LOCUS       sequence-401482-        5735 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  ATG3 mutant overexpression in E.Coli. 
            6His-TEVsite-ATG3-K62D/K64D-stop.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 5735)
  AUTHORS   Rao S, Skulsuppaisarn M, Strong LM, Ren X, Lazarou M, Hurley JH, 
            Hummer G
  TITLE     Three-step docking by WIPI2, ATG16L1, and ATG3 delivers LC3 to the 
            phagophore.
  JOURNAL   Sci Adv. 2024 Feb 9;10(6):eadj8027. doi: 10.1126/sciadv.adj8027. 
            Epub 2024 Feb 7.
  PUBMED    38324698
REFERENCE   2  (bases 1 to 5735)
  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"; journalName: "Sci Adv."; 
            date: "2024-02-9"; volume: "10(6):eadj8027. doi"; pages: " 
            10.1126/sciadv.adj8027. Epub 2024 Feb 7"
COMMENT     Sequence Label: His-ATG3-K62D/K64D
FEATURES             Location/Qualifiers
     source          1..5735
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     1423..1442
                     /label=pBRrevBam
                     /note="pBR322 vectors, tet region, downstream of BamHI, 
                     reverse primer"
     primer_bind     1526..1545
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        1526..1544
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     RBS             1633..1655
                     /note="efficient ribosome binding site from bacteriophage 
                     T7 gene 10 (Olins and Rangwala, 1989)"
     CDS             1675..1692
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     CDS             1693..1713
                     /codon_start=1
                     /product="tobacco etch virus (TEV) protease recognition and
                     cleavage site"
                     /label=TEV site
                     /translation="ENLYFQS"
     primer_bind     complement(2790..2808)
                     /label=T7 Term
                     /note="T7 terminator, reverse primer"
     terminator      2804..2851
                     /label=T7 terminator
                     /note="transcription terminator for bacteriophage T7 RNA 
                     polymerase"
     promoter        complement(join(3217..3222,3223..3239,3240..3245))
                     /label=tet promoter
                     /note="E. coli promoter for tetracycline efflux protein 
                     gene"
     primer_bind     complement(3272..3290)
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     promoter        3358..3462
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(3463..3531,3532..4323)
                     /codon_start=1
                     /gene="bla"
                     /product="beta-lactamase"
                     /label=AmpR
                     /note="confers resistance to ampicillin, carbenicillin, and
                     related antibiotics"
                     /translation="MSIQHFRVALIPFFAAFCLPVFAHPETLVKVKDAEDQLGARVGYI
                     ELDLNSGKILESFRPEERFPMMSTFKVLLCGAVLSRVDAGQEQLGRRIHYSQNDLVEYS
                     PVTEKHLTDGMTVRELCSAAITMSDNTAANLLLTTIGGPKELTAFLHNMGDHVTRLDRW
                     EPELNEAIPNDERDTTMPAAMATTLRKLLTGELLTLASRQQLIDWMEADKVAGPLLRSA
                     LPAGWFIADKSGAGERGSRGIIAALGPDGKPSRIVVIYTTGSQATMDERNRQIAEIGAS
                     LIKHW"
     primer_bind     complement(3681..3700)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      4494..5082
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     4983..5002
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     5236..5253
                     /label=L4440
                     /note="L4440 vector, forward primer"
     misc_feature    5268..5407
                     /label=bom
                     /note="basis of mobility region from pBR322"
     primer_bind     complement(5333..5352)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     5493..5515
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     CDS             complement(5509..5700)
                     /codon_start=1
                     /gene="rop"
                     /product="Rop protein, which maintains plasmids at low copy
                     number"
                     /label=rop
                     /translation="MTKQEKTALNMARFIRSQTLTLLEKLNELDADEQADICESLHDHA
                     DELYRSCLARFGDDGENL"
ORIGIN
        1 gggtaatgat accgatgaaa cgagagagga tgctcacgat acgggttact gatgatgaac
       61 atgcccggtt actggaacgt tgtgagggta aacaactggc ggtatggatg cggcgggacc
      121 agagaaaaat cactcagggt caatgccagc gcttcgttaa tacagatgta ggtgttccac
      181 agggtagcca gcagcatcct gcgatgcaga tccggaacat aatggtgcag ggcgctgact
      241 tccgcgtttc cagactttac gaaacacgga aaccgaagac cattcatgtt gttgctcagg
      301 tcgcagacgt tttgcagcag cagtcgcttc acgttcgctc gcgtatcggt gattcattct
      361 gctaaccagt aaggcaaccc cgccagccta gccgggtcct caacgacagg agcacgatca
      421 tgcgcacccg tggccaggac ccaacgctgc ccgagatgcg ccgcgtgcgg ctgctggaga
      481 tggcggacgc gatggatatg ttctgccaag ggttggtttg cgcattcaca gttctccgca
      541 agaattgatt ggctccaatt cttggagtgg tgaatccgtt agcgaggtgc cgccggcttc
      601 cattcaggtc gaggtggccc ggctccatgc accgcgacgc aacgcgggga ggcagacaag
      661 gtatagggcg gcgcctacaa tccatgccaa cccgttccat gtgctcgccg aggcggcata
      721 aatcgccgtg acgatcagcg gtccaatgat cgaagttagg ctggtaagag ccgcgagcga
      781 tccttgaagc tgtccctgat ggtcgtcatc tacctgcctg gacagcatgg cctgcaacgc
      841 gggcatcccg atgccgccgg aagcgagaag aatcataatg gggaaggcca tccagcctcg
      901 cgtcgcgaac gccagcaaga cgtagcccag cgcgtcggcc gccatgccgg cgataatggc
      961 ctgcttctcg ccgaaacgtt tggtggcggg accagtgacg aaggcttgag cgagggcgtg
     1021 caagattccg aataccgcaa gcgacaggcc gatcatcgtc gcgctccagc gaaagcggtc
     1081 ctcgccgaaa atgacccaga gcgctgccgg cacctgtcct acgagttgca tgataaagaa
     1141 gacagtcata agtgcggcga cgatagtcat gccccgcgcc caccggaagg agctgactgg
     1201 gttgaaggct ctcaagggca tcggtcgacg ctctccctta tgcgactcct gcattaggaa
     1261 gcagcccagt agtaggttga ggccgttgag caccgccgcc gcaaggaatg gtgcatgcaa
     1321 ggagatggcg cccaacagtc ccccggccac ggggcctgcc accataccca cgccgaaaca
     1381 agcgctcatg agcccgaagt ggcgagcccg atcttcccca tcggtgatgt cggcgatata
     1441 ggcgccagca accgcacctg tggcgccggt gatgccggcc acgatgcgtc cggcgtagag
     1501 gatcgagatc tcgatcccgc gaaattaata cgactcacta tagggagacc acaacggttt
     1561 ccctctagtg ccggctccgg agagctcttt aattaagcgg ccgccctgca ggactcgagt
     1621 tctagaaata attttgttta actttaagaa ggagatatac atatgaaatc ttctcaccat
     1681 caccatcacc atgaaaacct gtacttccaa tccaatgcaa tgcagaatgt gattaatact
     1741 gtgaagggaa aggcactgga agtggctgag tacctgaccc cggtcctcaa ggaatcaaag
     1801 tttaaggaaa caggtgtaat taccccagaa gagtttgtgg cagctggaga tcacctagtc
     1861 caccactgtc caacatggca atgggctaca ggggaagaat tggacgtgga cgcataccta
     1921 ccaacaggca aacaattttt ggtaaccaaa aatgtgccgt gctataagcg gtgcaaacag
     1981 atggaatatt cagatgaatt ggaagctatc attgaagaag atgatggtga tggcggatgg
     2041 gtagatacat atcacaacac aggtattaca ggaataacgg aagccgttaa agagatcaca
     2101 ctggaaaata aggacaatat aaggcttcaa gattgctcag cactatgtga agaggaagaa
     2161 gatgaagatg aaggagaagc tgcagatatg gaagaatatg aagagagtgg attgttggaa
     2221 acagatgagg ctaccctaga tacaaggaaa atagtagaag cttgtaaagc caaaactgat
     2281 gctggcggtg aagatgctat tttgcaaacc agaacttatg acctttacat cacttatgat
     2341 aaatattacc agactccacg attatggttg tttggctatg atgagcaacg gcagccttta
     2401 acagttgagc acatgtatga agacatcagt caggatcatg tgaagaaaac agtgaccatt
     2461 gaaaatcacc ctcatctgcc accacctccc atgtgttcag ttcacccatg caggcatgct
     2521 gaggtgatga agaaaatcat tgagactgtt gcagaaggag ggggagaact tggagttcat
     2581 atgtatcttc ttattttctt gaaatttgta caagctgtca ttccaacaat agaatatgac
     2641 tacacaagac acttcacaat gtaataacat tggaagtgga taacggatcc gcgatcgcgg
     2701 cgcgccacct ggtggccggc cggtaccacg cgtgcgcgct gatccggctg ctaacaaagc
     2761 ccgaaaggaa gctgagttgg ctgctgccac cgctgagcaa taactagcat aaccccttgg
     2821 ggcctctaaa cgggtcttga ggggtttttt gctgaaagga ggaactatat ccggatatcc
     2881 acaggacggg tgtggtcgcc atgatcgcgt agtcgatagt ggctccaagt agcgaagcga
     2941 gcaggactgg gcggcggcca aagcggtcgg acagtgctcc gagaacgggt gcgcatagaa
     3001 attgcatcaa cgcatatagc gctagcagca cgccatagtg actggcgatg ctgtcggaat
     3061 ggacgatatc ccgcaagagg cccggcagta ccggcataac caagcctatg cctacagcat
     3121 ccagggtgac ggtgccgagg atgacgatga gcgcattgtt agatttcata cacggtgcct
     3181 gactgcgtta gcaatttaac tgtgataaac taccgcatta aagcttatcg atgataagct
     3241 gtcaaacatg agaattcttg aagacgaaag ggcctcgtga tacgcctatt tttataggtt
     3301 aatgtcatga taataatggt ttcttagacg tcaggtggca cttttcgggg aaatgtgcgc
     3361 ggaaccccta tttgtttatt tttctaaata cattcaaata tgtatccgct catgagacaa
     3421 taaccctgat aaatgcttca ataacattga aaaaggaaga gtatgagtat tcaacatttc
     3481 cgtgtcgccc ttattccctt ttttgcggca ttttgccttc ctgtttttgc tcacccagaa
     3541 acgctggtga aagtaaaaga tgctgaagat cagttgggtg cacgagtggg ttacatcgaa
     3601 ctggatctca acagcggtaa gatccttgag agttttcgcc ccgaagaacg ttttccaatg
     3661 atgagcactt ttaaagttct gctatgtggc gcggtattat cccgtgttga cgccgggcaa
     3721 gagcaactcg gtcgccgcat acactattct cagaatgact tggttgagta ctcaccagtc
     3781 acagaaaagc atcttacgga tggcatgaca gtaagagaat tatgcagtgc tgccataacc
     3841 atgagtgata acactgcggc caacttactt ctgacaacga tcggaggacc gaaggagcta
     3901 accgcttttt tgcacaacat gggggatcat gtaactcgcc ttgatcgttg ggaaccggag
     3961 ctgaatgaag ccataccaaa cgacgagcgt gacaccacga tgcctgcagc aatggcaaca
     4021 acgttgcgca aactattaac tggcgaacta cttactctag cttcccggca acaattaata
     4081 gactggatgg aggcggataa agttgcagga ccacttctgc gctcggccct tccggctggc
     4141 tggtttattg ctgataaatc tggagccggt gagcgtgggt ctcgcggtat cattgcagca
     4201 ctggggccag atggtaagcc ctcccgtatc gtagttatct acacgacggg gagtcaggca
     4261 actatggatg aacgaaatag acagatcgct gagataggtg cctcactgat taagcattgg
     4321 taactgtcag accaagttta ctcatatata ctttagattg atttaaaact tcatttttaa
     4381 tttaaaagga tctaggtgaa gatccttttt gataatctca tgaccaaaat cccttaacgt
     4441 gagttttcgt tccactgagc gtcagacccc gtagaaaaga tcaaaggatc ttcttgagat
     4501 cctttttttc tgcgcgtaat ctgctgcttg caaacaaaaa aaccaccgct accagcggtg
     4561 gtttgtttgc cggatcaaga gctaccaact ctttttccga aggtaactgg cttcagcaga
     4621 gcgcagatac caaatactgt ccttctagtg tagccgtagt taggccacca cttcaagaac
     4681 tctgtagcac cgcctacata cctcgctctg ctaatcctgt taccagtggc tgctgccagt
     4741 ggcgataagt cgtgtcttac cgggttggac tcaagacgat agttaccgga taaggcgcag
     4801 cggtcgggct gaacgggggg ttcgtgcaca cagcccagct tggagcgaac gacctacacc
     4861 gaactgagat acctacagcg tgagctatga gaaagcgcca cgcttcccga agggagaaag
     4921 gcggacaggt atccggtaag cggcagggtc ggaacaggag agcgcacgag ggagcttcca
     4981 gggggaaacg cctggtatct ttatagtcct gtcgggtttc gccacctctg acttgagcgt
     5041 cgatttttgt gatgctcgtc aggggggcgg agcctatgga aaaacgccag caacgcggcc
     5101 tttttacggt tcctggcctt ttgctggcct tttgctcaca tgttctttcc tgcgttatcc
     5161 cctgattctg tggataaccg tattaccgcc tttgagtgag ctgataccgc tcgccgcagc
     5221 cgaacgaccg agcgcagcga gtcagtgagc gaggaagcgg aagagcgcct gatgcggtat
     5281 tttctcctta cgcatctgtg cggtatttca caccgcaatg gtgcactctc agtacaatct
     5341 gctctgatgc cgcatagtta agccagtata cactccgcta tcgctacgtg actgggtcat
     5401 ggctgcgccc cgacacccgc caacacccgc tgacgcgccc tgacgggctt gtctgctccc
     5461 ggcatccgct tacagacaag ctgtgaccgt ctccgggagc tgcatgtgtc agaggttttc
     5521 accgtcatca ccgaaacgcg cgaggcagct gcggtaaagc tcatcagcgt ggtcgtgaag
     5581 cgattcacag atgtctgcct gttcatccgc gtccagctcg ttgagtttct ccagaagcgt
     5641 taatgtctgg cttctgataa agcgggccat gttaagggcg gttttttcct gtttggtcac
     5701 tgatgcctcc gtgtaagggg gatttctgtt catgg
//
