LOCUS       sequence-248132-        5693 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Expresses pCMV 3xFLAG-ATG3 K295R in mammalian cells.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 5693)
  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 5693)
  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 K295R
FEATURES             Location/Qualifiers
     source          1..5693
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     rep_origin      complement(21..609)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(101..120)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(780..1571,1572..1640))
                     /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     1403..1422
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(1641..1745)
                     /gene="bla"
                     /label=AmpR promoter
     rep_origin      1772..2200
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(1859..1878)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     2069..2090
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     2366..2388
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     primer_bind     2380..2397
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     2381..2397
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     enhancer        2558..2937
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
     promoter        2938..3141
                     /label=CMV promoter
                     /note="human cytomegalovirus (CMV) immediate early 
                     promoter"
     primer_bind     3092..3112
                     /label=CMV-F
                     /note="Human CMV immediate early promoter, forward primer"
     CDS             3178..3243
                     /codon_start=1
                     /product="three tandem FLAG(R) epitope tags, followed by an
                     enterokinase cleavage site"
                     /label=3xFLAG
                     /translation="DYKDHDGDYKDHDIDYKDDDDK"
     protein_bind    3247..3271
                     /gene="mutant version of attB"
                     /label=attB1
                     /bound_moiety="BP Clonase(TM)"
                     /note="recombination site for the Gateway(R) BP reaction"
     protein_bind    complement(4220..4244)
                     /gene="mutant version of attB"
                     /label=attB2
                     /bound_moiety="BP Clonase(TM)"
                     /note="recombination site for the Gateway(R) BP reaction"
     polyA_signal    4262..4884
                     /label=hGH poly(A) signal
                     /note="human growth hormone polyadenylation signal"
     primer_bind     complement(4461..4480)
                     /label=hGH-PA-R
                     /note="Human growth hormone terminator, reverse primer"
     primer_bind     complement(4906..4926)
                     /label=pBABE 3'
                     /note="SV40 enhancer, reverse primer for pBABE vectors"
     promoter        4911..5241
                     /label=SV40 promoter
                     /note="SV40 enhancer and early promoter"
     rep_origin      5092..5227
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     primer_bind     complement(5279..5298)
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        complement(5280..5298)
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     primer_bind     complement(5313..5329)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(5313..5329)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(5326..5348)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    5337..5353
                     /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(5361..5367,5368..5385,5386..5391))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    5406..5427
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(5544..5561)
                     /label=L4440
                     /note="L4440 vector, forward primer"
ORIGIN
        1 aaggccgcgt tgctggcgtt tttccatagg ctccgccccc ctgacgagca tcacaaaaat
       61 cgacgctcaa gtcagaggtg gcgaaacccg acaggactat aaagatacca ggcgtttccc
      121 cctggaagct ccctcgtgcg ctctcctgtt ccgaccctgc cgcttaccgg atacctgtcc
      181 gcctttctcc cttcgggaag cgtggcgctt tctcaatgct cacgctgtag gtatctcagt
      241 tcggtgtagg tcgttcgctc caagctgggc tgtgtgcacg aaccccccgt tcagcccgac
      301 cgctgcgcct tatccggtaa ctatcgtctt gagtccaacc cggtaagaca cgacttatcg
      361 ccactggcag cagccactgg taacaggatt agcagagcga ggtatgtagg cggtgctaca
      421 gagttcttga agtggtggcc taactacggc tacactagaa ggacagtatt tggtatctgc
      481 gctctgctga agccagttac cttcggaaaa agagttggta gctcttgatc cggcaaacaa
      541 accaccgctg gtagcggtgg tttttttgtt tgcaagcagc agattacgcg cagaaaaaaa
      601 ggatctcaag aagatccttt gatcttttct acggggtctg acgctcagtg gaacgaaaac
      661 tcacgttaag ggattttggt catgagatta tcaaaaagga tcttcaccta gatcctttta
      721 aattaaaaat gaagttttaa atcaatctaa agtatatatg agtaaacttg gtctgacagt
      781 taccaatgct taatcagtga ggcacctatc tcagcgatct gtctatttcg ttcatccata
      841 gttgcctgac tccccgtcgt gtagataact acgatacggg agggcttacc atctggcccc
      901 agtgctgcaa tgataccgcg agacccacgc tcaccggctc cagatttatc agcaataaac
      961 cagccagccg gaagggccga gcgcagaagt ggtcctgcaa ctttatccgc ctccatccag
     1021 tctattaatt gttgccggga agctagagta agtagttcgc cagttaatag tttgcgcaac
     1081 gttgttgcca ttgctacagg catcgtggtg tcacgctcgt cgtttggtat ggcttcattc
     1141 agctccggtt cccaacgatc aaggcgagtt acatgatccc ccatgttgtg caaaaaagcg
     1201 gttagctcct tcggtcctcc gatcgttgtc agaagtaagt tggccgcagt gttatcactc
     1261 atggttatgg cagcactgca taattctctt actgtcatgc catccgtaag atgcttttct
     1321 gtgactggtg agtactcaac caagtcattc tgagaatagt gtatgcggcg accgagttgc
     1381 tcttgcccgg cgtcaatacg ggataatacc gcgccacata gcagaacttt aaaagtgctc
     1441 atcattggaa aacgttcttc ggggcgaaaa ctctcaagga tcttaccgct gttgagatcc
     1501 agttcgatgt aacccactcg tgcacccaac tgatcttcag catcttttac tttcaccagc
     1561 gtttctgggt gagcaaaaac aggaaggcaa aatgccgcaa aaaagggaat aagggcgaca
     1621 cggaaatgtt gaatactcat actcttcctt tttcaatatt attgaagcat ttatcagggt
     1681 tattgtctca tgagcggata catatttgaa tgtatttaga aaaataaaca aataggggtt
     1741 ccgcgcacat ttccccgaaa agtgccacct gacgcgccct gtagcggcgc attaagcgcg
     1801 gcgggtgtgg tggttacgcg cagcgtgacc gctacacttg ccagcgccct agcgcccgct
     1861 cctttcgctt tcttcccttc ctttctcgcc acgttcgccg gctttccccg tcaagctcta
     1921 aatcggggca tccctttagg gttccgattt agtgctttac ggcacctcga ccccaaaaaa
     1981 cttgattagg gtgatggttc acgtagtggg ccatcgccct gatagacggt ttttcgccct
     2041 ttgacgttgg agtccacgtt ctttaatagt ggactcttgt tccaaactgg aacaacactc
     2101 aaccctatct cggtctattc ttttgattta taagggattt tgccgatttc ggcctattgg
     2161 ttaaaaaatg agctgattta acaaaaattt aacgcgaatt ttaacaaaat attaacaaaa
     2221 tattaacgtt tacaatttcc ccattcgcca ttcaggctgc gcaactgttg ggaagggcga
     2281 tcggtgcggg cctcttcgct attacgccag ctggcgaaag ggggatgtgc tgcaaggcga
     2341 ttaagttggg taacgccagg gttttcccag tcacgacgtt gtaaaacgac ggccagtgcc
     2401 aagctgatct atacattgaa tcaatattgg caattagcca tattagtcat tggttatata
     2461 gcataaatca atattggcta ttggccattg catacgttgt atctatatca taatatgtac
     2521 atttatattg gctcatgtcc aatatgaccg ccatgttgac attgattatt gactagttat
     2581 taatagtaat caattacggg gtcattagtt catagcccat atatggagtt ccgcgttaca
     2641 taacttacgg taaatggccc gcctggctga ccgcccaacg acccccgccc attgacgtca
     2701 ataatgacgt atgttcccat agtaacgcca atagggactt tccattgacg tcaatgggtg
     2761 gagtatttac ggtaaactgc ccacttggca gtacatcaag tgtatcatat gccaagtccg
     2821 ccccctattg acgtcaatga cggtaaatgg cccgcctggc attatgccca gtacatgacc
     2881 ttacgggact ttcctacttg gcagtacatc tacgtattag tcatcgctat taccatggtg
     2941 atgcggtttt ggcagtacac caatgggcgt ggatagcggt ttgactcacg gggatttcca
     3001 agtctccacc ccattgacgt caatgggagt ttgttttggc accaaaatca acgggacttt
     3061 ccaaaatgtc gtaataaccc cgccccgttg acgcaaatgg gcggtaggcg tgtacggtgg
     3121 gaggtctata taagcagagc tcgtttagtg aaccgtcaga attgatctgg taccatggac
     3181 tacaaagacc atgacggtga ttataaagat catgacatcg attacaagga tgacgatgac
     3241 aagcttacaa gtttgtacaa aaaagcaggc ttaatgcaga atgtgattaa tactgtgaag
     3301 ggaaaggcac tggaagtggc tgagtacctg accccggtcc tcaaggaatc aaagtttaag
     3361 gaaacaggtg taattacccc agaagagttt gtggcagctg gagatcacct agtccaccac
     3421 tgtccaacat ggcaatgggc tacaggggaa gaattgaaag tgaaggcata cctaccaaca
     3481 ggcaaacaat ttttggtaac caaaaatgtg ccgtgctata agcggtgcaa acagatggaa
     3541 tattcagatg aattggaagc tatcattgaa gaagatgatg gtgatggcgg atgggtagat
     3601 acatatcaca acacaggtat tacaggaata acggaagccg ttaaagagat cacactggaa
     3661 aataaggaca atataaggct tcaagattgc tcagcactat gtgaagagga agaagatgaa
     3721 gatgaaggag aagctgcaga tatggaagaa tatgaagaga gtggattgtt ggaaacagat
     3781 gaggctaccc tagatacaag gaaaatagta gaagcttgta aagccaaaac tgatgctggc
     3841 ggtgaagatg ctattttgca aaccagaact tatgaccttt acatcactta tgataaatat
     3901 taccagactc cacgattatg gttgtttggc tatgatgagc aacggcagcc tttaacagtt
     3961 gagcacatgt atgaagacat cagtcaggat catgtgaaga aaacagtgac cattgaaaat
     4021 caccctcatc tgccaccacc tcccatgtgt tcagttcacc catgcaggca tgctgaggtg
     4081 atgaagaaaa tcattgagac tgttgcagaa ggagggggag aacttggagt tcatatgtat
     4141 cttcttattt tcttgagatt tgtacaagct gtcattccaa caatagaata tgactacaca
     4201 agacacttca caatgtaata cccagctttc ttgtacaaag tggtgtgatc tagaggatcc
     4261 cgggtggcat ccctgtgacc cctccccagt gcctctcctg gccctggaag ttgccactcc
     4321 agtgcccacc agccttgtcc taataaaatt aagttgcatc attttgtctg actaggtgtc
     4381 cttctataat attatggggt ggaggggggt ggtatggagc aaggggcaag ttgggaagac
     4441 aacctgtagg gcctgcgggg tctattggga accaagctgg agtgcagtgg cacaatcttg
     4501 gctcactgca atctccgcct cctgggttca agcgattctc ctgcctcagc ctcccgagtt
     4561 gttgggattc caggcatgca tgaccaggct cagctaattt ttgttttttt ggtagagacg
     4621 gggtttcacc atattggcca ggctggtctc caactcctaa tctcaggtga tctacccacc
     4681 ttggcctccc aaattgctgg gattacaggc gtgaaccact gctcccttcc ctgtccttct
     4741 gattttaaaa taactatacc agcaggagga cgtccagaca cagcataggc tacctgccat
     4801 ggcccaaccg gtgggacatt tgagttgctt gcttggcact gtcctctcat gcgttgggtc
     4861 cactcagtag atgcctgttg aattgggtac gcggccagct tctgtggaat gtgtgtcagt
     4921 tagggtgtgg aaagtcccca ggctccccag caggcagaag tatgcaaagc atgcatctca
     4981 attagtcagc aaccaggtgt ggaaaagtcc ccaggctccc cagcaggcag aagtatgcaa
     5041 agcatgcatc tcaattagtc agcaaccata gtcccgcccc taactccgcc catcccgccc
     5101 ctaactccgc ccagttccgc ccattctccg ccccatggct gactaatttt ttttatttat
     5161 gcagaggccg aggccgcctc ggcctctgag ctattccaga agtagtgagg aggctttttt
     5221 ggaggcctag gcttttgcaa aaagctcctc gaggaactga aaaaccagaa agttaattcc
     5281 ctatagtgag tcgtattaaa ttcgtaatca tggtcatagc tgtttcctgt gtgaaattgt
     5341 tatccgctca caattccaca caacatacga gccggaagca taaagtgtaa agcctggggt
     5401 gcctaatgag tgagctaact cacattaatt gcgttgcgct cactgcccgc tttccagtcg
     5461 ggaaacctgt cgtgccagct gcattaatga atcggccaac gcgcggggag aggcggtttg
     5521 cgtattgggc gctcttccgc ttcctcgctc actgactcgc tgcgctcggt cgttcggctg
     5581 cggcgagcgg tatcagctca ctcaaaggcg gtaatacggt tatccacaga atcaggggat
     5641 aacgcaggaa agaacatgtg agcaaaaggc cagcaaaagg ccaggaaccg taa
//
