggta gcggtggttt ttttgtttgc aagcagcaga ttacgcgcag aaaaaaagga
     5341 tctcaagaag atcctttgat cttttctacg gggtctgacg ctcagtggaa cgaaaactca
     5401 cgttaaggga ttttggtcat gagattatca aaaaggatct tcacctagat ccttttaaat
     5461 taaaaatgaa gttttaaatc aatctaaagt atatatgagt aaacttggtc tgacagttac
     5521 caatgcttaa tcagtgaggc acctatctca gcgatctgtc tatttcgttc atccatagtt
     5581 gcctgactcc ccgtcgtgta gataactacg atacgggagg gcttaccatc tggccccagt
     5641 gctgcaatga taccgcgaga cccacgctca ccggctccag atttatcagc aataaaccag
     5701 ccagccggaa gggccgagcg cagaagtggt cctgcaactt tatccgcctc catccagtct
     5761 attaattgtt gccgggaagc tagagtaagt agttcgccag ttaatagttt gcgcaacgtt
     5821 gttgccattg ctacaggcat cgtggtgtca cgctcgtcgt ttggtatggc ttcattcagc
     5881 tccggttccc aacgatcaag gcgagttaca tgatccccca tgttgtgcaa aaaagcggtt
     5941 agctccttcg gtcctccgat cgttgtcaga agtaagttgg ccgcagtgtt atcactcatg
     6001 gttatggcag cactgcataa ttctcttact gtcatgccat ccgtaagatg cttttctgtg
     6061 actggtgagt actcaaccaa gtcattctga gaatagtgta tgcggcgacc gagttgctct
     6121 tgcccggcgt caatacggga taataccgcg ccacatagca gaactttaaa agtgctcatc
     6181 attggaaaac gttcttcggg gcgaaaactc tcaaggatct taccgctgtt gagatccagt
     6241 tcgatgtaac ccactcgtgc acccaactga tcttcagcat cttttacttt caccagcgtt
     6301 tctgggtgag caaaaacagg aaggcaaaat gccgcaaaaa agggaataag ggcgacacgg
     6361 aaatgttgaa tactcatact cttccttttt caatattatt gaagcattta tcagggttat
     6421 tgtctcatga gcggatacat atttgaatgt atttagaaaa ataaacaaat aggggttccg
     6481 cgcacatttc cccgaaaagt gccacctgac gtctaagaaa ccattattat catgacatta
     6541 acctataaaa ataggcgtat cacgaggccc tttcgtctcg cgcgtttcgg tgatgacggt
     6601 gaaaacctct gacacatgca gctcccggag acggtcacag cttgtctgta agcggatgcc
     6661 gggagcagac aagcccgtca gggcgcgtca gcgggtgttg gcgggtgtcg gggctggctt
     6721 aactatgcgg catcagagca gattgtactg agagtgcacc ataacgcatt taagcataaa
     6781 cacgcactat gccgttcttc tcatgtatat atatatacag gcaacacgca gatataggtg
     6841 cgacgtgaac agtgagctgt atgtgcgcag ctcgcgttgc attttcggaa gcgctcgttt
     6901 tcggaaacgc tttgaagttc ctattccgaa gttcctattc tctagctaga aagtatagga
     6961 acttcagagc gcttttgaaa accaaaagcg ctctgaagac gcactttcaa aaaaccaaaa
     7021 acgcaccgga ctgtaacgag ctactaaaat attgcgaata ccgcttccac aaacattgct
     7081 caaaagtatc tctttgctat atatctctgt gctatatccc tatataacct acccatccac
     7141 ctttcgctcc ttgaacttgc atctaaactc gacctctaca ttttttatgt ttatctctag
     7201 tattactctt tagacaaaaa aattgtagta agaactattc atagagtgaa tcgaaaacaa
     7261 tacgaaaatg taaacatttc ctatacgtag tatatagaga caaaatagaa gaaaccgttc
     7321 ataattttct gaccaatgaa gaatcatcaa cgctatcact ttctgttcac aaagtatgcg
     7381 caatccacat cggtatagaa tataatcggg gatgccttta tcttgaaaaa atgcacccgc
     7441 agcttcgcta gtaatcagta aacgcgggaa gtggagtcag gcttttttta tggaagagaa
     7501 aatagacacc aaagtagcct tcttctaacc ttaacggacc tacagtgcaa aaagttatca
     7561 agagactgca ttatagagcg cacaaaggag aaaaaaagta atctaagatg ctttgttaga
     7621 aaaatagcgc tctcgggatg catttttgta gaacaaaaaa gaagtataga ttctttgttg
     7681 gtaaaatagc gctctcgcgt tgcatttctg ttctgtaaaa atgcagctca gattctttgt
     7741 ttgaaaaatt agcgctctcg cgttgcattt ttgttttaca aaaatgaagc acagattctt
     7801 cgttggtaaa atagcgcttt cgcgttgcat ttctgttctg taaaaatgca gctcagattc
     7861 tttgtttgaa aaattagcgc tctcgcgttg catttttgtt ctacaaaatg aagcacagat
     7921 gcttcgttgc ttg
//
