LOCUS       sequence-329422-        6479 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Expresses 6xHis tagged ATG13 [571–700] fragment mutated in F641G, 
            I645G with 14 scramble amino acid sequence DIKLIDTVDLESCN under a Cu
            promoter in yeast.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 6479)
  AUTHORS   Gatica D, Damasio A, Pascual C, Klionsky DJ, Ragusa MJ, Popelka H
  TITLE     The carboxy terminus of yeast Atg13 binds phospholipid membrane via 
            motifs that overlap with the Vac8-interacting domain.
  JOURNAL   Autophagy. 2020 Jun;16(6):1007-1020. doi: 
            10.1080/15548627.2019.1648117. Epub 2019 Aug 2.
  PUBMED    31352862
REFERENCE   2  (bases 1 to 6479)
  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: 
            "10.1080/15548627.2019.1648117"; journalName: "Autophagy"; date: 
            "2020-06"; volume: "16"; issue: "6"; pages: "1007-1020"
COMMENT     Sequence Label: pCu-His6-Atg13[571–700] F641G, I645G-Ss(424)
FEATURES             Location/Qualifiers
     source          1..6479
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     protein_bind    83..104
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     promoter        join(119..124,125..142,143..149)
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    157..173
                     /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     162..184
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     primer_bind     181..197
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     181..197
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     216..236
                     /label=T3
                     /note="T3 promoter, forward primer"
     promoter        218..236
                     /label=T3 promoter
                     /note="promoter for bacteriophage T3 RNA polymerase"
     promoter        255..489
                     /gene="S. cerevisiae CUP1"
                     /label=CUP1 promoter
                     /note="inducible metallothionein promoter, regulated by 
                     Cu2+"
     CDS             513..530
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     primer_bind     complement(938..954)
                     /label=KS primer
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(939..955)
                     /label=pBluescriptKS
                     /note="For pBluescript vector"
     terminator      957..1204
                     /gene="S. cerevisiae CYC1"
                     /label=CYC1 terminator
                     /note="transcription terminator for CYC1"
     primer_bind     complement(973..991)
                     /label=CYC1
                     /note="CYC1 transcription termination signal, reverse 
                     primer"
     primer_bind     complement(1222..1241)
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        complement(1223..1241)
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     primer_bind     complement(1251..1268)
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     complement(1251..1267)
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(1260..1282)
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     rep_origin      1408..1863
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(1495..1514)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     1705..1726
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     CDS             complement(1966..2640)
                     /codon_start=1
                     /gene="S. cerevisiae TRP1"
                     /product="phosphoribosylanthranilate isomerase, required 
                     for tryptophan biosynthesis"
                     /label=TRP1
                     /note="yeast auxotrophic marker"
                     /translation="MSVINFTGSSGPLVKVCGLQSTEAAECALDSDADLLGIICVPNRK
                     RTIDPVIARKISSLVKAYKNSSGTPKYLVGVFRNQPKEDVLALVNDYGIDIVQLHGDES
                     WQEYQEFLGLPVIKRLVFPKDCNILLSAASQKPHSFIPLFDSEAGGTGELLDWNSISDW
                     VGRQESPESLHFMLAGGLTPENVGDALRLNGVIGVDVSGGVETNGVKDSNKIANFVKNA
                     KK"
     promoter        complement(2641..2922)
                     /gene="S. cerevisiae TRP1"
                     /label=TRP1 promoter
     primer_bind     complement(2943..2962)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     3062..3084
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     primer_bind     complement(3122..3140)
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     rep_origin      3181..4523
                     /label=2u ori
                     /note="yeast 2u plasmid origin of replication"
     protein_bind    3513..3560
                     /label=FRT
                     /bound_moiety="FLP recombinase from the Saccharomyces 
                     cerevisiae 2u plasmid"
                     /note="FLP-mediated recombination occurs in the 8-bp core 
                     sequence TCTAGAAA (Turan and Bode, 2011)."
     promoter        4550..4654
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(4655..4723,4724..5515)
                     /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(4873..4892)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      5686..6274
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     6175..6194
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     6428..6445
                     /label=L4440
                     /note="L4440 vector, forward primer"
ORIGIN
        1 tccccgcgcg ttggccgatt cattaatgca gctggcacga caggtttccc gactggaaag
       61 cgggcagtga gcgcaacgca attaatgtga gttagctcac tcattaggca ccccaggctt
      121 tacactttat gcttccggct cgtatgttgt gtggaattgt gagcggataa caatttcaca
      181 caggaaacag ctatgaccat gattacgcca agcgcgcaat taaccctcac taaagggaac
      241 aaaagctgga gctcctagtt agaaaaagac atttttgctg tcagtcactg tcaagagatt
      301 cttttgctgg catttcttct agaagcaaaa agagcgatgc gtcttttccg ctgaaccgtt
      361 ccagcaaaaa agactaccaa cgcaatatgg attgtcagaa tcatataaaa gagaagcaaa
      421 taactccttg tcttgtatca attgcattat aatatcttct tgttagtgca atatcatata
      481 gaagtcatcg actagtggat ccccttaaga tgcaccatca ccatcatcac tctactagtg
      541 ccttgaatag tagaaggaat tctttagata agagctcaaa caagcagggt atgtcaggct
      601 tacctcctat ttttggtgga gagagtactt catatcacca cgacaacaaa atacaaaagt
      661 acaaccaatt aggagtagaa gaagatgatg atgacgagaa tgaccgtttg ctcaaccaaa
      721 tgggaaacag tgctacaaaa ggaaaaagtt caggatctcc aagatcaatt gatagcattt
      781 caagttcttt cataaaaagt aggataccta tcagacaacc ataccattac tctcaaccaa
      841 ctactgcgcc ctttcaagct caggcgaaat ttcataaacc tgcaaataag ttaatcgata
      901 atggtaatag gagtaatagt taggatatca agcttatcga taccgtcgac ctcgagtcat
      961 gtaattagtt atgtcacgct tacattcacg ccctcccccc acatccgctc taaccgaaaa
     1021 ggaaggagtt agacaacctg aagtctaggt ccctatttat ttttttatag ttatgttagt
     1081 attaagaacg ttatttatat ttcaaatttt tctttttttt ctgtacagac gcgtgtacgc
     1141 atgtaacatt atactgaaaa ccttgcttga gaaggttttg ggacgctcga aggctttaat
     1201 ttgcggccgg tacccaattc gccctatagt gagtcgtatt acgcgcgctc actggccgtc
     1261 gttttacaac gtcgtgactg ggaaaaccct ggcgttaccc aacttaatcg ccttgcagca
     1321 catccccctt tcgccagctg gcgtaatagc gaagaggccc gcaccgatcg cccttcccaa
     1381 cagttgcgca gcctgaatgg cgaatggacg cgccctgtag cggcgcatta agcgcggcgg
     1441 gtgtggtggt tacgcgcagc gtgaccgcta cacttgccag cgccctagcg cccgctcctt
     1501 tcgctttctt cccttccttt ctcgccacgt tcgccggctt tccccgtcaa gctctaaatc
     1561 gggggctccc tttagggttc cgatttagtg ctttacggca cctcgacccc aaaaaacttg
     1621 attagggtga tggttcacgt agtgggccat cgccctgata gacggttttt cgccctttga
     1681 cgttggagtc cacgttcttt aatagtggac tcttgttcca aactggaaca acactcaacc
     1741 atatctcggt ctattctttt gatttataag ggattttgcc gatttcggcc tattggttaa
     1801 aaaatgagct gatttaacaa aaatttaacg cgaattttaa caaaatatta acgcttacaa
     1861 tttcctgatg cggtattttc tccttacgca tctgtgcggt atttcacacc gcatagatcg
     1921 gcaagtgcac aaacaatact taaataaata ctactcagta ataacctatt tcttagcatt
     1981 tttgacgaaa tttgctattt tgttagagtc ttttacacca tttgtctcca cacctccgct
     2041 tacatcaaca ccaataacgc catttaatct aagcgcatca ccaacatttt ctggcgtcag
     2101 tccaccagct aacataaaat gtaagctttc ggggctctct tgccttccaa cccagtcaga
     2161 aatcgagttc caatccaaaa gttcacctgt cccacctgct tctgaatcaa acaagggaat
     2221 aaacgaatga ggtttctgtg aagctgcact gagtagtatg ttgcagtctt ttggaaatac
     2281 gagtctttta ataactggca aaccgaggaa ctcttggtat tcttgccacg actcatctcc
     2341 atgcagttgg acgatatcaa tgccgtaatc attgaccaga gccaaaacat cctccttagg
     2401 ttgattacga aacacgccaa ccaagtattt cggagtgcct gaactatttt tatatgcttt
     2461 tacaagactt gaaattttcc ttgcaataac cgggtcaatt gttctctttc tattgggcac
     2521 acatataata cccagcaagt cagcatcgga atctagagca cattctgcgg cctctgtgct
     2581 ctgcaagccg caaactttca ccaatggacc agaactacct gtgaaattaa taacagacat
     2641 actccaagct gcctttgtgt gcttaatcac gtatactcac gtgctcaata gtcaccaatg
     2701 ccctccctct tggccctctc cttttctttt ttcgaccgaa ttaattctta atcggcaaaa
     2761 aaagaaaagc tccggatcaa gattgtacgt aaggtgacaa gctatttttc aataaagaat
     2821 atcttccact actgccatct ggcgtcataa ctgcaaagta cacatatatt acgatgctgt
     2881 tctattaaat gcttcctata ttatatatat agtaatgtcg ttgatctatg gtgcactctc
     2941 agtacaatct gctctgatgc cgcatagtta agccagcccc gacacccgcc aacacccgct
     3001 gacgcgccct gacgggcttg tctgctcccg gcatccgctt acagacaagc tgtgaccgtc
     3061 tccgggagct gcatgtgtca gaggttttca ccgtcatcac cgaaacgcgc gagacgaaag
     3121 ggcctcgtga tacgcctatt tttataggtt aatgtcatga taataatggt ttcttagtat
     3181 gatccaatat caaaggaaat gatagcattg aaggatgaga ctaatccaat tgaggagtgg
     3241 cagcatatag aacagctaaa gggtagtgct gaaggaagca tacgataccc cgcatggaat
     3301 gggataatat cacaggaggt actagactac ctttcatcct acataaatag acgcatataa
     3361 gtacgcattt aagcataaac acgcactatg ccgttcttct catgtatata tatatacagg
     3421 caacacgcag atataggtgc gacgtgaaca gtgagctgta tgtgcgcagc tcgcgttgca
     3481 ttttcggaag cgctcgtttt cggaaacgct ttgaagttcc tattccgaag ttcctattct
     3541 ctagaaagta taggaacttc agagcgcttt tgaaaaccaa aagcgctctg aagacgcact
     3601 ttcaaaaaac caaaaacgca ccggactgta acgagctact aaaatattgc gaataccgct
     3661 tccacaaaca ttgctcaaaa gtatctcttt gctatatatc tctgtgctat atccctatat
     3721 aacctaccca tccacctttc gctccttgaa cttgcatcta aactcgacct ctacattttt
     3781 tatgtttatc tctagtatta ctctttagac aaaaaaattg tagtaagaac tattcataga
     3841 gtgaatcgaa aacaatacga aaatgtaaac atttcctata cgtagtatat agagacaaaa
     3901 tagaagaaac cgttcataat tttctgacca atgaagaatc atcaacgcta tcactttctg
     3961 ttcacaaagt atgcgcaatc cacatcggta tagaatataa tcggggatgc ctttatcttg
     4021 aaaaaatgca cccgcagctt cgctagtaat cagtaaacgc gggaagtgga gtcaggcttt
     4081 ttttatggaa gagaaaatag acaccaaagt agccttcttc taaccttaac ggacctacag
     4141 tgcaaaaagt tatcaagaga ctgcattata gagcgcacaa aggagaaaaa aagtaatcta
     4201 agatgctttg ttagaaaaat agcgctctcg ggatgcattt ttgtagaaca aaaaagaagt
     4261 atagattctt tgttggtaaa atagcgctct cgcgttgcat ttctgttctg taaaaatgca
     4321 gctcagattc tttgtttgaa aaattagcgc tctcgcgttg catttttgtt ttacaaaaat
     4381 gaagcacaga ttcttcgttg gtaaaatagc gctttcgcgt tgcatttctg ttctgtaaaa
     4441 atgcagctca gattctttgt ttgaaaaatt agcgctctcg cgttgcattt ttgttctaca
     4501 aaatgaagca cagatgcttc gttcaggtgg cacttttcgg ggaaatgtgc gcggaacccc
     4561 tatttgttta tttttctaaa tacattcaaa tatgtatccg ctcatgagac aataaccctg
     4621 ataaatgctt caataatatt gaaaaaggaa gagtatgagt attcaacatt tccgtgtcgc
     4681 ccttattccc ttttttgcgg cattttgcct tcctgttttt gctcacccag aaacgctggt
     4741 gaaagtaaaa gatgctgaag atcagttggg tgcacgagtg ggttacatcg aactggatct
     4801 caacagcggt aagatccttg agagttttcg ccccgaagaa cgttttccaa tgatgagcac
     4861 ttttaaagtt ctgctatgtg gcgcggtatt atcccgtatt gacgccgggc aagagcaact
     4921 cggtcgccgc atacactatt ctcagaatga cttggttgag tactcaccag tcacagaaaa
     4981 gcatcttacg gatggcatga cagtaagaga attatgcagt gctgccataa ccatgagtga
     5041 taacactgcg gccaacttac ttctgacaac gatcggagga ccgaaggagc taaccgcttt
     5101 tttgcacaac atgggggatc atgtaactcg ccttgatcgt tgggaaccgg agctgaatga
     5161 agccatacca aacgacgagc gtgacaccac gatgcctgta gcaatggcaa caacgttgcg
     5221 caaactatta actggcgaac tacttactct agcttcccgg caacaattaa tagactggat
     5281 ggaggcggat aaagttgcag gaccacttct gcgctcggcc cttccggctg gctggtttat
     5341 tgctgataaa tctggagccg gtgagcgtgg gtctcgcggt atcattgcag cactggggcc
     5401 agatggtaag ccctcccgta tcgtagttat ctacacgacg gggagtcagg caactatgga
     5461 tgaacgaaat agacagatcg ctgagatagg tgcctcactg attaagcatt ggtaactgtc
     5521 agaccaagtt tactcatata tactttagat tgatttaaaa cttcattttt aatttaaaag
     5581 gatctaggtg aagatccttt ttgataatct catgaccaaa atcccttaac gtgagttttc
     5641 gttccactga gcgtcagacc ccgtagaaaa gatcaaagga tcttcttgag atcctttttt
     5701 tctgcgcgta atctgctgct tgcaaacaaa aaaaccaccg ctaccagcgg tggtttgttt
     5761 gccggatcaa gagctaccaa ctctttttcc gaaggtaact ggcttcagca gagcgcagat
     5821 accaaatact gttcttctag tgtagccgta gttaggccac cacttcaaga actctgtagc
     5881 accgcctaca tacctcgctc tgctaatcct gttaccagtg gctgctgcca gtggcgataa
     5941 gtcgtgtctt accgggttgg actcaagacg atagttaccg gataaggcgc agcggtcggg
     6001 ctgaacgggg ggttcgtgca cacagcccag cttggagcga acgacctaca ccgaactgag
     6061 atacctacag cgtgagctat gagaaagcgc cacgcttccc gaagggagaa aggcggacag
     6121 gtatccggta agcggcaggg tcggaacagg agagcgcacg agggagcttc cagggggaaa
     6181 cgcctggtat ctttatagtc ctgtcgggtt tcgccacctc tgacttgagc gtcgattttt
     6241 gtgatgctcg tcaggggggc ggagcctatg gaaaaacgcc agcaacgcgg cctttttacg
     6301 gttcctggcc ttttgctggc cttttgctca catgttcttt cctgcgttat cccctgattc
     6361 tgtggataac cgtattaccg cctttgagtg agctgatacc gctcgccgca gccgaacgac
     6421 cgagcgcagc gagtcagtga gcgaggaagc ggaagagcgc ccaatacgca aaccgcctc
//
