LOCUS       sequence-252987-        5685 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Expresses 3xFLAG-ATG3 WT in mammalian cells.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 5685)
  AUTHORS   Agrotis A, von Chamier L, Oliver H, Kiso K, Singh T, Ketteler R
  TITLE     Human ATG4 autophagy proteases counteract attachment of 
            ubiquitin-like LC3/GABARAP proteins to other cellular proteins.
  JOURNAL   J Biol Chem. 2019 Jul 17. pii: AC119.009977. doi: 
            10.1074/jbc.AC119.009977.
  PUBMED    31315929
REFERENCE   2  (bases 1 to 5685)
  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: "J Biol 
            Chem. 2019 Jul 17. pii: AC119.009977. doi: 
            10.1074/jbc.AC119.009977."
COMMENT     Sequence Label: pCMV 3xFLAG-ATG3 WT
FEATURES             Location/Qualifiers
     source          1..5685
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     enhancer        116..495
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
     promoter        496..699
                     /label=CMV promoter
                     /note="human cytomegalovirus (CMV) immediate early 
                     promoter"
     primer_bind     650..670
                     /label=CMV-F
                     /note="Human CMV immediate early promoter, forward primer"
     CDS             736..801
                     /codon_start=1
                     /product="three tandem FLAG(R) epitope tags, followed by an
                     enterokinase cleavage site"
                     /label=3xFLAG
                     /translation="DYKDHDGDYKDHDIDYKDDDDK"
     protein_bind    805..829
                     /gene="mutant version of attB"
                     /label=attB1
                     /bound_moiety="BP Clonase(TM)"
                     /note="recombination site for the Gateway(R) BP reaction"
     protein_bind    complement(1778..1802)
                     /gene="mutant version of attB"
                     /label=attB2
                     /bound_moiety="BP Clonase(TM)"
                     /note="recombination site for the Gateway(R) BP reaction"
     polyA_signal    1820..2442
                     /label=hGH poly(A) signal
                     /note="human growth hormone polyadenylation signal"
     primer_bind     complement(2019..2038)
                     /label=hGH-PA-R
                     /note="Human growth hormone terminator, reverse primer"
     primer_bind     complement(2466..2486)
                     /label=pBABE 3'
                     /note="SV40 enhancer, reverse primer for pBABE vectors"
     promoter        2471..2800
                     /label=SV40 promoter
                     /note="SV40 enhancer and early promoter"
     rep_origin      2651..2786
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     primer_bind     complement(2842..2861)
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        complement(2843..2861)
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     primer_bind     complement(2875..2891)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(2875..2891)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(2888..2910)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    2899..2915
                     /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)."
     promoter        complement(join(2923..2929,2930..2947,2948..2953))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     primer_bind     complement(3106..3123)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(3277..3865)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(3357..3376)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(4036..4827,4828..4896))
                     /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     4659..4678
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(4897..5001)
                     /gene="bla"
                     /label=AmpR promoter
     rep_origin      5028..5483
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(5115..5134)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     5325..5346
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     5609..5631
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     primer_bind     5623..5640
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     5624..5640
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
ORIGIN
        1 ttagtcattg gttatatagc ataaatcaat attggctatt ggccattgca tacgttgtat
       61 ctatatcata atatgtacat ttatattggc tcatgtccaa tatgaccgcc atgttgacat
      121 tgattattga ctagttatta atagtaatca attacggggt cattagttca tagcccatat
      181 atggagttcc gcgttacata acttacggta aatggcccgc ctggctgacc gcccaacgac
      241 ccccgcccat tgacgtcaat aatgacgtat gttcccatag taacgccaat agggactttc
      301 cattgacgtc aatgggtgga gtatttacgg taaactgccc acttggcagt acatcaagtg
      361 tatcatatgc caagtccgcc ccctattgac gtcaatgacg gtaaatggcc cgcctggcat
      421 tatgcccagt acatgacctt acgggacttt cctacttggc agtacatcta cgtattagtc
      481 atcgctatta ccatggtgat gcggttttgg cagtacacca atgggcgtgg atagcggttt
      541 gactcacggg gatttccaag tctccacccc attgacgtca atgggagttt gttttggcac
      601 caaaatcaac gggactttcc aaaatgtcgt aataaccccg ccccgttgac gcaaatgggc
      661 ggtaggcgtg tacggtggga ggtctatata agcagagctc gtttagtgaa ccgtcagaat
      721 tgatctggta ccatggacta caaagaccat gacggtgatt ataaagatca tgacatcgat
      781 tacaaggatg acgatgacaa gcttacaagt ttgtacaaaa aagcaggctt aatgcagaat
      841 gtgattaata ctgtgaaggg aaaggcactg gaagtggctg agtacctgac cccggtcctc
      901 aaggaatcaa agtttaagga aacaggtgta attaccccag aagagtttgt ggcagctgga
      961 gatcacctag tccaccactg tccaacatgg caatgggcta caggggaaga attgaaagtg
     1021 aaggcatacc taccaacagg caaacaattt ttggtaacca aaaatgtgcc gtgctataag
     1081 cggtgcaaac agatggaata ttcagatgaa ttggaagcta tcattgaaga agatgatggt
     1141 gatggcggat gggtagatac atatcacaac acaggtatta caggaataac ggaagccgtt
     1201 aaagagatca cactggaaaa taaggacaat ataaggcttc aagattgctc agcactatgt
     1261 gaagaggaag aagatgaaga tgaaggagaa gctgcagata tggaagaata tgaagagagt
     1321 ggattgttgg aaacagatga ggctacccta gatacaagga aaatagtaga agcttgtaaa
     1381 gccaaaactg atgctggcgg tgaagatgct attttgcaaa ccagaactta tgacctttac
     1441 atcacttatg ataaatatta ccagactcca cgattatggt tgtttggcta tgatgagcaa
     1501 cggcagcctt taacagttga gcacatgtat gaagacatca gtcaggatca tgtgaagaaa
     1561 acagtgacca ttgaaaatca ccctcatctg ccaccacctc ccatgtgttc agttcaccca
     1621 tgcaggcatg ctgaggtgat gaagaaaatc attgagactg ttgcagaagg agggggagaa
     1681 cttggagttc atatgtatct tcttattttc ttgaaatttg tacaagctgt cattccaaca
     1741 atagaatatg actacacaag acacttcaca atgtaatacc cagctttctt gtacaaagtg
     1801 gtgtgatcta gaggatcccg ggtggcatcc ctgtgacccc tccccagtgc ctctcctggc
     1861 cctggaagtt gccactccag tgcccaccag ccttgtccta ataaaattaa gttgcatcat
     1921 tttgtctgac taggtgtcct tctataatat tatggggtgg aggggggtgg tatggagcaa
     1981 ggggcaagtt gggaagacaa cctgtagggc ctgcggggtc tattgggaac caagctggag
     2041 tgcagtggca caatcttggc tcactgcaat ctccgcctcc tgggttcaag cgattctcct
     2101 gcctcagcct cccgagttgt tgggattcca ggcatgcatg accaggctca gctaattttt
     2161 gtttttttgg tagagacggg gtttcaccat attggccagg ctggtctcca actcctaatc
     2221 tcaggtgatc tacccacctt ggcctcccaa attgctggga ttacaggcgt gaaccactgc
     2281 tcccttccct gtccttctga ttttaaaata actataccag caggaggacg tccagacaca
     2341 gcataggcta cctggccatg cccaaccggt gggacatttg agttgcttgc ttggcactgt
     2401 cctctcatgc gttgggtcca ctcagtagat gcctgttgaa ttgggtacgc ggccagcttg
     2461 gctgtggaat gtgtgtcagt tagggtgtgg aaagtcccca ggctccccag caggcagaag
     2521 tatgcaaagc atgcatctca attagtcagc aaccaggtgt ggaaagtccc caggctcccc
     2581 agcaggcaga agtatgcaaa gcatgcatct caattagtca gcaaccatag tcccgcccct
     2641 aactccgccc atcccgcccc taactccgcc cagttccgcc cattctccgc cccatggctg
     2701 actaattttt tttatttatg cagaggccga ggccgcctcg gcctctgagc tattccagaa
     2761 gtagtgagga ggcttttttg gaggcctagg cttttgcaaa aagctcctcg atcgaggaac
     2821 tgaaaaacca gaaagttaat tccctatagt gagtcgtatt aaattcgtaa tcatgtcata
     2881 gctgtttcct gtgtgaaatt gttatccgct cacaattcca cacaacatac gagccggaag
     2941 cataaagtgt aaagcctggg gtgcctaatg agcgagctaa ctcacattaa ttgcgttgcg
     3001 ctcactgccc gctttccagt cgggaaacct gtcgtgccag ctgcattaat gaatcggcca
     3061 acgcgcgggg agaggcggtt tgcgtattgg gcgctcttcc gcttcctcgc tcactgactc
     3121 gctgcgctcg gtcgttcggc tgcggcgagc ggtatcagct cactcaaagg cggtaatacg
     3181 gttatccaca gaatcagggg ataacgcagg aaagaacatg tgagcaaaag gccagcaaaa
     3241 ggccaggaac cgtaaaaagg ccgcgttgct ggcgtttttc cataggctcc gcccccctga
     3301 cgagcatcac aaaaatcgac gctcaagtca gaggtggcga aacccgacag gactataaag
     3361 ataccaggcg tttccccctg gaagctccct cgtgcgctct cctgttccga ccctgccgct
     3421 taccggatac ctgtccgcct ttctcccttc gggaagcgtg gcgctttctc atagctcacg
     3481 ctgtaggtat ctcagttcgg tgtaggtcgt tcgctccaag ctgggctgtg tgcacgaacc
     3541 ccccgttcag cccgaccgct gcgccttatc cggtaactat cgtcttgagt ccaacccggt
     3601 aagacacgac ttatcgccac tggcagcagc cactggtaac aggattagca gagcgaggta
     3661 tgtaggcggt gctacagagt tcttgaagtg gtggcctaac tacggctaca ctagaagaac
     3721 agtatttggt atctgcgctc tgctgaagcc agttaccttc ggaaaaagag ttggtagctc
     3781 ttgatccggc aaacaaacca ccgctggtag cggtggtttt tttgtttgca agcagcagat
     3841 tacgcgcaga aaaaaaggat ctcaagaaga tcctttgatc ttttctacgg ggtctgacgc
     3901 tcagtggaac gaaaactcac gttaagggat tttggtcatg agattatcaa aaaggatctt
     3961 cacctagatc cttttaaatt aaaaatgaag ttttaaatca atctaaagta tatatgagta
     4021 aacttggtct gacagttacc aatgcttaat cagtgaggca cctatctcag cgatctgtct
     4081 atttcgttca tccatagttg cctgactccc cgtcgtgtag ataactacga tacgggaggg
     4141 cttaccatct ggccccagtg ctgcaatgat accgcgagac ccacgctcac cggctccaga
     4201 tttatcagca ataaaccagc cagccggaag ggccgagcgc agaagtggtc ctgcaacttt
     4261 atccgcctcc atccagtcta ttaattgttg ccgggaagct agagtaagta gttcgccagt
     4321 taatagtttg cgcaacgttg ttgccattgc tacaggcatc gtggtgtcac gctcgtcgtt
     4381 tggtatggct tcattcagct ccggttccca acgatcaagg cgagttacat gatcccccat
     4441 gttgtgcaaa aaagcggtta gctccttcgg tcctccgatc gttgtcagaa gtaagttggc
     4501 cgcagtgtta tcactcatgg ttatggcagc actgcataat tctcttactg tcatgccatc
     4561 cgtaagatgc ttttctgtga ctggtgagta ctcaaccaag tcattctgag aatagtgtat
     4621 gcggcgaccg agttgctctt gcccggcgtc aatacgggat aataccgcgc cacatagcag
     4681 aactttaaaa gtgctcatca ttggaaaacg ttcttcgggg cgaaaactct caaggatctt
     4741 accgctgttg agatccagtt cgatgtaacc cactcgtgca cccaactgat cttcagcatc
     4801 ttttactttc accagcgttt ctgggtgagc aaaaacagga aggcaaaatg ccgcaaaaaa
     4861 gggaataagg gcgacacgga aatgttgaat actcatactc ttcctttttc aatattattg
     4921 aagcatttat cagggttatt gtctcatgag cggatacata tttgaatgta tttagaaaaa
     4981 taaacaaata ggggttccgc gcacatttcc ccgaaaagtg ccacctgacg cgccctgtag
     5041 cggcgcatta agcgcggcgg gtgtggtggt tacgcgcagc gtgaccgcta cacttgccag
     5101 cgccctagcg cccgctcctt tcgctttctt cccttccttt ctcgccacgt tcgccggctt
     5161 tccccgtcaa gctctaaatc gggggctccc tttagggttc cgatttagtg ctttacggca
     5221 cctcgacccc aaaaaacttg attagggtga tggttcacgt agtgggccat cgccctgata
     5281 gacggttttt cgccctttga cgttggagtc cacgttcttt aatagtggac tcttgttcca
     5341 aactggaaca acactcaacc ctatctcggt ctattctttt gatttataag ggattttgcc
     5401 gatttcggcc tattggttaa aaaatgagct gatttaacaa aaatttaacg cgaattttaa
     5461 caaaatatta acgcttacaa tttccattcg ccattcaggc tgcgcaactg ttgggaaggg
     5521 cgatcggtgc gggcctcttc gctattacgc cagctggcga aagggggatg tgctgcaagg
     5581 cgattaagtt gggtaacgcc agggttttcc cagtcacgac gttgtaaaac gacggccagt
     5641 gccaagctga tctatacatt gaatcaatat tggcaattag ccata
//
