LOCUS       sequence-253004-        5685 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Expresses pCMV 3xFLAG-ATG3 K243R 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 K243R
FEATURES             Location/Qualifiers
     source          1..5685
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     enhancer        85..464
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
     promoter        465..668
                     /label=CMV promoter
                     /note="human cytomegalovirus (CMV) immediate early 
                     promoter"
     primer_bind     619..639
                     /label=CMV-F
                     /note="Human CMV immediate early promoter, forward primer"
     CDS             705..770
                     /codon_start=1
                     /product="three tandem FLAG(R) epitope tags, followed by an
                     enterokinase cleavage site"
                     /label=3xFLAG
                     /translation="DYKDHDGDYKDHDIDYKDDDDK"
     protein_bind    774..798
                     /gene="mutant version of attB"
                     /label=attB1
                     /bound_moiety="BP Clonase(TM)"
                     /note="recombination site for the Gateway(R) BP reaction"
     protein_bind    complement(1747..1771)
                     /gene="mutant version of attB"
                     /label=attB2
                     /bound_moiety="BP Clonase(TM)"
                     /note="recombination site for the Gateway(R) BP reaction"
     polyA_signal    1789..2411
                     /label=hGH poly(A) signal
                     /note="human growth hormone polyadenylation signal"
     primer_bind     complement(1988..2007)
                     /label=hGH-PA-R
                     /note="Human growth hormone terminator, reverse primer"
     primer_bind     complement(2435..2455)
                     /label=pBABE 3'
                     /note="SV40 enhancer, reverse primer for pBABE vectors"
     promoter        2440..2769
                     /label=SV40 promoter
                     /note="SV40 enhancer and early promoter"
     rep_origin      2620..2755
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     primer_bind     complement(2811..2830)
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        complement(2812..2830)
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     primer_bind     complement(2844..2860)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(2844..2860)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(2857..2879)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    2868..2884
                     /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(2892..2898,2899..2916,2917..2922))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     primer_bind     complement(3075..3092)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(3246..3834)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(3326..3345)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(4005..4796,4797..4865))
                     /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     4628..4647
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(4866..4970)
                     /gene="bla"
                     /label=AmpR promoter
     rep_origin      4997..5452
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(5084..5103)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     5294..5315
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     5578..5600
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     primer_bind     5592..5609
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     5593..5609
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
ORIGIN
        1 ttggctattg gccattgcat acgttgtatc tatatcataa tatgtacatt tatattggct
       61 catgtccaat atgaccgcca tgttgacatt gattattgac tagttattaa tagtaatcaa
      121 ttacggggtc attagttcat agcccatata tggagttccg cgttacataa cttacggtaa
      181 atggcccgcc tggctgaccg cccaacgacc cccgcccatt gacgtcaata atgacgtatg
      241 ttcccatagt aacgccaata gggactttcc attgacgtca atgggtggag tatttacggt
      301 aaactgccca cttggcagta catcaagtgt atcatatgcc aagtccgccc cctattgacg
      361 tcaatgacgg taaatggccc gcctggcatt atgcccagta catgacctta cgggactttc
      421 ctacttggca gtacatctac gtattagtca tcgctattac catggtgatg cggttttggc
      481 agtacaccaa tgggcgtgga tagcggtttg actcacgggg atttccaagt ctccacccca
      541 ttgacgtcaa tgggagtttg ttttggcacc aaaatcaacg ggactttcca aaatgtcgta
      601 ataaccccgc cccgttgacg caaatgggcg gtaggcgtgt acggtgggag gtctatataa
      661 gcagagctcg tttagtgaac cgtcagaatt gatctggtac catggactac aaagaccatg
      721 acggtgatta taaagatcat gacatcgatt acaaggatga cgatgacaag cttacaagtt
      781 tgtacaaaaa agcaggctta atgcagaatg tgattaatac tgtgaaggga aaggcactgg
      841 aagtggctga gtacctgacc ccggtcctca aggaatcaaa gtttaaggaa acaggtgtaa
      901 ttaccccaga agagtttgtg gcagctggag atcacctagt ccaccactgt ccaacatggc
      961 aatgggctac aggggaagaa ttgaaagtga aggcatacct accaacaggc aaacaatttt
     1021 tggtaaccaa aaatgtgccg tgctataagc ggtgcaaaca gatggaatat tcagatgaat
     1081 tggaagctat cattgaagaa gatgatggtg atggcggatg ggtagataca tatcacaaca
     1141 caggtattac aggaataacg gaagccgtta aagagatcac actggaaaat aaggacaata
     1201 taaggcttca agattgctca gcactatgtg aagaggaaga agatgaagat gaaggagaag
     1261 ctgcagatat ggaagaatat gaagagagtg gattgttgga aacagatgag gctaccctag
     1321 atacaaggaa aatagtagaa gcttgtaaag ccaaaactga tgctggcggt gaagatgcta
     1381 ttttgcaaac cagaacttat gacctttaca tcacttatga taaatattac cagactccac
     1441 gattatggtt gtttggctat gatgagcaac ggcagccttt aacagttgag cacatgtatg
     1501 aagacatcag tcaggatcat gtgaagagaa cagtgaccat tgaaaatcac cctcatctgc
     1561 caccacctcc catgtgttca gttcacccat gcaggcatgc tgaggtgatg aagaaaatca
     1621 ttgagactgt tgcagaagga gggggagaac ttggagttca tatgtatctt cttattttct
     1681 tgaaatttgt acaagctgtc attccaacaa tagaatatga ctacacaaga cacttcacaa
     1741 tgtaataccc agctttcttg tacaaagtgg tgtgatctag aggatcccgg gtggcatccc
     1801 tgtgacccct ccccagtgcc tctcctggcc ctggaagttg ccactccagt gcccaccagc
     1861 cttgtcctaa taaaattaag ttgcatcatt ttgtctgact aggtgtcctt ctataatatt
     1921 atggggtgga ggggggtggt atggagcaag gggcaagttg ggaagacaac ctgtagggcc
     1981 tgcggggtct attgggaacc aagctggagt gcagtggcac aatcttggct cactgcaatc
     2041 tccgcctcct gggttcaagc gattctcctg cctcagcctc ccgagttgtt gggattccag
     2101 gcatgcatga ccaggctcag ctaatttttg tttttttggt agagacgggg tttcaccata
     2161 ttggccaggc tggtctccaa ctcctaatct caggtgatct acccaccttg gcctcccaaa
     2221 ttgctgggat tacaggcgtg aaccactgct cccttccctg tccttctgat tttaaaataa
     2281 ctataccagc aggaggacgt ccagacacag cataggctac ctggccatgc ccaaccggtg
     2341 ggacatttga gttgcttgct tggcactgtc ctctcatgcg ttgggtccac tcagtagatg
     2401 cctgttgaat tgggtacgcg gccagcttgg ctgtggaatg tgtgtcagtt agggtgtgga
     2461 aagtccccag gctccccagc aggcagaagt atgcaaagca tgcatctcaa ttagtcagca
     2521 accaggtgtg gaaagtcccc aggctcccca gcaggcagaa gtatgcaaag catgcatctc
     2581 aattagtcag caaccatagt cccgccccta actccgccca tcccgcccct aactccgccc
     2641 agttccgccc attctccgcc ccatggctga ctaatttttt ttatttatgc agaggccgag
     2701 gccgcctcgg cctctgagct attccagaag tagtgaggag gcttttttgg aggcctaggc
     2761 ttttgcaaaa agctcctcga tcgaggaact gaaaaaccag aaagttaatt ccctatagtg
     2821 agtcgtatta aattcgtaat catgtcatag ctgtttcctg tgtgaaattg ttatccgctc
     2881 acaattccac acaacatacg agccggaagc ataaagtgta aagcctgggg tgcctaatga
     2941 gcgagctaac tcacattaat tgcgttgcgc tcactgcccg ctttccagtc gggaaacctg
     3001 tcgtgccagc tgcattaatg aatcggccaa cgcgcgggga gaggcggttt gcgtattggg
     3061 cgctcttccg cttcctcgct cactgactcg ctgcgctcgg tcgttcggct gcggcgagcg
     3121 gtatcagctc actcaaaggc ggtaatacgg ttatccacag aatcagggga taacgcagga
     3181 aagaacatgt gagcaaaagg ccagcaaaag gccaggaacc gtaaaaaggc cgcgttgctg
     3241 gcgtttttcc ataggctccg cccccctgac gagcatcaca aaaatcgacg ctcaagtcag
     3301 aggtggcgaa acccgacagg actataaaga taccaggcgt ttccccctgg aagctccctc
     3361 gtgcgctctc ctgttccgac cctgccgctt accggatacc tgtccgcctt tctcccttcg
     3421 ggaagcgtgg cgctttctca tagctcacgc tgtaggtatc tcagttcggt gtaggtcgtt
     3481 cgctccaagc tgggctgtgt gcacgaaccc cccgttcagc ccgaccgctg cgccttatcc
     3541 ggtaactatc gtcttgagtc caacccggta agacacgact tatcgccact ggcagcagcc
     3601 actggtaaca ggattagcag agcgaggtat gtaggcggtg ctacagagtt cttgaagtgg
     3661 tggcctaact acggctacac tagaagaaca gtatttggta tctgcgctct gctgaagcca
     3721 gttaccttcg gaaaaagagt tggtagctct tgatccggca aacaaaccac cgctggtagc
     3781 ggtggttttt ttgtttgcaa gcagcagatt acgcgcagaa aaaaaggatc tcaagaagat
     3841 cctttgatct tttctacggg gtctgacgct cagtggaacg aaaactcacg ttaagggatt
     3901 ttggtcatga gattatcaaa aaggatcttc acctagatcc ttttaaatta aaaatgaagt
     3961 tttaaatcaa tctaaagtat atatgagtaa acttggtctg acagttacca atgcttaatc
     4021 agtgaggcac ctatctcagc gatctgtcta tttcgttcat ccatagttgc ctgactcccc
     4081 gtcgtgtaga taactacgat acgggagggc ttaccatctg gccccagtgc tgcaatgata
     4141 ccgcgagacc cacgctcacc ggctccagat ttatcagcaa taaaccagcc agccggaagg
     4201 gccgagcgca gaagtggtcc tgcaacttta tccgcctcca tccagtctat taattgttgc
     4261 cgggaagcta gagtaagtag ttcgccagtt aatagtttgc gcaacgttgt tgccattgct
     4321 acaggcatcg tggtgtcacg ctcgtcgttt ggtatggctt cattcagctc cggttcccaa
     4381 cgatcaaggc gagttacatg atcccccatg ttgtgcaaaa aagcggttag ctccttcggt
     4441 cctccgatcg ttgtcagaag taagttggcc gcagtgttat cactcatggt tatggcagca
     4501 ctgcataatt ctcttactgt catgccatcc gtaagatgct tttctgtgac tggtgagtac
     4561 tcaaccaagt cattctgaga atagtgtatg cggcgaccga gttgctcttg cccggcgtca
     4621 atacgggata ataccgcgcc acatagcaga actttaaaag tgctcatcat tggaaaacgt
     4681 tcttcggggc gaaaactctc aaggatctta ccgctgttga gatccagttc gatgtaaccc
     4741 actcgtgcac ccaactgatc ttcagcatct tttactttca ccagcgtttc tgggtgagca
     4801 aaaacaggaa ggcaaaatgc cgcaaaaaag ggaataaggg cgacacggaa atgttgaata
     4861 ctcatactct tcctttttca atattattga agcatttatc agggttattg tctcatgagc
     4921 ggatacatat ttgaatgtat ttagaaaaat aaacaaatag gggttccgcg cacatttccc
     4981 cgaaaagtgc cacctgacgc gccctgtagc ggcgcattaa gcgcggcggg tgtggtggtt
     5041 acgcgcagcg tgaccgctac acttgccagc gccctagcgc ccgctccttt cgctttcttc
     5101 ccttcctttc tcgccacgtt cgccggcttt ccccgtcaag ctctaaatcg ggggctccct
     5161 ttagggttcc gatttagtgc tttacggcac ctcgacccca aaaaacttga ttagggtgat
     5221 ggttcacgta gtgggccatc gccctgatag acggtttttc gccctttgac gttggagtcc
     5281 acgttcttta atagtggact cttgttccaa actggaacaa cactcaaccc tatctcggtc
     5341 tattcttttg atttataagg gattttgccg atttcggcct attggttaaa aaatgagctg
     5401 atttaacaaa aatttaacgc gaattttaac aaaatattaa cgcttacaat ttccattcgc
     5461 cattcaggct gcgcaactgt tgggaagggc gatcggtgcg ggcctcttcg ctattacgcc
     5521 agctggcgaa agggggatgt gctgcaaggc gattaagttg ggtaacgcca gggttttccc
     5581 agtcacgacg ttgtaaaacg acggccagtg ccaagctgat ctatacattg aatcaatatt
     5641 ggcaattagc catattagtc attggttata tagcataaat caata
//
