LOCUS       sequence-253000-        5685 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Expresses pCMV 3xFLAG-ATG3 1-13 KR (first thirteen 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 1-13 KR
FEATURES             Location/Qualifiers
     source          1..5685
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     74..96
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     primer_bind     88..105
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     89..105
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     enhancer        266..645
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
     promoter        646..849
                     /label=CMV promoter
                     /note="human cytomegalovirus (CMV) immediate early 
                     promoter"
     primer_bind     800..820
                     /label=CMV-F
                     /note="Human CMV immediate early promoter, forward primer"
     CDS             886..951
                     /codon_start=1
                     /product="three tandem FLAG(R) epitope tags, followed by an
                     enterokinase cleavage site"
                     /label=3xFLAG
                     /translation="DYKDHDGDYKDHDIDYKDDDDK"
     protein_bind    955..979
                     /gene="mutant version of attB"
                     /label=attB1
                     /bound_moiety="BP Clonase(TM)"
                     /note="recombination site for the Gateway(R) BP reaction"
     protein_bind    complement(1928..1952)
                     /gene="mutant version of attB"
                     /label=attB2
                     /bound_moiety="BP Clonase(TM)"
                     /note="recombination site for the Gateway(R) BP reaction"
     polyA_signal    1970..2592
                     /label=hGH poly(A) signal
                     /note="human growth hormone polyadenylation signal"
     primer_bind     complement(2169..2188)
                     /label=hGH-PA-R
                     /note="Human growth hormone terminator, reverse primer"
     primer_bind     complement(2616..2636)
                     /label=pBABE 3'
                     /note="SV40 enhancer, reverse primer for pBABE vectors"
     promoter        2621..2950
                     /label=SV40 promoter
                     /note="SV40 enhancer and early promoter"
     rep_origin      2801..2936
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     primer_bind     complement(2992..3011)
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        complement(2993..3011)
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     primer_bind     complement(3025..3041)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(3025..3041)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(3038..3060)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    3049..3065
                     /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(3073..3079,3080..3097,3098..3103))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     primer_bind     complement(3256..3273)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(3427..4015)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(3507..3526)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(4186..4977,4978..5046))
                     /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     4809..4828
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(5047..5151)
                     /gene="bla"
                     /label=AmpR promoter
     rep_origin      5178..5633
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(5265..5284)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     5475..5496
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
ORIGIN
        1 tcttcgctat tacgccagct ggcgaaaggg ggatgtgctg caaggcgatt aagttgggta
       61 acgccagggt tttcccagtc acgacgttgt aaaacgacgg ccagtgccaa gctgatctat
      121 acattgaatc aatattggca attagccata ttagtcattg gttatatagc ataaatcaat
      181 attggctatt ggccattgca tacgttgtat ctatatcata atatgtacat ttatattggc
      241 tcatgtccaa tatgaccgcc atgttgacat tgattattga ctagttatta atagtaatca
      301 attacggggt cattagttca tagcccatat atggagttcc gcgttacata acttacggta
      361 aatggcccgc ctggctgacc gcccaacgac ccccgcccat tgacgtcaat aatgacgtat
      421 gttcccatag taacgccaat agggactttc cattgacgtc aatgggtgga gtatttacgg
      481 taaactgccc acttggcagt acatcaagtg tatcatatgc caagtccgcc ccctattgac
      541 gtcaatgacg gtaaatggcc cgcctggcat tatgcccagt acatgacctt acgggacttt
      601 cctacttggc agtacatcta cgtattagtc atcgctatta ccatggtgat gcggttttgg
      661 cagtacacca atgggcgtgg atagcggttt gactcacggg gatttccaag tctccacccc
      721 attgacgtca atgggagttt gttttggcac caaaatcaac gggactttcc aaaatgtcgt
      781 aataaccccg ccccgttgac gcaaatgggc ggtaggcgtg tacggtggga ggtctatata
      841 agcagagctc gtttagtgaa ccgtcagaat tgatctggta ccatggacta caaagaccat
      901 gacggtgatt ataaagatca tgacatcgat tacaaggatg acgatgacaa gcttacaagt
      961 ttgtacaaaa aagcaggctt aatgcagaat gtgattaata ctgtgagggg aagggcactg
     1021 gaagtggctg agtacctgac cccggtcctc agggaatcaa ggtttaggga aacaggtgta
     1081 attaccccag aagagtttgt ggcagctgga gatcacctag tccaccactg tccaacatgg
     1141 caatgggcta caggggaaga attgagagtg agggcatacc taccaacagg cagacaattt
     1201 ttggtaacca gaaatgtgcc gtgctatagg cggtgcagac agatggaata ttcagatgaa
     1261 ttggaagcta tcattgaaga agatgatggt gatggcggat gggtagatac atatcacaac
     1321 acaggtatta caggaataac ggaagccgtt agagagatca cactggaaaa tagggacaat
     1381 ataaggcttc aagattgctc agcactatgt gaagaggaag aagatgaaga tgaaggagaa
     1441 gctgcagata tggaagaata tgaagagagt ggattgttgg aaacagatga ggctacccta
     1501 gatacaagga aaatagtaga agcttgtaaa gccaaaactg atgctggcgg tgaagatgct
     1561 attttgcaaa ccagaactta tgacctttac atcacttatg ataaatatta ccagactcca
     1621 cgattatggt tgtttggcta tgatgagcaa cggcagcctt taacagttga gcacatgtat
     1681 gaagacatca gtcaggatca tgtgaagaaa acagtgacca ttgaaaatca ccctcatctg
     1741 ccaccacctc ccatgtgttc agttcaccca tgcaggcatg ctgaggtgat gaagaaaatc
     1801 attgagactg ttgcagaagg agggggagaa cttggagttc atatgtatct tcttattttc
     1861 ttgaaatttg tacaagctgt cattccaaca atagaatatg actacacaag acacttcaca
     1921 atgtaatacc cagctttctt gtacaaagtg gtgtgatcta gaggatcccg ggtggcatcc
     1981 ctgtgacccc tccccagtgc ctctcctggc cctggaagtt gccactccag tgcccaccag
     2041 ccttgtccta ataaaattaa gttgcatcat tttgtctgac taggtgtcct tctataatat
     2101 tatggggtgg aggggggtgg tatggagcaa ggggcaagtt gggaagacaa cctgtagggc
     2161 ctgcggggtc tattgggaac caagctggag tgcagtggca caatcttggc tcactgcaat
     2221 ctccgcctcc tgggttcaag cgattctcct gcctcagcct cccgagttgt tgggattcca
     2281 ggcatgcatg accaggctca gctaattttt gtttttttgg tagagacggg gtttcaccat
     2341 attggccagg ctggtctcca actcctaatc tcaggtgatc tacccacctt ggcctcccaa
     2401 attgctggga ttacaggcgt gaaccactgc tcccttccct gtccttctga ttttaaaata
     2461 actataccag caggaggacg tccagacaca gcataggcta cctggccatg cccaaccggt
     2521 gggacatttg agttgcttgc ttggcactgt cctctcatgc gttgggtcca ctcagtagat
     2581 gcctgttgaa ttgggtacgc ggccagcttg gctgtggaat gtgtgtcagt tagggtgtgg
     2641 aaagtcccca ggctccccag caggcagaag tatgcaaagc atgcatctca attagtcagc
     2701 aaccaggtgt ggaaagtccc caggctcccc agcaggcaga agtatgcaaa gcatgcatct
     2761 caattagtca gcaaccatag tcccgcccct aactccgccc atcccgcccc taactccgcc
     2821 cagttccgcc cattctccgc cccatggctg actaattttt tttatttatg cagaggccga
     2881 ggccgcctcg gcctctgagc tattccagaa gtagtgagga ggcttttttg gaggcctagg
     2941 cttttgcaaa aagctcctcg atcgaggaac tgaaaaacca gaaagttaat tccctatagt
     3001 gagtcgtatt aaattcgtaa tcatgtcata gctgtttcct gtgtgaaatt gttatccgct
     3061 cacaattcca cacaacatac gagccggaag cataaagtgt aaagcctggg gtgcctaatg
     3121 agcgagctaa ctcacattaa ttgcgttgcg ctcactgccc gctttccagt cgggaaacct
     3181 gtcgtgccag ctgcattaat gaatcggcca acgcgcgggg agaggcggtt tgcgtattgg
     3241 gcgctcttcc gcttcctcgc tcactgactc gctgcgctcg gtcgttcggc tgcggcgagc
     3301 ggtatcagct cactcaaagg cggtaatacg gttatccaca gaatcagggg ataacgcagg
     3361 aaagaacatg tgagcaaaag gccagcaaaa ggccaggaac cgtaaaaagg ccgcgttgct
     3421 ggcgtttttc cataggctcc gcccccctga cgagcatcac aaaaatcgac gctcaagtca
     3481 gaggtggcga aacccgacag gactataaag ataccaggcg tttccccctg gaagctccct
     3541 cgtgcgctct cctgttccga ccctgccgct taccggatac ctgtccgcct ttctcccttc
     3601 gggaagcgtg gcgctttctc atagctcacg ctgtaggtat ctcagttcgg tgtaggtcgt
     3661 tcgctccaag ctgggctgtg tgcacgaacc ccccgttcag cccgaccgct gcgccttatc
     3721 cggtaactat cgtcttgagt ccaacccggt aagacacgac ttatcgccac tggcagcagc
     3781 cactggtaac aggattagca gagcgaggta tgtaggcggt gctacagagt tcttgaagtg
     3841 gtggcctaac tacggctaca ctagaagaac agtatttggt atctgcgctc tgctgaagcc
     3901 agttaccttc ggaaaaagag ttggtagctc ttgatccggc aaacaaacca ccgctggtag
     3961 cggtggtttt tttgtttgca agcagcagat tacgcgcaga aaaaaaggat ctcaagaaga
     4021 tcctttgatc ttttctacgg ggtctgacgc tcagtggaac gaaaactcac gttaagggat
     4081 tttggtcatg agattatcaa aaaggatctt cacctagatc cttttaaatt aaaaatgaag
     4141 ttttaaatca atctaaagta tatatgagta aacttggtct gacagttacc aatgcttaat
     4201 cagtgaggca cctatctcag cgatctgtct atttcgttca tccatagttg cctgactccc
     4261 cgtcgtgtag ataactacga tacgggaggg cttaccatct ggccccagtg ctgcaatgat
     4321 accgcgagac ccacgctcac cggctccaga tttatcagca ataaaccagc cagccggaag
     4381 ggccgagcgc agaagtggtc ctgcaacttt atccgcctcc atccagtcta ttaattgttg
     4441 ccgggaagct agagtaagta gttcgccagt taatagtttg cgcaacgttg ttgccattgc
     4501 tacaggcatc gtggtgtcac gctcgtcgtt tggtatggct tcattcagct ccggttccca
     4561 acgatcaagg cgagttacat gatcccccat gttgtgcaaa aaagcggtta gctccttcgg
     4621 tcctccgatc gttgtcagaa gtaagttggc cgcagtgtta tcactcatgg ttatggcagc
     4681 actgcataat tctcttactg tcatgccatc cgtaagatgc ttttctgtga ctggtgagta
     4741 ctcaaccaag tcattctgag aatagtgtat gcggcgaccg agttgctctt gcccggcgtc
     4801 aatacgggat aataccgcgc cacatagcag aactttaaaa gtgctcatca ttggaaaacg
     4861 ttcttcgggg cgaaaactct caaggatctt accgctgttg agatccagtt cgatgtaacc
     4921 cactcgtgca cccaactgat cttcagcatc ttttactttc accagcgttt ctgggtgagc
     4981 aaaaacagga aggcaaaatg ccgcaaaaaa gggaataagg gcgacacgga aatgttgaat
     5041 actcatactc ttcctttttc aatattattg aagcatttat cagggttatt gtctcatgag
     5101 cggatacata tttgaatgta tttagaaaaa taaacaaata ggggttccgc gcacatttcc
     5161 ccgaaaagtg ccacctgacg cgccctgtag cggcgcatta agcgcggcgg gtgtggtggt
     5221 tacgcgcagc gtgaccgcta cacttgccag cgccctagcg cccgctcctt tcgctttctt
     5281 cccttccttt ctcgccacgt tcgccggctt tccccgtcaa gctctaaatc gggggctccc
     5341 tttagggttc cgatttagtg ctttacggca cctcgacccc aaaaaacttg attagggtga
     5401 tggttcacgt agtgggccat cgccctgata gacggttttt cgccctttga cgttggagtc
     5461 cacgttcttt aatagtggac tcttgttcca aactggaaca acactcaacc ctatctcggt
     5521 ctattctttt gatttataag ggattttgcc gatttcggcc tattggttaa aaaatgagct
     5581 gatttaacaa aaatttaacg cgaattttaa caaaatatta acgcttacaa tttccattcg
     5641 ccattcaggc tgcgcaactg ttgggaaggg cgatcggtgc gggcc
//
