LOCUS       sequence-236905-        7300 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Expressing GFP-Atg17.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 7300)
  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 7300)
  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: pCu-GFP-Atg17(416) (L105D)
FEATURES             Location/Qualifiers
     source          1..7300
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     protein_bind    62..83
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     promoter        join(98..103,104..121,122..128)
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    136..152
                     /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)."
     primer_bind     141..163
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     primer_bind     160..176
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     160..176
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     195..215
                     /label=T3
                     /note="T3 promoter, forward primer"
     promoter        197..215
                     /label=T3 promoter
                     /note="promoter for bacteriophage T3 RNA polymerase"
     promoter        234..468
                     /gene="S. cerevisiae CUP1"
                     /label=CUP1 promoter
                     /note="inducible metallothionein promoter, regulated by 
                     Cu2+"
     CDS             476..1189
                     /codon_start=1
                     /product="S65T variant of Aequorea victoria green 
                     fluorescent protein (Heim et al., 1995)"
                     /label=GFP (S65T)
                     /note="excitable with blue light"
                     /translation="MSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTLK
                     FICTTGKLPVPWPTLVTTFTYGVQCFSRYPDHMKRHDFFKSAMPEGYVQERTIFFKDDG
                     NYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIKV
                     NFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSALSKDPNEKRDHMVLLE
                     FVTAAGITHGMDELYK"
     primer_bind     complement(506..534)
                     /label=GFP-R
                     /note="GFP, reverse primer. Does NOT anneal to EGFP"
     primer_bind     1129..1149
                     /label=GFP-F
                     /note="GFP, forward primer. Does NOT anneal to EGFP"
     RBS             2272..2283
                     /note="strong bacterial ribosome binding site (Elowitz and 
                     Leibler, 2000)"
     terminator      2492..2739
                     /gene="S. cerevisiae CYC1"
                     /label=CYC1 terminator
                     /note="transcription terminator for CYC1"
     primer_bind     complement(2508..2526)
                     /label=CYC1
                     /note="CYC1 transcription termination signal, reverse 
                     primer"
     primer_bind     complement(2757..2776)
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        complement(2758..2776)
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     primer_bind     complement(2786..2803)
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     complement(2786..2802)
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(2795..2817)
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     rep_origin      2943..3398
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(3030..3049)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     3240..3261
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     CDS             complement(3529..4332)
                     /codon_start=1
                     /gene="S. cerevisiae URA3"
                     /product="orotidine-5'-phosphate decarboxylase, required 
                     for uracil biosynthesis"
                     /label=URA3
                     /note="yeast auxotrophic marker, counterselectable with 
                     5-fluoroorotic acid (5-FOA)"
                     /translation="MSKATYKERAATHPSPVAAKLFNIMHEKQTNLCASLDVRTTKELL
                     ELVEALGPKICLLKTHVDILTDFSMEGTVKPLKALSAKYNFLLFEDRKFADIGNTVKLQ
                     YSAGVYRIAEWADITNAHGVVGPGIVSGLKQAAEEVTKEPRGLLMLAELSCKGSLSTGE
                     YTKGTVDIAKSDKDFVIGFIAQRDMGGRDEGYDWLIMTPGVGLDDKGDALGQQYRTVDD
                     VVSTGSDIIIVGRGLFAKGRDAKVEGERYRKAGWEAYLRRCGQQN"
     promoter        complement(4333..4548)
                     /gene="S. cerevisiae URA3"
                     /label=URA3 promoter
     primer_bind     complement(4572..4591)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     4691..4713
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     primer_bind     complement(4751..4769)
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     misc_feature    4810..5313
                     /label=CEN/ARS
                     /note="S. cerevisiae CEN6 centromere fused to an 
                     autonomously replicating sequence"
     promoter        5350..5454
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(5455..5523,5524..6315)
                     /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     complement(5673..5692)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      6486..7074
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     6975..6994
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     7228..7245
                     /label=L4440
                     /note="L4440 vector, forward primer"
ORIGIN
        1 attaatgcag ctggcacgac aggtttcccg actggaaagc gggcagtgag cgcaacgcaa
       61 ttaatgtgag ttagctcact cattaggcac cccaggcttt acactttatg cttccggctc
      121 gtatgttgtg tggaattgtg agcggataac aatttcacac aggaaacagc tatgaccatg
      181 attacgccaa gcgcgcaatt aaccctcact aaagggaaca aaagctggag ctcctagtta
      241 gaaaaagaca tttttgctgt cagtcactgt caagagattc ttttgctggc atttcttcta
      301 gaagcaaaaa gagcgatgcg tcttttccgc tgaaccgttc cagcaaaaaa gactaccaac
      361 gcaatatgga ttgtcagaat catataaaag agaagcaaat aactccttgt cttgtatcaa
      421 ttgcattata atatcttctt gttagtgcaa tatcatatag aagtcatcga ctagtatgag
      481 taaaggagaa gaacttttca ctggagttgt cccaattctt gttgaattag atggtgatgt
      541 taatgggcac aaattttctg tcagtggaga gggtgaaggt gatgcaacat acggaaaact
      601 tacccttaaa tttatttgca ctactggaaa actacctgtt ccatggccaa cacttgtcac
      661 tactttcact tatggtgttc aatgcttttc aagataccca gatcatatga aacggcatga
      721 ctttttcaag agtgccatgc ccgaaggtta tgtacaggaa agaactatat ttttcaaaga
      781 tgacgggaac tacaagacac gtgctgaagt caagtttgaa ggtgataccc ttgttaatag
      841 aatcgagtta aaaggtattg attttaaaga agatggaaac attcttggac acaaattgga
      901 atacaactat aactcacaca atgtatacat catggcagac aaacaaaaga atggaatcaa
      961 agttaacttc aaaattagac acaacattga agatggaagc gttcaactag cagaccatta
     1021 tcaacaaaat actccaattg gcgatggccc tgtcctttta ccagacaacc attacctgtc
     1081 cacacaatct gccctttcga aagatcccaa cgaaaagaga gaccacatgg tccttcttga
     1141 gtttgtaaca gctgctggga ttacacatgg catggatgaa ctatacaaac ccgggctgca
     1201 ggaattcgat atcaagctta tcgatatgaa cgaagcagat gttacaaaat ttgttaataa
     1261 tgccaggaaa acgctgaccg atgctcaact tttatgttca agtgctaatt taaggattgt
     1321 agatattaag aaaaaattgt catcttggca gttgagtatt tcaaaactca attttctaat
     1381 agttggcttg agacagcaag gaaagtttct ttacactatt ttaaaggaag gcattgggac
     1441 aaagctaatc cagaaacaat ggaatcaagc tgtcttagtg gtcttagttg atgagatgaa
     1501 gtactggcaa tatgaaatca cttctaaagt acaaagggac gatggtatag taaatgaatt
     1561 gagcatatca gaaaaagatg ataccgatcc ctctaaacta ggagattaca tctcgagaga
     1621 caacgttaac ttattgaatg acaaactgaa agaagtgcca gtaattgagc gtcaaattga
     1681 aaacattaag cttcaatacg aaaatatggt cagaaaagtt aacaaagaac tgattgatac
     1741 caagttgacg gacgtaactc aaaaattcca aagtaaattt ggtatagata acctgatgga
     1801 aacaaatgtt gcagagcagt ttagcaggga actaacggac cttgaaaaag atttagcaga
     1861 gataatgaat tcattgacac agcactttga taagacattg ctgttacaag ataaaaaaat
     1921 tgataacgat gaacgtgagg agctgtttaa ggtggtacaa ggcgacgaca aagaactata
     1981 caacattttc aaaactctgc atggggtaat tgatgacgtg gacaaaacaa ttcttaactt
     2041 gggtcaattt ttgcaggcaa aaataaagga aaagacagaa ctacacagcg aagtttctga
     2101 aataataaac gatttcaata gaaatttgga atatctatta atctttaaag atatttccaa
     2161 tctgattgat agcttcaaaa attcctgtac acaagatatt caaacaacta aggaactttg
     2221 tgaattttat gataattttg aagaaagcta cggtaactta gttctagaag caaagaggag
     2281 aaaggatgtg gcaaacagaa tgaaaactat attgaaagat tgtgaaaagc agttgcagaa
     2341 tttagatgct caggaccagg aagaacgtca gaattttata gcggaaaatg gaacttatct
     2401 tcctgagaca atctggcccg gtaaaattga cgatttttct tccctgtaca ctttaaatta
     2461 caacgtgaag aatccttagg tcgacctcga gtcatgtaat tagttatgtc acgcttacat
     2521 tcacgccctc cccccacatc cgctctaacc gaaaaggaag gagttagaca acctgaagtc
     2581 taggtcccta tttatttttt tatagttatg ttagtattaa gaacgttatt tatatttcaa
     2641 atttttcttt tttttctgta cagacgcgtg tacgcatgta acattatact gaaaaccttg
     2701 cttgagaagg ttttgggacg ctcgaaggct ttaatttgcg gccggtaccc aattcgccct
     2761 atagtgagtc gtattacgcg cgctcactgg ccgtcgtttt acaacgtcgt gactgggaaa
     2821 accctggcgt tacccaactt aatcgccttg cagcacatcc ccctttcgcc agctggcgta
     2881 atagcgaaga ggcccgcacc gatcgccctt cccaacagtt gcgcagcctg aatggcgaat
     2941 ggacgcgccc tgtagcggcg cattaagcgc ggcgggtgtg gtggttacgc gcagcgtgac
     3001 cgctacactt gccagcgccc tagcgcccgc tcctttcgct ttcttccctt cctttctcgc
     3061 cacgttcgcc ggctttcccc gtcaagctct aaatcggggg ctccctttag ggttccgatt
     3121 tagtgcttta cggcacctcg accccaaaaa acttgattag ggtgatggtt cacgtagtgg
     3181 gccatcgccc tgatagacgg tttttcgccc tttgacgttg gagtccacgt tctttaatag
     3241 tggactcttg ttccaaactg gaacaacact caaccctatc tcggtctatt cttttgattt
     3301 ataagggatt ttgccgattt cggcctattg gttaaaaaat gagctgattt aacaaaaatt
     3361 taacgcgaat tttaacaaaa tattaacgct tacaatttcc tgatgcggta ttttctcctt
     3421 acgcatctgt gcggtatttc acaccgcata gggtaataac tgatataatt aaattgaagc
     3481 tctaatttgt gagtttagta tacatgcatt tacttataat acagtttttt agttttgctg
     3541 gccgcatctt ctcaaatatg cttcccagcc tgcttttctg taacgttcac cctctacctt
     3601 agcatccctt ccctttgcaa atagtcctct tccaacaata ataatgtcag atcctgtaga
     3661 gaccacatca tccacggttc tatactgttg acccaatgcg tctcccttgt catctaaacc
     3721 cacaccgggt gtcataatca accaatcgta accttcatct cttccaccca tgtctctttg
     3781 agcaataaag ccgataacaa aatctttgtc gctcttcgca atgtcaacag tacccttagt
     3841 atattctcca gtagataggg agcccttgca tgacaattct gctaacatca aaaggcctct
     3901 aggttccttt gttacttctt ctgccgcctg cttcaaaccg ctaacaatac ctgggcccac
     3961 cacaccgtgt gcattcgtaa tgtctgccca ttctgctatt ctgtatacac ccgcagagta
     4021 ctgcaatttg actgtattac caatgtcagc aaattttctg tcttcgaaga gtaaaaaatt
     4081 gtacttggcg gataatgcct ttagcggctt aactgtgccc tccatggaaa aatcagtcaa
     4141 gatatccaca tgtgttttta gtaaacaaat tttgggacct aatgcttcaa ctaactccag
     4201 taattccttg gtggtacgaa catccaatga agcacacaag tttgtttgct tttcgtgcat
     4261 gatattaaat agcttggcag caacaggact aggatgagta gcagcacgtt ccttatatgt
     4321 agctttcgac atgatttatc ttcgtttcct gcaggttttt gttctgtgca gttgggttaa
     4381 gaatactggg caatttcatg tttcttcaac actacatatg cgtatatata ccaatctaag
     4441 tctgtgctcc ttccttcgtt cttccttctg ttcggagatt accgaatcaa aaaaatttca
     4501 aggaaaccga aatcaaaaaa aagaataaaa aaaaaatgat gaattgaaaa ggtggtatgg
     4561 tgcactctca gtacaatctg ctctgatgcc gcatagttaa gccagccccg acacccgcca
     4621 acacccgctg acgcgccctg acgggcttgt ctgctcccgg catccgctta cagacaagct
     4681 gtgaccgtct ccgggagctg catgtgtcag aggttttcac cgtcatcacc gaaacgcgcg
     4741 agacgaaagg gcctcgtgat acgcctattt ttataggtta atgtcatgat aataatggtt
     4801 tcttagacgg atcgcttgcc tgtaacttac acgcgcctcg tatcttttaa tgatggaata
     4861 atttgggaat ttactctgtg tttatttatt tttatgtttt gtatttggat tttagaaagt
     4921 aaataaagaa ggtagaagag ttacggaatg aagaaaaaaa aataaacaaa ggtttaaaaa
     4981 atttcaacaa aaagcgtact ttacatatat atttattaga caagaaaagc agattaaata
     5041 gatatacatt cgattaacga taagtaaaat gtaaaatcac aggattttcg tgtgtggtct
     5101 tctacacaga caagatgaaa caattcggca ttaatacctg agagcaggaa gagcaagata
     5161 aaaggtagta tttgttggcg atccccctag agtcttttac atcttcggaa aacaaaaact
     5221 attttttctt taatttcttt ttttactttc tatttttaat ttatatattt atattaaaaa
     5281 atttaaatta taattatttt tatagcacgt gatgaaaagg acccaggtgg cacttttcgg
     5341 ggaaatgtgc gcggaacccc tatttgttta tttttctaaa tacattcaaa tatgtatccg
     5401 ctcatgagac aataaccctg ataaatgctt caataatatt gaaaaaggaa gagtatgagt
     5461 attcaacatt tccgtgtcgc ccttattccc ttttttgcgg cattttgcct tcctgttttt
     5521 gctcacccag aaacgctggt gaaagtaaaa gatgctgaag atcagttggg tgcacgagtg
     5581 ggttacatcg aactggatct caacagcggt aagatccttg agagttttcg ccccgaagaa
     5641 cgttttccaa tgatgagcac ttttaaagtt ctgctatgtg gcgcggtatt atcccgtatt
     5701 gacgccgggc aagagcaact cggtcgccgc atacactatt ctcagaatga cttggttgag
     5761 tactcaccag tcacagaaaa gcatcttacg gatggcatga cagtaagaga attatgcagt
     5821 gctgccataa ccatgagtga taacactgcg gccaacttac ttctgacaac gatcggagga
     5881 ccgaaggagc taaccgcttt tttgcacaac atgggggatc atgtaactcg ccttgatcgt
     5941 tgggaaccgg agctgaatga agccatacca aacgacgagc gtgacaccac gatgcctgta
     6001 gcaatggcaa caacgttgcg caaactatta actggcgaac tacttactct agcttcccgg
     6061 caacaattaa tagactggat ggaggcggat aaagttgcag gaccacttct gcgctcggcc
     6121 cttccggctg gctggtttat tgctgataaa tctggagccg gtgagcgtgg gtctcgcggt
     6181 atcattgcag cactggggcc agatggtaag ccctcccgta tcgtagttat ctacacgacg
     6241 gggagtcagg caactatgga tgaacgaaat agacagatcg ctgagatagg tgcctcactg
     6301 attaagcatt ggtaactgtc agaccaagtt tactcatata tactttagat tgatttaaaa
     6361 cttcattttt aatttaaaag gatctaggtg aagatccttt ttgataatct catgaccaaa
     6421 atcccttaac gtgagttttc gttccactga gcgtcagacc ccgtagaaaa gatcaaagga
     6481 tcttcttgag atcctttttt tctgcgcgta atctgctgct tgcaaacaaa aaaaccaccg
     6541 ctaccagcgg tggtttgttt gccggatcaa gagctaccaa ctctttttcc gaaggtaact
     6601 ggcttcagca gagcgcagat accaaatact gttcttctag tgtagccgta gttaggccac
     6661 cacttcaaga actctgtagc accgcctaca tacctcgctc tgctaatcct gttaccagtg
     6721 gctgctgcca gtggcgataa gtcgtgtctt accgggttgg actcaagacg atagttaccg
     6781 gataaggcgc agcggtcggg ctgaacgggg ggttcgtgca cacagcccag cttggagcga
     6841 acgacctaca ccgaactgag atacctacag cgtgagctat gagaaagcgc cacgcttccc
     6901 gaagggagaa aggcggacag gtatccggta agcggcaggg tcggaacagg agagcgcacg
     6961 agggagcttc cagggggaaa cgcctggtat ctttatagtc ctgtcgggtt tcgccacctc
     7021 tgacttgagc gtcgattttt gtgatgctcg tcaggggggc ggagcctatg gaaaaacgcc
     7081 agcaacgcgg cctttttacg gttcctggcc ttttgctggc cttttgctca catgttcttt
     7141 cctgcgttat cccctgattc tgtggataac cgtattaccg cctttgagtg agctgatacc
     7201 gctcgccgca gccgaacgac cgagcgcagc gagtcagtga gcgaggaagc ggaagagcgc
     7261 ccaatacgca aaccgcctct ccccgcgcgt tggccgattc
//
