LOCUS       sequence-236194-        7888 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 7888)
  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 7888)
  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(∆266-280)
FEATURES             Location/Qualifiers
     source          1..7888
                     /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             1860..1871
                     /note="strong bacterial ribosome binding site (Elowitz and 
                     Leibler, 2000)"
     terminator      2471..2658
                     /gene="S. cerevisiae ADH1"
                     /label=ADH1 terminator
                     /note="transcription terminator for the S. cerevisiae 
                     alcohol dehydrogenase 1 (ADH1) gene"
     primer_bind     complement(2526..2550)
                     /label=pHybLex-R
                     /note="ADH terminator, reverse primer"
     CDS             complement(2779..3873)
                     /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(3874..4279)
                     /gene="S. cerevisiae LEU2"
                     /label=LEU2 promoter
     primer_bind     complement(4321..4337)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(4321..4337)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(4334..4356)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    4345..4361
                     /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(4369..4375,4376..4393,4394..4399))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    4414..4435
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(4552..4569)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(4723..5311)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(4803..4822)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(5482..6273,6274..6342))
                     /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     6105..6124
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(6343..6447)
                     /gene="bla"
                     /label=AmpR promoter
     primer_bind     6515..6533
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     primer_bind     complement(6571..6593)
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     primer_bind     6693..6712
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     rep_origin      join(6729..7888,1..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 gtgaaggaaa agacagaact acacagcgaa
     1681 gtttctgaaa taataaacga tttcaataga aatttggaat atctattaat ctttaaagat
     1741 atttccaatc tgattgatag cttcaaaaat tcctgtacac aagatattca aacaactaag
     1801 gaactttgtg aattttatga taattttgaa gaaagctacg gtaacttagt tctagaagca
     1861 aagaggagaa aggatgtggc aaacagaatg aaaactatat tgaaagattg tgaaaagcag
     1921 ttgcagaatt tagatgctca ggaccaggaa gaacgtcaga attttatagc ggaaaatgga
     1981 acttatcttc ctgagacaat ctggcccggt aaaattgacg atttttcttc cctgtacact
     2041 ttaaattaca acgtgaagaa tccttagaaa aaaggaaaaa aaggctgcag agatctatga
     2101 atcgtagata ctgaaaaacc ccgcaagttc acttcaactg tgcatcgtgc accatctcaa
     2161 tttctttcat ttatacatcg ttttgccttc ttttatgtaa ctatactcct ctaagtttca
     2221 atcttggcca tgtaacctct gatctataga attttttaaa tgactagaat taatgcccat
     2281 cttttttttg gacctaaatt cttcatgaaa atatattacg agggcttatt cagaagcttt
     2341 ggacttcttc gccagaggtt tggtcaagtc tccaatcaag gttgtcggct tgtctacctt
     2401 gccagaaatt tacgaaaaga tggaaaaggg tcaaatcgtt ggtagatacg ttgttgacac
     2461 ttctaaataa gcgaatttct tatgatttat gatttttatt attaaataag ttataaaaaa
     2521 aataagtgta tacaaatttt aaagtgactc ttaggtttta aaacgaaaat tcttattctt
     2581 gagtaactct ttcctgtagg tcaggttgct ttctcaggta tagcatgagg tcgctcttat
     2641 tgaccacacc tctaccggca tgccggtcga aattccccta ccctatgaac atattccatt
     2701 ttgtaatttc gtgtcgtttc tattatgaat ttcatttata aagtttatgt acaaatatca
     2761 taaaaaaaga gaatcttttt aagcaaggat tttcttaacc tcttcggcga cagcatcacc
     2821 gacttcggtg gtactgttgg aaccacctaa atcaccagtt ctgatacctg catccaaaac
     2881 ctttttaact gcatcttcaa tggccttacc ttcttcaggc aagttcaatg acaatttcaa
     2941 catcattgca gcagacaaga tagtggcgat agggttgacc ttattctttg gcaaatctgg
     3001 agcagaaccg tggcatggtt cgtacaaacc aaatgcggtg ttcttgtctg gcaaagaggc
     3061 caaggacgca gatggcaaca aacccaagga acctgggata acggaggctt catcggagac
     3121 gatatcacca aacatgttgc tggtgattat aataccattt aggtgggttg ggttcctaac
     3181 taggatcatg gcggcagaat caatcaattg atgttgaacc ttcaatgtag gaaattcgtt
     3241 cttgatggtt tcctccacag tttttctcca taatcttgaa gaggccaaaa cattagcttt
     3301 atccaaggac caaataggca atggtggctc atgttgtagg gccatgaaag cggccattct
     3361 tgtgattctt tgcacttctg gaacggtgta ttgttcacta tcccaagcga caccatcacc
     3421 atcgtcttcc tttctcttac caaagtaaat acctcccact aattctctga caacaacgaa
     3481 gtcagtacct ttagcaaatt gtggcttgat tggagataag tctaaaagag agtcggatgc
     3541 aaagttacat ggtcttaagt tggcgtacaa ttgaagttct ttacggattt ttagtaaacc
     3601 ttgttcaggt ctaacactac ctgtacccca tttaggacca cccacagcac ctaacaaaac
     3661 ggcatcagcc ttcttggagg cttccagcgc ctcatctgga agtgggacac ctgtagcttc
     3721 gatagcagca ccaccaatta aatgattttc gaaatcgaac ttgacattgg aacgaacatc
     3781 agaaatagct ttaagaacct taatggcttc ggctgtgatt tcttgaccaa cgtggtcacc
     3841 tggcaaaacg acgatcttct taggggcaga cattagaatg gtatatcctt gaaatatata
     3901 tatatattgc tgaaatgtaa aaggtaagaa aagttagaaa gtaagacgat tgctaaccac
     3961 ctattggaaa aaacaatagg tccttaaata atattgtcaa cttcaagtat tgtgatgcaa
     4021 gcatttagtc atgaacgctt ctctattcta tatgaaaagc cggttccggc gctctcacct
     4081 ttcctttttc tcccaatttt tcagttgaaa aaggtatatg cgtcaggcga cctctgaaat
     4141 taacaaaaaa tttccagtca tcgaatttga ttctgtgcga tagcgcccct gtgtgttctc
     4201 gttatgttga ggaaaaaaat aatggttgct aagagattcg aactcttgca tcttacgata
     4261 cctgagtatt cccacagttg ggggatctcg actctagcta gaggatcaat tcgtaatcat
     4321 gtcatagctg tttcctgtgt gaaattgtta tccgctcaca attccacaca acatacgagc
     4381 cggaagcata aagtgtaaag cctggggtgc ctaatgagtg agctaactca cattaattgc
     4441 gttgcgctca ctgcccgctt tccagtcggg aaacctgtcg tgccagctgc attaatgaat
     4501 cggccaacgc gcggggagag gcggtttgcg tattgggcgc tcttccgctt cctcgctcac
     4561 tgactcgctg cgctcggtcg ttcggctgcg gcgagcggta tcagctcact caaaggcggt
     4621 aatacggtta tccacagaat caggggataa cgcaggaaag aacatgtgag caaaaggcca
     4681 gcaaaaggcc aggaaccgta aaaaggccgc gttgctggcg tttttccata ggctccgccc
     4741 ccctgacgag catcacaaaa atcgacgctc aagtcagagg tggcgaaacc cgacaggact
     4801 ataaagatac caggcgtttc cccctggaag ctccctcgtg cgctctcctg ttccgaccct
     4861 gccgcttacc ggatacctgt ccgcctttct cccttcggga agcgtggcgc tttctcatag
     4921 ctcacgctgt aggtatctca gttcggtgta ggtcgttcgc tccaagctgg gctgtgtgca
     4981 cgaacccccc gttcagcccg accgctgcgc cttatccggt aactatcgtc ttgagtccaa
     5041 cccggtaaga cacgacttat cgccactggc agcagccact ggtaacagga ttagcagagc
     5101 gaggtatgta ggcggtgcta cagagttctt gaagtggtgg cctaactacg gctacactag
     5161 aagaacagta tttggtatct gcgctctgct gaagccagtt accttcggaa aaagagttgg
     5221 tagctcttga tccggcaaac aaaccaccgc tggtagcggt ggtttttttg tttgcaagca
     5281 gcagattacg cgcagaaaaa aaggatctca agaagatcct ttgatctttt ctacggggtc
     5341 tgacgctcag tggaacgaaa actcacgtta agggattttg gtcatgagat tatcaaaaag
     5401 gatcttcacc tagatccttt taaattaaaa atgaagtttt aaatcaatct aaagtatata
     5461 tgagtaaact tggtctgaca gttaccaatg cttaatcagt gaggcaccta tctcagcgat
     5521 ctgtctattt cgttcatcca tagttgcctg actccccgtc gtgtagataa ctacgatacg
     5581 ggagggctta ccatctggcc ccagtgctgc aatgataccg cgagacccac gctcaccggc
     5641 tccagattta tcagcaataa accagccagc cggaagggcc gagcgcagaa gtggtcctgc
     5701 aactttatcc gcctccatcc agtctattaa ttgttgccgg gaagctagag taagtagttc
     5761 gccagttaat agtttgcgca acgttgttgc cattgctaca ggcatcgtgg tgtcacgctc
     5821 gtcgtttggt atggcttcat tcagctccgg ttcccaacga tcaaggcgag ttacatgatc
     5881 ccccatgttg tgcaaaaaag cggttagctc cttcggtcct ccgatcgttg tcagaagtaa
     5941 gttggccgca gtgttatcac tcatggttat ggcagcactg cataattctc ttactgtcat
     6001 gccatccgta agatgctttt ctgtgactgg tgagtactca accaagtcat tctgagaata
     6061 gtgtatgcgg cgaccgagtt gctcttgccc ggcgtcaata cgggataata ccgcgccaca
     6121 tagcagaact ttaaaagtgc tcatcattgg aaaacgttct tcggggcgaa aactctcaag
     6181 gatcttaccg ctgttgagat ccagttcgat gtaacccact cgtgcaccca actgatcttc
     6241 agcatctttt actttcacca gcgtttctgg gtgagcaaaa acaggaaggc aaaatgccgc
     6301 aaaaaaggga ataagggcga cacggaaatg ttgaatactc atactcttcc tttttcaata
     6361 ttattgaagc atttatcagg gttattgtct catgagcgga tacatatttg aatgtattta
     6421 gaaaaataaa caaatagggg ttccgcgcac atttccccga aaagtgccac ctgacgtcta
     6481 agaaaccatt attatcatga cattaaccta taaaaatagg cgtatcacga ggccctttcg
     6541 tctcgcgcgt ttcggtgatg acggtgaaaa cctctgacac atgcagctcc cggagacggt
     6601 cacagcttgt ctgtaagcgg atgccgggag cagacaagcc cgtcagggcg cgtcagcggg
     6661 tgttggcggg tgtcggggct ggcttaacta tgcggcatca gagcagattg tactgagagt
     6721 gcaccataac gcatttaagc ataaacacgc actatgccgt tcttctcatg tatatatata
     6781 tacaggcaac acgcagatat aggtgcgacg tgaacagtga gctgtatgtg cgcagctcgc
     6841 gttgcatttt cggaagcgct cgttttcgga aacgctttga agttcctatt ccgaagttcc
     6901 tattctctag ctagaaagta taggaacttc agagcgcttt tgaaaaccaa aagcgctctg
     6961 aagacgcact ttcaaaaaac caaaaacgca ccggactgta acgagctact aaaatattgc
     7021 gaataccgct tccacaaaca ttgctcaaaa gtatctcttt gctatatatc tctgtgctat
     7081 atccctatat aacctaccca tccacctttc gctccttgaa cttgcatcta aactcgacct
     7141 ctacattttt tatgtttatc tctagtatta ctctttagac aaaaaaattg tagtaagaac
     7201 tattcataga gtgaatcgaa aacaatacga aaatgtaaac atttcctata cgtagtatat
     7261 agagacaaaa tagaagaaac cgttcataat tttctgacca atgaagaatc atcaacgcta
     7321 tcactttctg ttcacaaagt atgcgcaatc cacatcggta tagaatataa tcggggatgc
     7381 ctttatcttg aaaaaatgca cccgcagctt cgctagtaat cagtaaacgc gggaagtgga
     7441 gtcaggcttt ttttatggaa gagaaaatag acaccaaagt agccttcttc taaccttaac
     7501 ggacctacag tgcaaaaagt tatcaagaga ctgcattata gagcgcacaa aggagaaaaa
     7561 aagtaatcta agatgctttg ttagaaaaat agcgctctcg ggatgcattt ttgtagaaca
     7621 aaaaagaagt atagattctt tgttggtaaa atagcgctct cgcgttgcat ttctgttctg
     7681 taaaaatgca gctcagattc tttgtttgaa aaattagcgc tctcgcgttg catttttgtt
     7741 ttacaaaaat gaagcacaga ttcttcgttg gtaaaatagc gctttcgcgt tgcatttctg
     7801 ttctgtaaaa atgcagctca gattctttgt ttgaaaaatt agcgctctcg cgttgcattt
     7861 ttgttctaca aaatgaagca cagatgct
//
