LOCUS       sequence-253002-        5685 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Expresses pCMV 3xFLAG-ATG3 14-22 KR (last nine lysines mutated to 
            arginine) 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 14-22 KR
FEATURES             Location/Qualifiers
     source          1..5685
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     4..26
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     primer_bind     18..35
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     19..35
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     enhancer        196..575
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
     promoter        576..779
                     /label=CMV promoter
                     /note="human cytomegalovirus (CMV) immediate early 
                     promoter"
     primer_bind     730..750
                     /label=CMV-F
                     /note="Human CMV immediate early promoter, forward primer"
     CDS             816..881
                     /codon_start=1
                     /product="three tandem FLAG(R) epitope tags, followed by an
                     enterokinase cleavage site"
                     /label=3xFLAG
                     /translation="DYKDHDGDYKDHDIDYKDDDDK"
     protein_bind    885..909
                     /gene="mutant version of attB"
                     /label=attB1
                     /bound_moiety="BP Clonase(TM)"
                     /note="recombination site for the Gateway(R) BP reaction"
     protein_bind    complement(1858..1882)
                     /gene="mutant version of attB"
                     /label=attB2
                     /bound_moiety="BP Clonase(TM)"
                     /note="recombination site for the Gateway(R) BP reaction"
     polyA_signal    1900..2522
                     /label=hGH poly(A) signal
                     /note="human growth hormone polyadenylation signal"
     primer_bind     complement(2099..2118)
                     /label=hGH-PA-R
                     /note="Human growth hormone terminator, reverse primer"
     primer_bind     complement(2546..2566)
                     /label=pBABE 3'
                     /note="SV40 enhancer, reverse primer for pBABE vectors"
     promoter        2551..2880
                     /label=SV40 promoter
                     /note="SV40 enhancer and early promoter"
     rep_origin      2731..2866
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     primer_bind     complement(2922..2941)
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        complement(2923..2941)
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     primer_bind     complement(2955..2971)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(2955..2971)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(2968..2990)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    2979..2995
                     /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(3003..3009,3010..3027,3028..3033))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     primer_bind     complement(3186..3203)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(3357..3945)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(3437..3456)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(4116..4907,4908..4976))
                     /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     4739..4758
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(4977..5081)
                     /gene="bla"
                     /label=AmpR promoter
     rep_origin      5108..5563
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(5195..5214)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     5405..5426
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
ORIGIN
        1 tttcccagtc acgacgttgt aaaacgacgg ccagtgccaa gctgatctat acattgaatc
       61 aatattggca attagccata ttagtcattg gttatatagc ataaatcaat attggctatt
      121 ggccattgca tacgttgtat ctatatcata atatgtacat ttatattggc tcatgtccaa
      181 tatgaccgcc atgttgacat tgattattga ctagttatta atagtaatca attacggggt
      241 cattagttca tagcccatat atggagttcc gcgttacata acttacggta aatggcccgc
      301 ctggctgacc gcccaacgac ccccgcccat tgacgtcaat aatgacgtat gttcccatag
      361 taacgccaat agggactttc cattgacgtc aatgggtgga gtatttacgg taaactgccc
      421 acttggcagt acatcaagtg tatcatatgc caagtccgcc ccctattgac gtcaatgacg
      481 gtaaatggcc cgcctggcat tatgcccagt acatgacctt acgggacttt cctacttggc
      541 agtacatcta cgtattagtc atcgctatta ccatggtgat gcggttttgg cagtacacca
      601 atgggcgtgg atagcggttt gactcacggg gatttccaag tctccacccc attgacgtca
      661 atgggagttt gttttggcac caaaatcaac gggactttcc aaaatgtcgt aataaccccg
      721 ccccgttgac gcaaatgggc ggtaggcgtg tacggtggga ggtctatata agcagagctc
      781 gtttagtgaa ccgtcagaat tgatctggta ccatggacta caaagaccat gacggtgatt
      841 ataaagatca tgacatcgat tacaaggatg acgatgacaa gcttacaagt ttgtacaaaa
      901 aagcaggctt aatgcagaat gtgattaata ctgtgaaggg aaaggcactg gaagtggctg
      961 agtacctgac cccggtcctc aaggaatcaa agtttaagga aacaggtgta attaccccag
     1021 aagagtttgt ggcagctgga gatcacctag tccaccactg tccaacatgg caatgggcta
     1081 caggggaaga attgaaagtg aaggcatacc taccaacagg caaacaattt ttggtaacca
     1141 aaaatgtgcc gtgctataag cggtgcaaac agatggaata ttcagatgaa ttggaagcta
     1201 tcattgaaga agatgatggt gatggcggat gggtagatac atatcacaac acaggtatta
     1261 caggaataac ggaagccgtt aaagagatca cactggaaaa taaggacaat ataaggcttc
     1321 aagattgctc agcactatgt gaagaggaag aagatgaaga tgaaggagaa gctgcagata
     1381 tggaagaata tgaagagagt ggattgttgg aaacagatga ggctacccta gatacaagga
     1441 gaatagtaga agcttgtaga gccagaactg atgctggcgg tgaagatgct attttgcaaa
     1501 ccagaactta tgacctttac atcacttatg atagatatta ccagactcca cgattatggt
     1561 tgtttggcta tgatgagcaa cggcagcctt taacagttga gcacatgtat gaagacatca
     1621 gtcaggatca tgtgaggaga acagtgacca ttgaaaatca ccctcatctg ccaccacctc
     1681 ccatgtgttc agttcaccca tgcaggcatg ctgaggtgat gaggagaatc attgagactg
     1741 ttgcagaagg agggggagaa cttggagttc atatgtatct tcttattttc ttgagatttg
     1801 tacaagctgt cattccaaca atagaatatg actacacaag acacttcaca atgtaatacc
     1861 cagctttctt gtacaaagtg gtgtgatcta gaggatcccg ggtggcatcc ctgtgacccc
     1921 tccccagtgc ctctcctggc cctggaagtt gccactccag tgcccaccag ccttgtccta
     1981 ataaaattaa gttgcatcat tttgtctgac taggtgtcct tctataatat tatggggtgg
     2041 aggggggtgg tatggagcaa ggggcaagtt gggaagacaa cctgtagggc ctgcggggtc
     2101 tattgggaac caagctggag tgcagtggca caatcttggc tcactgcaat ctccgcctcc
     2161 tgggttcaag cgattctcct gcctcagcct cccgagttgt tgggattcca ggcatgcatg
     2221 accaggctca gctaattttt gtttttttgg tagagacggg gtttcaccat attggccagg
     2281 ctggtctcca actcctaatc tcaggtgatc tacccacctt ggcctcccaa attgctggga
     2341 ttacaggcgt gaaccactgc tcccttccct gtccttctga ttttaaaata actataccag
     2401 caggaggacg tccagacaca gcataggcta cctggccatg cccaaccggt gggacatttg
     2461 agttgcttgc ttggcactgt cctctcatgc gttgggtcca ctcagtagat gcctgttgaa
     2521 ttgggtacgc ggccagcttg gctgtggaat gtgtgtcagt tagggtgtgg aaagtcccca
     2581 ggctccccag caggcagaag tatgcaaagc atgcatctca attagtcagc aaccaggtgt
     2641 ggaaagtccc caggctcccc agcaggcaga agtatgcaaa gcatgcatct caattagtca
     2701 gcaaccatag tcccgcccct aactccgccc atcccgcccc taactccgcc cagttccgcc
     2761 cattctccgc cccatggctg actaattttt tttatttatg cagaggccga ggccgcctcg
     2821 gcctctgagc tattccagaa gtagtgagga ggcttttttg gaggcctagg cttttgcaaa
     2881 aagctcctcg atcgaggaac tgaaaaacca gaaagttaat tccctatagt gagtcgtatt
     2941 aaattcgtaa tcatgtcata gctgtttcct gtgtgaaatt gttatccgct cacaattcca
     3001 cacaacatac gagccggaag cataaagtgt aaagcctggg gtgcctaatg agcgagctaa
     3061 ctcacattaa ttgcgttgcg ctcactgccc gctttccagt cgggaaacct gtcgtgccag
     3121 ctgcattaat gaatcggcca acgcgcgggg agaggcggtt tgcgtattgg gcgctcttcc
     3181 gcttcctcgc tcactgactc gctgcgctcg gtcgttcggc tgcggcgagc ggtatcagct
     3241 cactcaaagg cggtaatacg gttatccaca gaatcagggg ataacgcagg aaagaacatg
     3301 tgagcaaaag gccagcaaaa ggccaggaac cgtaaaaagg ccgcgttgct ggcgtttttc
     3361 cataggctcc gcccccctga cgagcatcac aaaaatcgac gctcaagtca gaggtggcga
     3421 aacccgacag gactataaag ataccaggcg tttccccctg gaagctccct cgtgcgctct
     3481 cctgttccga ccctgccgct taccggatac ctgtccgcct ttctcccttc gggaagcgtg
     3541 gcgctttctc atagctcacg ctgtaggtat ctcagttcgg tgtaggtcgt tcgctccaag
     3601 ctgggctgtg tgcacgaacc ccccgttcag cccgaccgct gcgccttatc cggtaactat
     3661 cgtcttgagt ccaacccggt aagacacgac ttatcgccac tggcagcagc cactggtaac
     3721 aggattagca gagcgaggta tgtaggcggt gctacagagt tcttgaagtg gtggcctaac
     3781 tacggctaca ctagaagaac agtatttggt atctgcgctc tgctgaagcc agttaccttc
     3841 ggaaaaagag ttggtagctc ttgatccggc aaacaaacca ccgctggtag cggtggtttt
     3901 tttgtttgca agcagcagat tacgcgcaga aaaaaaggat ctcaagaaga tcctttgatc
     3961 ttttctacgg ggtctgacgc tcagtggaac gaaaactcac gttaagggat tttggtcatg
     4021 agattatcaa aaaggatctt cacctagatc cttttaaatt aaaaatgaag ttttaaatca
     4081 atctaaagta tatatgagta aacttggtct gacagttacc aatgcttaat cagtgaggca
     4141 cctatctcag cgatctgtct atttcgttca tccatagttg cctgactccc cgtcgtgtag
     4201 ataactacga tacgggaggg cttaccatct ggccccagtg ctgcaatgat accgcgagac
     4261 ccacgctcac cggctccaga tttatcagca ataaaccagc cagccggaag ggccgagcgc
     4321 agaagtggtc ctgcaacttt atccgcctcc atccagtcta ttaattgttg ccgggaagct
     4381 agagtaagta gttcgccagt taatagtttg cgcaacgttg ttgccattgc tacaggcatc
     4441 gtggtgtcac gctcgtcgtt tggtatggct tcattcagct ccggttccca acgatcaagg
     4501 cgagttacat gatcccccat gttgtgcaaa aaagcggtta gctccttcgg tcctccgatc
     4561 gttgtcagaa gtaagttggc cgcagtgtta tcactcatgg ttatggcagc actgcataat
     4621 tctcttactg tcatgccatc cgtaagatgc ttttctgtga ctggtgagta ctcaaccaag
     4681 tcattctgag aatagtgtat gcggcgaccg agttgctctt gcccggcgtc aatacgggat
     4741 aataccgcgc cacatagcag aactttaaaa gtgctcatca ttggaaaacg ttcttcgggg
     4801 cgaaaactct caaggatctt accgctgttg agatccagtt cgatgtaacc cactcgtgca
     4861 cccaactgat cttcagcatc ttttactttc accagcgttt ctgggtgagc aaaaacagga
     4921 aggcaaaatg ccgcaaaaaa gggaataagg gcgacacgga aatgttgaat actcatactc
     4981 ttcctttttc aatattattg aagcatttat cagggttatt gtctcatgag cggatacata
     5041 tttgaatgta tttagaaaaa taaacaaata ggggttccgc gcacatttcc ccgaaaagtg
     5101 ccacctgacg cgccctgtag cggcgcatta agcgcggcgg gtgtggtggt tacgcgcagc
     5161 gtgaccgcta cacttgccag cgccctagcg cccgctcctt tcgctttctt cccttccttt
     5221 ctcgccacgt tcgccggctt tccccgtcaa gctctaaatc gggggctccc tttagggttc
     5281 cgatttagtg ctttacggca cctcgacccc aaaaaacttg attagggtga tggttcacgt
     5341 agtgggccat cgccctgata gacggttttt cgccctttga cgttggagtc cacgttcttt
     5401 aatagtggac tcttgttcca aactggaaca acactcaacc ctatctcggt ctattctttt
     5461 gatttataag ggattttgcc gatttcggcc tattggttaa aaaatgagct gatttaacaa
     5521 aaatttaacg cgaattttaa caaaatatta acgcttacaa tttccattcg ccattcaggc
     5581 tgcgcaactg ttgggaaggg cgatcggtgc gggcctcttc gctattacgc cagctggcga
     5641 aagggggatg tgctgcaagg cgattaagtt gggtaacgcc agggt
//
