LOCUS       sequence-252998-        5685 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Expresses 3xFLAG-ATG3 C264A 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 C264A
FEATURES             Location/Qualifiers
     source          1..5685
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     enhancer        118..497
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
     promoter        498..701
                     /label=CMV promoter
                     /note="human cytomegalovirus (CMV) immediate early 
                     promoter"
     primer_bind     652..672
                     /label=CMV-F
                     /note="Human CMV immediate early promoter, forward primer"
     CDS             738..803
                     /codon_start=1
                     /product="three tandem FLAG(R) epitope tags, followed by an
                     enterokinase cleavage site"
                     /label=3xFLAG
                     /translation="DYKDHDGDYKDHDIDYKDDDDK"
     protein_bind    807..831
                     /gene="mutant version of attB"
                     /label=attB1
                     /bound_moiety="BP Clonase(TM)"
                     /note="recombination site for the Gateway(R) BP reaction"
     protein_bind    complement(1780..1804)
                     /gene="mutant version of attB"
                     /label=attB2
                     /bound_moiety="BP Clonase(TM)"
                     /note="recombination site for the Gateway(R) BP reaction"
     polyA_signal    1822..2444
                     /label=hGH poly(A) signal
                     /note="human growth hormone polyadenylation signal"
     primer_bind     complement(2021..2040)
                     /label=hGH-PA-R
                     /note="Human growth hormone terminator, reverse primer"
     primer_bind     complement(2468..2488)
                     /label=pBABE 3'
                     /note="SV40 enhancer, reverse primer for pBABE vectors"
     promoter        2473..2802
                     /label=SV40 promoter
                     /note="SV40 enhancer and early promoter"
     rep_origin      2653..2788
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     primer_bind     complement(2844..2863)
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        complement(2845..2863)
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     primer_bind     complement(2877..2893)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(2877..2893)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(2890..2912)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    2901..2917
                     /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(2925..2931,2932..2949,2950..2955))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     primer_bind     complement(3108..3125)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(3279..3867)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(3359..3378)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(4038..4829,4830..4898))
                     /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     4661..4680
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(4899..5003)
                     /gene="bla"
                     /label=AmpR promoter
     rep_origin      5030..5485
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(5117..5136)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     5327..5348
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     5611..5633
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     primer_bind     5625..5642
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     5626..5642
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
ORIGIN
        1 tattagtcat tggttatata gcataaatca atattggcta ttggccattg catacgttgt
       61 atctatatca taatatgtac atttatattg gctcatgtcc aatatgaccg ccatgttgac
      121 attgattatt gactagttat taatagtaat caattacggg gtcattagtt catagcccat
      181 atatggagtt ccgcgttaca taacttacgg taaatggccc gcctggctga ccgcccaacg
      241 acccccgccc attgacgtca ataatgacgt atgttcccat agtaacgcca atagggactt
      301 tccattgacg tcaatgggtg gagtatttac ggtaaactgc ccacttggca gtacatcaag
      361 tgtatcatat gccaagtccg ccccctattg acgtcaatga cggtaaatgg cccgcctggc
      421 attatgccca gtacatgacc ttacgggact ttcctacttg gcagtacatc tacgtattag
      481 tcatcgctat taccatggtg atgcggtttt ggcagtacac caatgggcgt ggatagcggt
      541 ttgactcacg gggatttcca agtctccacc ccattgacgt caatgggagt ttgttttggc
      601 accaaaatca acgggacttt ccaaaatgtc gtaataaccc cgccccgttg acgcaaatgg
      661 gcggtaggcg tgtacggtgg gaggtctata taagcagagc tcgtttagtg aaccgtcaga
      721 attgatctgg taccatggac tacaaagacc atgacggtga ttataaagat catgacatcg
      781 attacaagga tgacgatgac aagcttacaa gtttgtacaa aaaagcaggc ttaatgcaga
      841 atgtgattaa tactgtgaag ggaaaggcac tggaagtggc tgagtacctg accccggtcc
      901 tcaaggaatc aaagtttaag gaaacaggtg taattacccc agaagagttt gtggcagctg
      961 gagatcacct agtccaccac tgtccaacat ggcaatgggc tacaggggaa gaattgaaag
     1021 tgaaggcata cctaccaaca ggcaaacaat ttttggtaac caaaaatgtg ccgtgctata
     1081 agcggtgcaa acagatggaa tattcagatg aattggaagc tatcattgaa gaagatgatg
     1141 gtgatggcgg atgggtagat acatatcaca acacaggtat tacaggaata acggaagccg
     1201 ttaaagagat cacactggaa aataaggaca atataaggct tcaagattgc tcagcactat
     1261 gtgaagagga agaagatgaa gatgaaggag aagctgcaga tatggaagaa tatgaagaga
     1321 gtggattgtt ggaaacagat gaggctaccc tagatacaag gaaaatagta gaagcttgta
     1381 aagccaaaac tgatgctggc ggtgaagatg ctattttgca aaccagaact tatgaccttt
     1441 acatcactta tgataaatat taccagactc cacgattatg gttgtttggc tatgatgagc
     1501 aacggcagcc tttaacagtt gagcacatgt atgaagacat cagtcaggat catgtgaaga
     1561 aaacagtgac cattgaaaat caccctcatc tgccaccacc tcccatgtgt tcagttcacc
     1621 cagccaggca tgctgaggtg atgaagaaaa tcattgagac tgttgcagaa ggagggggag
     1681 aacttggagt tcatatgtat cttcttattt tcttgaaatt tgtacaagct gtcattccaa
     1741 caatagaata tgactacaca agacacttca caatgtaata cccagctttc ttgtacaaag
     1801 tggtgtgatc tagaggatcc cgggtggcat ccctgtgacc cctccccagt gcctctcctg
     1861 gccctggaag ttgccactcc agtgcccacc agccttgtcc taataaaatt aagttgcatc
     1921 attttgtctg actaggtgtc cttctataat attatggggt ggaggggggt ggtatggagc
     1981 aaggggcaag ttgggaagac aacctgtagg gcctgcgggg tctattggga accaagctgg
     2041 agtgcagtgg cacaatcttg gctcactgca atctccgcct cctgggttca agcgattctc
     2101 ctgcctcagc ctcccgagtt gttgggattc caggcatgca tgaccaggct cagctaattt
     2161 ttgttttttt ggtagagacg gggtttcacc atattggcca ggctggtctc caactcctaa
     2221 tctcaggtga tctacccacc ttggcctccc aaattgctgg gattacaggc gtgaaccact
     2281 gctcccttcc ctgtccttct gattttaaaa taactatacc agcaggagga cgtccagaca
     2341 cagcataggc tacctggcca tgcccaaccg gtgggacatt tgagttgctt gcttggcact
     2401 gtcctctcat gcgttgggtc cactcagtag atgcctgttg aattgggtac gcggccagct
     2461 tggctgtgga atgtgtgtca gttagggtgt ggaaagtccc caggctcccc agcaggcaga
     2521 agtatgcaaa gcatgcatct caattagtca gcaaccaggt gtggaaagtc cccaggctcc
     2581 ccagcaggca gaagtatgca aagcatgcat ctcaattagt cagcaaccat agtcccgccc
     2641 ctaactccgc ccatcccgcc cctaactccg cccagttccg cccattctcc gccccatggc
     2701 tgactaattt tttttattta tgcagaggcc gaggccgcct cggcctctga gctattccag
     2761 aagtagtgag gaggcttttt tggaggccta ggcttttgca aaaagctcct cgatcgagga
     2821 actgaaaaac cagaaagtta attccctata gtgagtcgta ttaaattcgt aatcatgtca
     2881 tagctgtttc ctgtgtgaaa ttgttatccg ctcacaattc cacacaacat acgagccgga
     2941 agcataaagt gtaaagcctg gggtgcctaa tgagcgagct aactcacatt aattgcgttg
     3001 cgctcactgc ccgctttcca gtcgggaaac ctgtcgtgcc agctgcatta atgaatcggc
     3061 caacgcgcgg ggagaggcgg tttgcgtatt gggcgctctt ccgcttcctc gctcactgac
     3121 tcgctgcgct cggtcgttcg gctgcggcga gcggtatcag ctcactcaaa ggcggtaata
     3181 cggttatcca cagaatcagg ggataacgca ggaaagaaca tgtgagcaaa aggccagcaa
     3241 aaggccagga accgtaaaaa ggccgcgttg ctggcgtttt tccataggct ccgcccccct
     3301 gacgagcatc acaaaaatcg acgctcaagt cagaggtggc gaaacccgac aggactataa
     3361 agataccagg cgtttccccc tggaagctcc ctcgtgcgct ctcctgttcc gaccctgccg
     3421 cttaccggat acctgtccgc ctttctccct tcgggaagcg tggcgctttc tcatagctca
     3481 cgctgtaggt atctcagttc ggtgtaggtc gttcgctcca agctgggctg tgtgcacgaa
     3541 ccccccgttc agcccgaccg ctgcgcctta tccggtaact atcgtcttga gtccaacccg
     3601 gtaagacacg acttatcgcc actggcagca gccactggta acaggattag cagagcgagg
     3661 tatgtaggcg gtgctacaga gttcttgaag tggtggccta actacggcta cactagaaga
     3721 acagtatttg gtatctgcgc tctgctgaag ccagttacct tcggaaaaag agttggtagc
     3781 tcttgatccg gcaaacaaac caccgctggt agcggtggtt tttttgtttg caagcagcag
     3841 attacgcgca gaaaaaaagg atctcaagaa gatcctttga tcttttctac ggggtctgac
     3901 gctcagtgga acgaaaactc acgttaaggg attttggtca tgagattatc aaaaaggatc
     3961 ttcacctaga tccttttaaa ttaaaaatga agttttaaat caatctaaag tatatatgag
     4021 taaacttggt ctgacagtta ccaatgctta atcagtgagg cacctatctc agcgatctgt
     4081 ctatttcgtt catccatagt tgcctgactc cccgtcgtgt agataactac gatacgggag
     4141 ggcttaccat ctggccccag tgctgcaatg ataccgcgag acccacgctc accggctcca
     4201 gatttatcag caataaacca gccagccgga agggccgagc gcagaagtgg tcctgcaact
     4261 ttatccgcct ccatccagtc tattaattgt tgccgggaag ctagagtaag tagttcgcca
     4321 gttaatagtt tgcgcaacgt tgttgccatt gctacaggca tcgtggtgtc acgctcgtcg
     4381 tttggtatgg cttcattcag ctccggttcc caacgatcaa ggcgagttac atgatccccc
     4441 atgttgtgca aaaaagcggt tagctccttc ggtcctccga tcgttgtcag aagtaagttg
     4501 gccgcagtgt tatcactcat ggttatggca gcactgcata attctcttac tgtcatgcca
     4561 tccgtaagat gcttttctgt gactggtgag tactcaacca agtcattctg agaatagtgt
     4621 atgcggcgac cgagttgctc ttgcccggcg tcaatacggg ataataccgc gccacatagc
     4681 agaactttaa aagtgctcat cattggaaaa cgttcttcgg ggcgaaaact ctcaaggatc
     4741 ttaccgctgt tgagatccag ttcgatgtaa cccactcgtg cacccaactg atcttcagca
     4801 tcttttactt tcaccagcgt ttctgggtga gcaaaaacag gaaggcaaaa tgccgcaaaa
     4861 aagggaataa gggcgacacg gaaatgttga atactcatac tcttcctttt tcaatattat
     4921 tgaagcattt atcagggtta ttgtctcatg agcggataca tatttgaatg tatttagaaa
     4981 aataaacaaa taggggttcc gcgcacattt ccccgaaaag tgccacctga cgcgccctgt
     5041 agcggcgcat taagcgcggc gggtgtggtg gttacgcgca gcgtgaccgc tacacttgcc
     5101 agcgccctag cgcccgctcc tttcgctttc ttcccttcct ttctcgccac gttcgccggc
     5161 tttccccgtc aagctctaaa tcgggggctc cctttagggt tccgatttag tgctttacgg
     5221 cacctcgacc ccaaaaaact tgattagggt gatggttcac gtagtgggcc atcgccctga
     5281 tagacggttt ttcgcccttt gacgttggag tccacgttct ttaatagtgg actcttgttc
     5341 caaactggaa caacactcaa ccctatctcg gtctattctt ttgatttata agggattttg
     5401 ccgatttcgg cctattggtt aaaaaatgag ctgatttaac aaaaatttaa cgcgaatttt
     5461 aacaaaatat taacgcttac aatttccatt cgccattcag gctgcgcaac tgttgggaag
     5521 ggcgatcggt gcgggcctct tcgctattac gccagctggc gaaaggggga tgtgctgcaa
     5581 ggcgattaag ttgggtaacg ccagggtttt cccagtcacg acgttgtaaa acgacggcca
     5641 gtgccaagct gatctataca ttgaatcaat attggcaatt agcca
//
