LOCUS       sequence-401346-        5738 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  ATG3 mutant overexpression in E.Coli. 6His-TEVsite-ATG3-H266A-stop.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 5738)
  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 5738)
  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-H266A
FEATURES             Location/Qualifiers
     source          1..5738
                     /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(2793..2811)
                     /label=T7 Term
                     /note="T7 terminator, reverse primer"
     terminator      2807..2854
                     /label=T7 terminator
                     /note="transcription terminator for bacteriophage T7 RNA 
                     polymerase"
     promoter        complement(join(3220..3225,3226..3242,3243..3248))
                     /label=tet promoter
                     /note="E. coli promoter for tetracycline efflux protein 
                     gene"
     primer_bind     complement(3275..3293)
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     promoter        3361..3465
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(3466..3534,3535..4326)
                     /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(3684..3703)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      4497..5085
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     4986..5005
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     5239..5256
                     /label=L4440
                     /note="L4440 vector, forward primer"
     misc_feature    5271..5410
                     /label=bom
                     /note="basis of mobility region from pBR322"
     primer_bind     complement(5336..5355)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     5496..5518
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     CDS             complement(5512..5703)
                     /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 tgaaagtgaa ggcataccta
     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 cagggcagct
     2521 gaggtgatga agaaaatcat tgagactgtt gcagaaggag ggggagaact tggagttcat
     2581 atgtatcttc ttattttctt gaaatttgta caagctgtca ttccaacaat agaatatgac
     2641 tacacaagac acttcacaat gtaataataa cattggaagt ggataacgga tccgcgatcg
     2701 cggcgcgcca cctggtggcc ggccggtacc acgcgtgcgc gctgatccgg ctgctaacaa
     2761 agcccgaaag gaagctgagt tggctgctgc caccgctgag caataactag cataacccct
     2821 tggggcctct aaacgggtct tgaggggttt tttgctgaaa ggaggaacta tatccggata
     2881 tccacaggac gggtgtggtc gccatgatcg cgtagtcgat agtggctcca agtagcgaag
     2941 cgagcaggac tgggcggcgg ccaaagcggt cggacagtgc tccgagaacg ggtgcgcata
     3001 gaaattgcat caacgcatat agcgctagca gcacgccata gtgactggcg atgctgtcgg
     3061 aatggacgat atcccgcaag aggcccggca gtaccggcat aaccaagcct atgcctacag
     3121 catccagggt gacggtgccg aggatgacga tgagcgcatt gttagatttc atacacggtg
     3181 cctgactgcg ttagcaattt aactgtgata aactaccgca ttaaagctta tcgatgataa
     3241 gctgtcaaac atgagaattc ttgaagacga aagggcctcg tgatacgcct atttttatag
     3301 gttaatgtca tgataataat ggtttcttag acgtcaggtg gcacttttcg gggaaatgtg
     3361 cgcggaaccc ctatttgttt atttttctaa atacattcaa atatgtatcc gctcatgaga
     3421 caataaccct gataaatgct tcaataacat tgaaaaagga agagtatgag tattcaacat
     3481 ttccgtgtcg cccttattcc cttttttgcg gcattttgcc ttcctgtttt tgctcaccca
     3541 gaaacgctgg tgaaagtaaa agatgctgaa gatcagttgg gtgcacgagt gggttacatc
     3601 gaactggatc tcaacagcgg taagatcctt gagagttttc gccccgaaga acgttttcca
     3661 atgatgagca cttttaaagt tctgctatgt ggcgcggtat tatcccgtgt tgacgccggg
     3721 caagagcaac tcggtcgccg catacactat tctcagaatg acttggttga gtactcacca
     3781 gtcacagaaa agcatcttac ggatggcatg acagtaagag aattatgcag tgctgccata
     3841 accatgagtg ataacactgc ggccaactta cttctgacaa cgatcggagg accgaaggag
     3901 ctaaccgctt ttttgcacaa catgggggat catgtaactc gccttgatcg ttgggaaccg
     3961 gagctgaatg aagccatacc aaacgacgag cgtgacacca cgatgcctgc agcaatggca
     4021 acaacgttgc gcaaactatt aactggcgaa ctacttactc tagcttcccg gcaacaatta
     4081 atagactgga tggaggcgga taaagttgca ggaccacttc tgcgctcggc ccttccggct
     4141 ggctggttta ttgctgataa atctggagcc ggtgagcgtg ggtctcgcgg tatcattgca
     4201 gcactggggc cagatggtaa gccctcccgt atcgtagtta tctacacgac ggggagtcag
     4261 gcaactatgg atgaacgaaa tagacagatc gctgagatag gtgcctcact gattaagcat
     4321 tggtaactgt cagaccaagt ttactcatat atactttaga ttgatttaaa acttcatttt
     4381 taatttaaaa ggatctaggt gaagatcctt tttgataatc tcatgaccaa aatcccttaa
     4441 cgtgagtttt cgttccactg agcgtcagac cccgtagaaa agatcaaagg atcttcttga
     4501 gatccttttt ttctgcgcgt aatctgctgc ttgcaaacaa aaaaaccacc gctaccagcg
     4561 gtggtttgtt tgccggatca agagctacca actctttttc cgaaggtaac tggcttcagc
     4621 agagcgcaga taccaaatac tgtccttcta gtgtagccgt agttaggcca ccacttcaag
     4681 aactctgtag caccgcctac atacctcgct ctgctaatcc tgttaccagt ggctgctgcc
     4741 agtggcgata agtcgtgtct taccgggttg gactcaagac gatagttacc ggataaggcg
     4801 cagcggtcgg gctgaacggg gggttcgtgc acacagccca gcttggagcg aacgacctac
     4861 accgaactga gatacctaca gcgtgagcta tgagaaagcg ccacgcttcc cgaagggaga
     4921 aaggcggaca ggtatccggt aagcggcagg gtcggaacag gagagcgcac gagggagctt
     4981 ccagggggaa acgcctggta tctttatagt cctgtcgggt ttcgccacct ctgacttgag
     5041 cgtcgatttt tgtgatgctc gtcagggggg cggagcctat ggaaaaacgc cagcaacgcg
     5101 gcctttttac ggttcctggc cttttgctgg ccttttgctc acatgttctt tcctgcgtta
     5161 tcccctgatt ctgtggataa ccgtattacc gcctttgagt gagctgatac cgctcgccgc
     5221 agccgaacga ccgagcgcag cgagtcagtg agcgaggaag cggaagagcg cctgatgcgg
     5281 tattttctcc ttacgcatct gtgcggtatt tcacaccgca atggtgcact ctcagtacaa
     5341 tctgctctga tgccgcatag ttaagccagt atacactccg ctatcgctac gtgactgggt
     5401 catggctgcg ccccgacacc cgccaacacc cgctgacgcg ccctgacggg cttgtctgct
     5461 cccggcatcc gcttacagac aagctgtgac cgtctccggg agctgcatgt gtcagaggtt
     5521 ttcaccgtca tcaccgaaac gcgcgaggca gctgcggtaa agctcatcag cgtggtcgtg
     5581 aagcgattca cagatgtctg cctgttcatc cgcgtccagc tcgttgagtt tctccagaag
     5641 cgttaatgtc tggcttctga taaagcgggc catgttaagg gcggtttttt cctgtttggt
     5701 cactgatgcc tccgtgtaag ggggatttct gttcatgg
//
