LOCUS       sequence-236361-        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) (I325D)
FEATURES             Location/Qualifiers
     source          1..7300
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     protein_bind    128..149
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     promoter        join(164..169,170..187,188..194)
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    202..218
                     /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     207..229
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     primer_bind     226..242
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     226..242
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     261..281
                     /label=T3
                     /note="T3 promoter, forward primer"
     promoter        263..281
                     /label=T3 promoter
                     /note="promoter for bacteriophage T3 RNA polymerase"
     promoter        300..534
                     /gene="S. cerevisiae CUP1"
                     /label=CUP1 promoter
                     /note="inducible metallothionein promoter, regulated by 
                     Cu2+"
     CDS             542..1255
                     /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(572..600)
                     /label=GFP-R
                     /note="GFP, reverse primer. Does NOT anneal to EGFP"
     primer_bind     1195..1215
                     /label=GFP-F
                     /note="GFP, forward primer. Does NOT anneal to EGFP"
     RBS             2338..2349
                     /note="strong bacterial ribosome binding site (Elowitz and 
                     Leibler, 2000)"
     terminator      2558..2805
                     /gene="S. cerevisiae CYC1"
                     /label=CYC1 terminator
                     /note="transcription terminator for CYC1"
     primer_bind     complement(2574..2592)
                     /label=CYC1
                     /note="CYC1 transcription termination signal, reverse 
                     primer"
     primer_bind     complement(2823..2842)
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        complement(2824..2842)
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     primer_bind     complement(2852..2869)
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     complement(2852..2868)
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(2861..2883)
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     rep_origin      3009..3464
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(3096..3115)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     3306..3327
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     CDS             complement(3595..4398)
                     /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(4399..4614)
                     /gene="S. cerevisiae URA3"
                     /label=URA3 promoter
     primer_bind     complement(4638..4657)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     4757..4779
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     primer_bind     complement(4817..4835)
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     misc_feature    4876..5379
                     /label=CEN/ARS
                     /note="S. cerevisiae CEN6 centromere fused to an 
                     autonomously replicating sequence"
     promoter        5416..5520
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(5521..5589,5590..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     complement(5739..5758)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      6552..7140
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     7041..7060
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     join(7294..7300,1..11)
                     /label=L4440
                     /note="L4440 vector, forward primer"
ORIGIN
        1 cagtgagcga ggaagcggaa gagcgcccaa tacgcaaacc gcctctcccc gcgcgttggc
       61 cgattcatta atgcagctgg cacgacaggt ttcccgactg gaaagcgggc agtgagcgca
      121 acgcaattaa tgtgagttag ctcactcatt aggcacccca ggctttacac tttatgcttc
      181 cggctcgtat gttgtgtgga attgtgagcg gataacaatt tcacacagga aacagctatg
      241 accatgatta cgccaagcgc gcaattaacc ctcactaaag ggaacaaaag ctggagctcc
      301 tagttagaaa aagacatttt tgctgtcagt cactgtcaag agattctttt gctggcattt
      361 cttctagaag caaaaagagc gatgcgtctt ttccgctgaa ccgttccagc aaaaaagact
      421 accaacgcaa tatggattgt cagaatcata taaaagagaa gcaaataact ccttgtcttg
      481 tatcaattgc attataatat cttcttgtta gtgcaatatc atatagaagt catcgactag
      541 tatgagtaaa ggagaagaac ttttcactgg agttgtccca attcttgttg aattagatgg
      601 tgatgttaat gggcacaaat tttctgtcag tggagagggt gaaggtgatg caacatacgg
      661 aaaacttacc cttaaattta tttgcactac tggaaaacta cctgttccat ggccaacact
      721 tgtcactact ttcacttatg gtgttcaatg cttttcaaga tacccagatc atatgaaacg
      781 gcatgacttt ttcaagagtg ccatgcccga aggttatgta caggaaagaa ctatattttt
      841 caaagatgac gggaactaca agacacgtgc tgaagtcaag tttgaaggtg atacccttgt
      901 taatagaatc gagttaaaag gtattgattt taaagaagat ggaaacattc ttggacacaa
      961 attggaatac aactataact cacacaatgt atacatcatg gcagacaaac aaaagaatgg
     1021 aatcaaagtt aacttcaaaa ttagacacaa cattgaagat ggaagcgttc aactagcaga
     1081 ccattatcaa caaaatactc caattggcga tggccctgtc cttttaccag acaaccatta
     1141 cctgtccaca caatctgccc tttcgaaaga tcccaacgaa aagagagacc acatggtcct
     1201 tcttgagttt gtaacagctg ctgggattac acatggcatg gatgaactat acaaacccgg
     1261 gctgcaggaa ttcgatatca agcttatcga tatgaacgaa gcagatgtta caaaatttgt
     1321 taataatgcc aggaaaacgc tgaccgatgc tcaactttta tgttcaagtg ctaatttaag
     1381 gattgtagat attaagaaaa aattgtcatc ttggcagttg agtatttcaa aactcaattt
     1441 tctaatagtt ggcttgagac agcaaggaaa gtttctttac actattttaa aggaaggcat
     1501 tgggacaaag ctaatccaga aacaatggaa tcaagctgtc ttagtggtct tagttgatga
     1561 gatgaagtac tggcaatatg aaatcacttc taaagtacaa aggttggatg gtatagtaaa
     1621 tgaattgagc atatcagaaa aagatgatac cgatccctct aaactaggag attacatctc
     1681 gagagacaac gttaacttat tgaatgacaa actgaaagaa gtgccagtaa ttgagcgtca
     1741 aattgaaaac attaagcttc aatacgaaaa tatggtcaga aaagttaaca aagaactgat
     1801 tgataccaag ttgacggacg taactcaaaa attccaaagt aaatttggta tagataacct
     1861 gatggaaaca aatgttgcag agcagtttag cagggaacta acggaccttg aaaaagattt
     1921 agcagagata atgaattcat tgacacagca ctttgataag acattgctgt tacaagataa
     1981 aaaaattgat aacgatgaac gtgaggagct gtttaaggtg gtacaaggcg acgacaaaga
     2041 actatacaac attttcaaaa ctctgcatga ggtaattgat gacgtggaca aaacaattct
     2101 taacttgggt caatttttgc aggcaaaaat aaaggaaaag acagaactac acagcgaagt
     2161 ttctgaaata ataaacgatt tcaatagaaa tttggaatat ctattaatct ttaaagatat
     2221 ttccaatctg attgatagct tcaaaaattc ctgtacacaa gatgatcaaa caactaagga
     2281 actttgtgaa ttttatgata attttgaaga aagctacggt aacttagttc tagaagcaaa
     2341 gaggagaaag gatgtggcaa acagaatgaa aactatattg aaagattgtg aaaagcagtt
     2401 gcagaattta gatgctcagg accaggaaga acgtcagaat tttatagcgg aaaatggaac
     2461 ttatcttcct gagacaatct ggcccggtaa aattgacgat ttttcttccc tgtacacttt
     2521 aaattacaac gtgaagaatc cttaggtcga cctcgagtca tgtaattagt tatgtcacgc
     2581 ttacattcac gccctccccc cacatccgct ctaaccgaaa aggaaggagt tagacaacct
     2641 gaagtctagg tccctattta tttttttata gttatgttag tattaagaac gttatttata
     2701 tttcaaattt ttcttttttt tctgtacaga cgcgtgtacg catgtaacat tatactgaaa
     2761 accttgcttg agaaggtttt gggacgctcg aaggctttaa tttgcggccg gtacccaatt
     2821 cgccctatag tgagtcgtat tacgcgcgct cactggccgt cgttttacaa cgtcgtgact
     2881 gggaaaaccc tggcgttacc caacttaatc gccttgcagc acatccccct ttcgccagct
     2941 ggcgtaatag cgaagaggcc cgcaccgatc gcccttccca acagttgcgc agcctgaatg
     3001 gcgaatggac gcgccctgta gcggcgcatt aagcgcggcg ggtgtggtgg ttacgcgcag
     3061 cgtgaccgct acacttgcca gcgccctagc gcccgctcct ttcgctttct tcccttcctt
     3121 tctcgccacg ttcgccggct ttccccgtca agctctaaat cgggggctcc ctttagggtt
     3181 ccgatttagt gctttacggc acctcgaccc caaaaaactt gattagggtg atggttcacg
     3241 tagtgggcca tcgccctgat agacggtttt tcgccctttg acgttggagt ccacgttctt
     3301 taatagtgga ctcttgttcc aaactggaac aacactcaac cctatctcgg tctattcttt
     3361 tgatttataa gggattttgc cgatttcggc ctattggtta aaaaatgagc tgatttaaca
     3421 aaaatttaac gcgaatttta acaaaatatt aacgcttaca atttcctgat gcggtatttt
     3481 ctccttacgc atctgtgcgg tatttcacac cgcatagggt aataactgat ataattaaat
     3541 tgaagctcta atttgtgagt ttagtataca tgcatttact tataatacag ttttttagtt
     3601 ttgctggccg catcttctca aatatgcttc ccagcctgct tttctgtaac gttcaccctc
     3661 taccttagca tcccttccct ttgcaaatag tcctcttcca acaataataa tgtcagatcc
     3721 tgtagagacc acatcatcca cggttctata ctgttgaccc aatgcgtctc ccttgtcatc
     3781 taaacccaca ccgggtgtca taatcaacca atcgtaacct tcatctcttc cacccatgtc
     3841 tctttgagca ataaagccga taacaaaatc tttgtcgctc ttcgcaatgt caacagtacc
     3901 cttagtatat tctccagtag atagggagcc cttgcatgac aattctgcta acatcaaaag
     3961 gcctctaggt tcctttgtta cttcttctgc cgcctgcttc aaaccgctaa caatacctgg
     4021 gcccaccaca ccgtgtgcat tcgtaatgtc tgcccattct gctattctgt atacacccgc
     4081 agagtactgc aatttgactg tattaccaat gtcagcaaat tttctgtctt cgaagagtaa
     4141 aaaattgtac ttggcggata atgcctttag cggcttaact gtgccctcca tggaaaaatc
     4201 agtcaagata tccacatgtg tttttagtaa acaaattttg ggacctaatg cttcaactaa
     4261 ctccagtaat tccttggtgg tacgaacatc caatgaagca cacaagtttg tttgcttttc
     4321 gtgcatgata ttaaatagct tggcagcaac aggactagga tgagtagcag cacgttcctt
     4381 atatgtagct ttcgacatga tttatcttcg tttcctgcag gtttttgttc tgtgcagttg
     4441 ggttaagaat actgggcaat ttcatgtttc ttcaacacta catatgcgta tatataccaa
     4501 tctaagtctg tgctccttcc ttcgttcttc cttctgttcg gagattaccg aatcaaaaaa
     4561 atttcaagga aaccgaaatc aaaaaaaaga ataaaaaaaa aatgatgaat tgaaaaggtg
     4621 gtatggtgca ctctcagtac aatctgctct gatgccgcat agttaagcca gccccgacac
     4681 ccgccaacac ccgctgacgc gccctgacgg gcttgtctgc tcccggcatc cgcttacaga
     4741 caagctgtga ccgtctccgg gagctgcatg tgtcagaggt tttcaccgtc atcaccgaaa
     4801 cgcgcgagac gaaagggcct cgtgatacgc ctatttttat aggttaatgt catgataata
     4861 atggtttctt agacggatcg cttgcctgta acttacacgc gcctcgtatc ttttaatgat
     4921 ggaataattt gggaatttac tctgtgttta tttattttta tgttttgtat ttggatttta
     4981 gaaagtaaat aaagaaggta gaagagttac ggaatgaaga aaaaaaaata aacaaaggtt
     5041 taaaaaattt caacaaaaag cgtactttac atatatattt attagacaag aaaagcagat
     5101 taaatagata tacattcgat taacgataag taaaatgtaa aatcacagga ttttcgtgtg
     5161 tggtcttcta cacagacaag atgaaacaat tcggcattaa tacctgagag caggaagagc
     5221 aagataaaag gtagtatttg ttggcgatcc ccctagagtc ttttacatct tcggaaaaca
     5281 aaaactattt tttctttaat ttcttttttt actttctatt tttaatttat atatttatat
     5341 taaaaaattt aaattataat tatttttata gcacgtgatg aaaaggaccc aggtggcact
     5401 tttcggggaa atgtgcgcgg aacccctatt tgtttatttt tctaaataca ttcaaatatg
     5461 tatccgctca tgagacaata accctgataa atgcttcaat aatattgaaa aaggaagagt
     5521 atgagtattc aacatttccg tgtcgccctt attccctttt ttgcggcatt ttgccttcct
     5581 gtttttgctc acccagaaac gctggtgaaa gtaaaagatg ctgaagatca gttgggtgca
     5641 cgagtgggtt acatcgaact ggatctcaac agcggtaaga tccttgagag ttttcgcccc
     5701 gaagaacgtt ttccaatgat gagcactttt aaagttctgc tatgtggcgc ggtattatcc
     5761 cgtattgacg ccgggcaaga gcaactcggt cgccgcatac actattctca gaatgacttg
     5821 gttgagtact caccagtcac agaaaagcat cttacggatg gcatgacagt aagagaatta
     5881 tgcagtgctg ccataaccat gagtgataac actgcggcca acttacttct gacaacgatc
     5941 ggaggaccga aggagctaac cgcttttttg cacaacatgg gggatcatgt aactcgcctt
     6001 gatcgttggg aaccggagct gaatgaagcc ataccaaacg acgagcgtga caccacgatg
     6061 cctgtagcaa tggcaacaac gttgcgcaaa ctattaactg gcgaactact tactctagct
     6121 tcccggcaac aattaataga ctggatggag gcggataaag ttgcaggacc acttctgcgc
     6181 tcggcccttc cggctggctg gtttattgct gataaatctg gagccggtga gcgtgggtct
     6241 cgcggtatca ttgcagcact ggggccagat ggtaagccct cccgtatcgt agttatctac
     6301 acgacgggga gtcaggcaac tatggatgaa cgaaatagac agatcgctga gataggtgcc
     6361 tcactgatta agcattggta actgtcagac caagtttact catatatact ttagattgat
     6421 ttaaaacttc atttttaatt taaaaggatc taggtgaaga tcctttttga taatctcatg
     6481 accaaaatcc cttaacgtga gttttcgttc cactgagcgt cagaccccgt agaaaagatc
     6541 aaaggatctt cttgagatcc tttttttctg cgcgtaatct gctgcttgca aacaaaaaaa
     6601 ccaccgctac cagcggtggt ttgtttgccg gatcaagagc taccaactct ttttccgaag
     6661 gtaactggct tcagcagagc gcagatacca aatactgttc ttctagtgta gccgtagtta
     6721 ggccaccact tcaagaactc tgtagcaccg cctacatacc tcgctctgct aatcctgtta
     6781 ccagtggctg ctgccagtgg cgataagtcg tgtcttaccg ggttggactc aagacgatag
     6841 ttaccggata aggcgcagcg gtcgggctga acggggggtt cgtgcacaca gcccagcttg
     6901 gagcgaacga cctacaccga actgagatac ctacagcgtg agctatgaga aagcgccacg
     6961 cttcccgaag ggagaaaggc ggacaggtat ccggtaagcg gcagggtcgg aacaggagag
     7021 cgcacgaggg agcttccagg gggaaacgcc tggtatcttt atagtcctgt cgggtttcgc
     7081 cacctctgac ttgagcgtcg atttttgtga tgctcgtcag gggggcggag cctatggaaa
     7141 aacgccagca acgcggcctt tttacggttc ctggcctttt gctggccttt tgctcacatg
     7201 ttctttcctg cgttatcccc tgattctgtg gataaccgta ttaccgcctt tgagtgagct
     7261 gataccgctc gccgcagccg aacgaccgag cgcagcgagt
//
