LOCUS       sequence-329426-        6479 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Expresses 6xHis tagged ATG13 [571–700] fragment mutated in K683E, 
            H685E, K686E 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] K683E, H685E, K686E-Ss...
FEATURES             Location/Qualifiers
     source          1..6479
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     166..185
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     419..436
                     /label=L4440
                     /note="L4440 vector, forward primer"
     protein_bind    553..574
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     promoter        join(589..594,595..612,613..619)
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    627..643
                     /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     632..654
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     primer_bind     651..667
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     651..667
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     686..706
                     /label=T3
                     /note="T3 promoter, forward primer"
     promoter        688..706
                     /label=T3 promoter
                     /note="promoter for bacteriophage T3 RNA polymerase"
     promoter        725..959
                     /gene="S. cerevisiae CUP1"
                     /label=CUP1 promoter
                     /note="inducible metallothionein promoter, regulated by 
                     Cu2+"
     CDS             983..1000
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     primer_bind     complement(1408..1424)
                     /label=KS primer
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(1409..1425)
                     /label=pBluescriptKS
                     /note="For pBluescript vector"
     terminator      1427..1674
                     /gene="S. cerevisiae CYC1"
                     /label=CYC1 terminator
                     /note="transcription terminator for CYC1"
     primer_bind     complement(1443..1461)
                     /label=CYC1
                     /note="CYC1 transcription termination signal, reverse 
                     primer"
     primer_bind     complement(1692..1711)
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        complement(1693..1711)
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     primer_bind     complement(1721..1738)
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     complement(1721..1737)
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(1730..1752)
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     rep_origin      1878..2333
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(1965..1984)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     2175..2196
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     CDS             complement(2436..3110)
                     /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(3111..3392)
                     /gene="S. cerevisiae TRP1"
                     /label=TRP1 promoter
     primer_bind     complement(3413..3432)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     3532..3554
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     primer_bind     complement(3592..3610)
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     rep_origin      3651..4993
                     /label=2u ori
                     /note="yeast 2u plasmid origin of replication"
     protein_bind    3983..4030
                     /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        5020..5124
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(5125..5193,5194..5985)
                     /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(5343..5362)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      join(6156..6479,1..265)
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
ORIGIN
        1 gggttcgtgc acacagccca gcttggagcg aacgacctac accgaactga gatacctaca
       61 gcgtgagcta tgagaaagcg ccacgcttcc cgaagggaga aaggcggaca ggtatccggt
      121 aagcggcagg gtcggaacag gagagcgcac gagggagctt ccagggggaa acgcctggta
      181 tctttatagt cctgtcgggt ttcgccacct ctgacttgag cgtcgatttt tgtgatgctc
      241 gtcagggggg cggagcctat ggaaaaacgc cagcaacgcg gcctttttac ggttcctggc
      301 cttttgctgg ccttttgctc acatgttctt tcctgcgtta tcccctgatt ctgtggataa
      361 ccgtattacc gcctttgagt gagctgatac cgctcgccgc agccgaacga ccgagcgcag
      421 cgagtcagtg agcgaggaag cggaagagcg cccaatacgc aaaccgcctc tccccgcgcg
      481 ttggccgatt cattaatgca gctggcacga caggtttccc gactggaaag cgggcagtga
      541 gcgcaacgca attaatgtga gttagctcac tcattaggca ccccaggctt tacactttat
      601 gcttccggct cgtatgttgt gtggaattgt gagcggataa caatttcaca caggaaacag
      661 ctatgaccat gattacgcca agcgcgcaat taaccctcac taaagggaac aaaagctgga
      721 gctcctagtt agaaaaagac atttttgctg tcagtcactg tcaagagatt cttttgctgg
      781 catttcttct agaagcaaaa agagcgatgc gtcttttccg ctgaaccgtt ccagcaaaaa
      841 agactaccaa cgcaatatgg attgtcagaa tcatataaaa gagaagcaaa taactccttg
      901 tcttgtatca attgcattat aatatcttct tgttagtgca atatcatata gaagtcatcg
      961 actagtggat ccccttaaga tgcaccatca ccatcatcac tctactagtg ccttgaatag
     1021 tagaaggaat tctttagata agagctcaaa caagcagggt atgtcaggct tacctcctat
     1081 ttttggtgga gagagtactt catatcacca cgacaacaaa atacaaaagt acaaccaatt
     1141 aggagtagaa gaagatgatg atgacgagaa tgaccgtttg ctcaaccaaa tgggaaacag
     1201 tgctacaaaa ttcaaaagtt caatatctcc aagatcaatt gatagcattt caagttcttt
     1261 cataaaaagt aggataccta tcagacaacc ataccattac tctcaaccaa ctactgcgcc
     1321 ctttcaagct caggcggaat ttgaagaacc tgcaaataag ttaatcgata atggtaatag
     1381 gagtaatagt taggatatca agcttatcga taccgtcgac ctcgagtcat gtaattagtt
     1441 atgtcacgct tacattcacg ccctcccccc acatccactc taaccgaaaa ggaaggagtt
     1501 agacaacctg aagtctaggt ccctatttat ttttttatag ttatgttagt attaagaacg
     1561 ttatttatat ttcaaatttt tctttttttt ctgtacagac gcgtgtacgc atgtaacatt
     1621 atactgaaaa ccttgcttga gaaggttttg ggacgctcga aggctttaat ttgcggccgg
     1681 tacccaattc gccctatagt gagtcgtatt acgcgcgctc actggccgtc gttttacaac
     1741 gtcgtgactg ggaaaaccct ggcgttaccc aacttaatcg ccttgcagca catccccctt
     1801 tcgccagctg gcgtaatagc gaagaggccc gcaccgatcg cccttcccaa cagttgcgca
     1861 gcctgaatgg cgaatggacg cgccctgtag cggcgcatta agcgcggcgg gtgtggtggt
     1921 tacgcgcagc gtgaccgcta cacttgccag cgccctagcg cccgctcctt tcgctttctt
     1981 cccttccttt ctcgccacgt tcgccggctt tccccgtcaa gctctaaatc gggggctccc
     2041 tttagggttc cgatttagtg ctttacggca cctcgacccc aaaaaacttg attagggtga
     2101 tggttcacgt agtgggccat cgccctgata gacggttttt cgccctttga cgttggagtc
     2161 cacgttcttt aatagtggac tcttgttcca aactggaaca acactcaacc ctatctcggt
     2221 ctattctttt gatttataag ggattttgcc gatttcggcc tattggttaa aaaatgagct
     2281 gatttaacaa aaatttaacg cgaattttaa caaaatatta acgcttacaa tttcctgatg
     2341 cggtattttc tccttacgca tctgtgcggt atttcacacc gcatagatcg gcaagtgcac
     2401 aaacaatact taaataaata ctactcagta ataacctatt tcttagcatt tttgacgaaa
     2461 tttgctattt tgttagagtc ttttacacca tttgtctcca cacctccgct tacatcaaca
     2521 ccaataacgc catttaatct aagcgcatca ccaacatttt ctggcgtcag tccaccagct
     2581 aacataaaat gtaagctttc ggggctctct tgccttccaa cccagtcaga aatcgagttc
     2641 caatccaaaa gttcacctgt cccacctgct tctgaatcaa acaagggaat aaacgaatga
     2701 ggtttctgtg aagctgcact gagtagtatg ttgcagtctt ttggaaatac gagtctttta
     2761 ataactggca aaccgaggaa ctcttggtat tcttgccacg actcatctcc atgcagttgg
     2821 acgatatcaa tgccgtaatc attgaccaga gccaaaacat cctccttagg ttgattacga
     2881 aacacgccaa ccaagtattt cggagtgcct gaactatttt tatatgcttt tacaagactt
     2941 gaaattttcc ttgcaataac cgggtcaatt gttctctttc tattgggcac acatataata
     3001 cccagcaagt cagcatcgga atctagagca cattctgcgg cctctgtgct ctgcaagccg
     3061 caaactttca ccaatggacc agaactacct gtgaaattaa taacagacat actccaagct
     3121 gcctttgtgt gcttaatcac gtatactcac gtgctcaata gtcaccaatg ccctccctct
     3181 tggccctctc cttttctttt ttcgaccgaa ttaattctta atcggcaaaa aaagaaaagc
     3241 tccggatcaa gattgtacgt aaggtgacaa gctatttttc aataaagaat atcttccact
     3301 actgccatct ggcgtcataa ctgcaaagta cacatatatt acgatgctgt tctattaaat
     3361 gcttcctata ttatatatat agtaatgtcg ttgatctatg gtgcactctc agtacaatct
     3421 gctctgatgc cgcatagtta agccagcccc gacacccgcc aacacccgct gacgcgccct
     3481 gacgggcttg tctgctcccg gcatccgctt acagacaagc tgtgaccgtc tccgggagct
     3541 gcatgtgtca gaggttttca ccgtcatcac cgaaacgcgc gagacgaaag ggcctcgtga
     3601 tacgcctatt tttataggtt aatgtcatga taataatggt ttcttagtat gatccaatat
     3661 caaaggaaat gatagcattg aaggatgaga ctaatccaat tgaggagtgg cagcatatag
     3721 aacagctaaa gggtagtgct gaaggaagca tacgataccc cgcatggaat gggataatat
     3781 cacaggaggt actagactac ctttcatcct acataaatag acgcatataa gtacgcattt
     3841 aagcataaac acgcactatg ccgttcttct catgtatata tatatacagg caacacgcag
     3901 atataggtgc gacgtgaaca gtgagctgta tgtgcgcagc tcgcgttgca ttttcggaag
     3961 cgctcgtttt cggaaacgct ttgaagttcc tattccgaag ttcctattct ctagaaagta
     4021 taggaacttc agagcgcttt tgaaaaccaa aagcgctctg aagacgcact ttcaaaaaac
     4081 caaaaacgca ccggactgta acgagctact aaaatattgc gaataccgct tccacaaaca
     4141 ttgctcaaaa gtatctcttt gctatatatc tctgtgctat atccctatat aacctaccca
     4201 tccacctttc gctccttgaa cttgcatcta aactcgacct ctacattttt tatgtttatc
     4261 tctagtatta ctctttagac aaaaaaattg tagtaagaac tattcataga gtgaatcgaa
     4321 aacaatacga aaatgtaaac atttcctata cgtagtatat agagacaaaa tagaagaaac
     4381 cgttcataat tttctgacca atgaagaatc atcaacgcta tcactttctg ttcacaaagt
     4441 atgcgcaatc cacatcggta tagaatataa tcggggatgc ctttatcttg aaaaaatgca
     4501 cccgcagctt cgctagtaat cagtaaacgc gggaagtgga gtcaggcttt ttttatggaa
     4561 gagaaaatag acaccaaagt agccttcttc taaccttaac ggacctacag tgcaaaaagt
     4621 tatcaagaga ctgcattata gagcgcacaa aggagaaaaa aagtaatcta agatgctttg
     4681 ttagaaaaat agcgctctcg ggatgcattt ttgtagaaca aaaaagaagt atagattctt
     4741 tgttggtaaa atagcgctct cgcgttgcat ttctgttctg taaaaatgca gctcagattc
     4801 tttgtttgaa aaattagcgc tctcgcgttg catttttgtt ttacaaaaat gaagcacaga
     4861 ttcttcgttg gtaaaatagc gctttcgcgt tgcatttctg ttctgtaaaa atgcagctca
     4921 gattctttgt ttgaaaaatt agcgctctcg cgttgcattt ttgttctaca aaatgaagca
     4981 cagatgcttc gttcaggtgg cacttttcgg ggaaatgtgc gcggaacccc tatttgttta
     5041 tttttctaaa tacattcaaa tatgtatccg ctcatgagac aataaccctg ataaatgctt
     5101 caataatatt gaaaaaggaa gagtatgagt attcaacatt tccgtgtcgc ccttattccc
     5161 ttttttgcgg cattttgcct tcctgttttt gctcacccag aaacgctggt gaaagtaaaa
     5221 gatgctgaag atcagttggg tgcacgagtg ggttacatcg aactggatct caacagcggt
     5281 aagatccttg agagttttcg ccccgaagaa cgttttccaa tgatgagcac ttttaaagtt
     5341 ctgctatgtg gcgcggtatt atcccgtatt gacgccgggc aagagcaact cggtcgccgc
     5401 atacactatt ctcagaatga cttggttgag tactcaccag tcacagaaaa gcatcttacg
     5461 gatggcatga cagtaagaga attatgcagt gctgccataa ccatgagtga taacactgcg
     5521 gccaacttac ttctgacaac gatcggagga ccgaaggagc taaccgcttt tttgcacaac
     5581 atgggggatc atgtaactcg ccttgatcgt tgggaaccgg agctgaatga agccatacca
     5641 aacgacgagc gtgacaccac gatgcctgta gcaatggcaa caacgttgcg caaactatta
     5701 actggcgaac tacttactct agcttcccgg caacaattaa tagactggat ggaggcggat
     5761 aaagttgcag gaccacttct gcgctcggcc cttccggctg gctggtttat tgctgataaa
     5821 tctggagccg gtgagcgtgg gtctcgcggt atcattgcag cactggggcc agatggtaag
     5881 ccctcccgta tcgtagttat ctacacgacg gggagtcagg caactatgga tgaacgaaat
     5941 agacagatcg ctgagatagg tgcctcactg attaagcatt ggtaactgtc agaccaagtt
     6001 tactcatata tactttagat tgatttaaaa cttcattttt aatttaaaag gatctaggtg
     6061 aagatccttt ttgataatct catgaccaaa atcccttaac gtgagttttc gttccactga
     6121 gcgtcagacc ccgtagaaaa gatcaaagga tcttcttgag atcctttttt tctgcgcgta
     6181 atctgctgct tgcaaacaaa aaaaccaccg ctaccagcgg tggtttgttt gccggatcaa
     6241 gagctaccaa ctctttttcc gaaggtaact ggcttcagca gagcgcagat accaaatact
     6301 gttcttctag tgtagccgta gttaggccac cacttcaaga actctgtagc accgcctaca
     6361 tacctcgctc tgctaatcct gttaccagtg gctgctgcca gtggcgataa gtcgtgtctt
     6421 accgggttgg actcaagacg atagttaccg gataaggcgc agcggtcggg ctgaacggg
//
