LOCUS       sequence-252994-        5685 bp    DNA     circular SYN 15-APR-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 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 Apr 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..5685
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     enhancer        115..494
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
     promoter        495..698
                     /label=CMV promoter
                     /note="human cytomegalovirus (CMV) immediate early 
                     promoter"
     primer_bind     649..669
                     /label=CMV-F
                     /note="Human CMV immediate early promoter, forward primer"
     CDS             735..800
                     /codon_start=1
                     /product="three tandem FLAG(R) epitope tags, followed by an
                     enterokinase cleavage site"
                     /label=3xFLAG
                     /translation="DYKDHDGDYKDHDIDYKDDDDK"
     protein_bind    804..828
                     /gene="mutant version of attB"
                     /label=attB1
                     /bound_moiety="BP Clonase(TM)"
                     /note="recombination site for the Gateway(R) BP reaction"
     protein_bind    complement(1777..1801)
                     /gene="mutant version of attB"
                     /label=attB2
                     /bound_moiety="BP Clonase(TM)"
                     /note="recombination site for the Gateway(R) BP reaction"
     polyA_signal    1819..2441
                     /label=hGH poly(A) signal
                     /note="human growth hormone polyadenylation signal"
     primer_bind     complement(2018..2037)
                     /label=hGH-PA-R
                     /note="Human growth hormone terminator, reverse primer"
     primer_bind     complement(2465..2485)
                     /label=pBABE 3'
                     /note="SV40 enhancer, reverse primer for pBABE vectors"
     promoter        2470..2799
                     /label=SV40 promoter
                     /note="SV40 enhancer and early promoter"
     rep_origin      2650..2785
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     primer_bind     complement(2841..2860)
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        complement(2842..2860)
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     primer_bind     complement(2874..2890)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(2874..2890)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(2887..2909)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    2898..2914
                     /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(2922..2928,2929..2946,2947..2952))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     primer_bind     complement(3105..3122)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(3276..3864)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(3356..3375)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(4035..4826,4827..4895))
                     /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     4658..4677
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(4896..5000)
                     /gene="bla"
                     /label=AmpR promoter
     rep_origin      5027..5482
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(5114..5133)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     5324..5345
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     5608..5630
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     primer_bind     5622..5639
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     5623..5639
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
ORIGIN
        1 tagtcattgg ttatatagca taaatcaata ttggctattg gccattgcat acgttgtatc
       61 tatatcataa tatgtacatt tatattggct catgtccaat atgaccgcca tgttgacatt
      121 gattattgac tagttattaa tagtaatcaa ttacggggtc attagttcat agcccatata
      181 tggagttccg cgttacataa cttacggtaa atggcccgcc tggctgaccg cccaacgacc
      241 cccgcccatt gacgtcaata atgacgtatg ttcccatagt aacgccaata gggactttcc
      301 attgacgtca atgggtggag tatttacggt aaactgccca cttggcagta catcaagtgt
      361 atcatatgcc aagtccgccc cctattgacg tcaatgacgg taaatggccc gcctggcatt
      421 atgcccagta catgacctta cgggactttc ctacttggca gtacatctac gtattagtca
      481 tcgctattac catggtgatg cggttttggc agtacaccaa tgggcgtgga tagcggtttg
      541 actcacgggg atttccaagt ctccacccca ttgacgtcaa tgggagtttg ttttggcacc
      601 aaaatcaacg ggactttcca aaatgtcgta ataaccccgc cccgttgacg caaatgggcg
      661 gtaggcgtgt acggtgggag gtctatataa gcagagctcg tttagtgaac cgtcagaatt
      721 gatctggtac catggactac aaagaccatg acggtgatta taaagatcat gacatcgatt
      781 acaaggatga cgatgacaag cttacaagtt tgtacaaaaa agcaggctta atgcagaatg
      841 tgattaatac tgtgaaggga aaggcactgg aagtggctga gtacctgacc ccggtcctca
      901 aggaatcaaa gtttaaggaa acaggtgtaa ttaccccaga agagtttgtg gcagctggag
      961 atcacctagt ccaccactgt ccaacatggc aatgggctac aggggaagaa ttgaaagtga
     1021 aggcatacct accaacaggc aaacaatttt tggtaaccaa aaatgtgccg tgctataagc
     1081 ggtgcaaaca gatggaatat tcagatgaat tggaagctat cattgaagaa gatgatggtg
     1141 atggcggatg ggtagataca tatcacaaca caggtattac aggaataacg gaagccgtta
     1201 aagagatcac actggaaaat aaggacaata taaggcttca agattgctca gcactatgtg
     1261 aagaggaaga agatgaagat gaaggagaag ctgcagatat ggaagaatat gaagagagtg
     1321 gattgttgga aacagatgag gctaccctag atacaaggaa aatagtagaa gcttgtaaag
     1381 ccaaaactga tgctggcggt gaagatgcta ttttgcaaac cagaacttat gacctttaca
     1441 tcacttatga taaatattac cagactccac gattatggtt gtttggctat gatgagcaac
     1501 ggcagccttt aacagttgag cacatgtatg aagacatcag tcaggatcat gtgaagaaaa
     1561 cagtgaccat tgaaaatcac cctcatctgc caccacctcc catgtgttca gttcacccat
     1621 gcaggcatgc tgaggtgatg aagaaaatca ttgagactgt tgcagaagga gggggagaac
     1681 ttggagttca tatgtatctt cttattttct tgagatttgt acaagctgtc attccaacaa
     1741 tagaatatga ctacacaaga cacttcacaa tgtaataccc agctttcttg tacaaagtgg
     1801 tgtgatctag aggatcccgg gtggcatccc tgtgacccct ccccagtgcc tctcctggcc
     1861 ctggaagttg ccactccagt gcccaccagc cttgtcctaa taaaattaag ttgcatcatt
     1921 ttgtctgact aggtgtcctt ctataatatt atggggtgga ggggggtggt atggagcaag
     1981 gggcaagttg ggaagacaac ctgtagggcc tgcggggtct attgggaacc aagctggagt
     2041 gcagtggcac aatcttggct cactgcaatc tccgcctcct gggttcaagc gattctcctg
     2101 cctcagcctc ccgagttgtt gggattccag gcatgcatga ccaggctcag ctaatttttg
     2161 tttttttggt agagacgggg tttcaccata ttggccaggc tggtctccaa ctcctaatct
     2221 caggtgatct acccaccttg gcctcccaaa ttgctgggat tacaggcgtg aaccactgct
     2281 cccttccctg tccttctgat tttaaaataa ctataccagc aggaggacgt ccagacacag
     2341 cataggctac ctggccatgc ccaaccggtg ggacatttga gttgcttgct tggcactgtc
     2401 ctctcatgcg ttgggtccac tcagtagatg cctgttgaat tgggtacgcg gccagcttgg
     2461 ctgtggaatg tgtgtcagtt agggtgtgga aagtccccag gctccccagc aggcagaagt
     2521 atgcaaagca tgcatctcaa ttagtcagca accaggtgtg gaaagtcccc aggctcccca
     2581 gcaggcagaa gtatgcaaag catgcatctc aattagtcag caaccatagt cccgccccta
     2641 actccgccca tcccgcccct aactccgccc agttccgccc attctccgcc ccatggctga
     2701 ctaatttttt ttatttatgc agaggccgag gccgcctcgg cctctgagct attccagaag
     2761 tagtgaggag gcttttttgg aggcctaggc ttttgcaaaa agctcctcga tcgaggaact
     2821 gaaaaaccag aaagttaatt ccctatagtg agtcgtatta aattcgtaat catgtcatag
     2881 ctgtttcctg tgtgaaattg ttatccgctc acaattccac acaacatacg agccggaagc
     2941 ataaagtgta aagcctgggg tgcctaatga gcgagctaac tcacattaat tgcgttgcgc
     3001 tcactgcccg ctttccagtc gggaaacctg tcgtgccagc tgcattaatg aatcggccaa
     3061 cgcgcgggga gaggcggttt gcgtattggg cgctcttccg cttcctcgct cactgactcg
     3121 ctgcgctcgg tcgttcggct gcggcgagcg gtatcagctc actcaaaggc ggtaatacgg
     3181 ttatccacag aatcagggga taacgcagga aagaacatgt gagcaaaagg ccagcaaaag
     3241 gccaggaacc gtaaaaaggc cgcgttgctg gcgtttttcc ataggctccg cccccctgac
     3301 gagcatcaca aaaatcgacg ctcaagtcag aggtggcgaa acccgacagg actataaaga
     3361 taccaggcgt ttccccctgg aagctccctc gtgcgctctc ctgttccgac cctgccgctt
     3421 accggatacc tgtccgcctt tctcccttcg ggaagcgtgg cgctttctca tagctcacgc
     3481 tgtaggtatc tcagttcggt gtaggtcgtt cgctccaagc tgggctgtgt gcacgaaccc
     3541 cccgttcagc ccgaccgctg cgccttatcc ggtaactatc gtcttgagtc caacccggta
     3601 agacacgact tatcgccact ggcagcagcc actggtaaca ggattagcag agcgaggtat
     3661 gtaggcggtg ctacagagtt cttgaagtgg tggcctaact acggctacac tagaagaaca
     3721 gtatttggta tctgcgctct gctgaagcca gttaccttcg gaaaaagagt tggtagctct
     3781 tgatccggca aacaaaccac cgctggtagc ggtggttttt ttgtttgcaa gcagcagatt
     3841 acgcgcagaa aaaaaggatc tcaagaagat cctttgatct tttctacggg gtctgacgct
     3901 cagtggaacg aaaactcacg ttaagggatt ttggtcatga gattatcaaa aaggatcttc
     3961 acctagatcc ttttaaatta aaaatgaagt tttaaatcaa tctaaagtat atatgagtaa
     4021 acttggtctg acagttacca atgcttaatc agtgaggcac ctatctcagc gatctgtcta
     4081 tttcgttcat ccatagttgc ctgactcccc gtcgtgtaga taactacgat acgggagggc
     4141 ttaccatctg gccccagtgc tgcaatgata ccgcgagacc cacgctcacc ggctccagat
     4201 ttatcagcaa taaaccagcc agccggaagg gccgagcgca gaagtggtcc tgcaacttta
     4261 tccgcctcca tccagtctat taattgttgc cgggaagcta gagtaagtag ttcgccagtt
     4321 aatagtttgc gcaacgttgt tgccattgct acaggcatcg tggtgtcacg ctcgtcgttt
     4381 ggtatggctt cattcagctc cggttcccaa cgatcaaggc gagttacatg atcccccatg
     4441 ttgtgcaaaa aagcggttag ctccttcggt cctccgatcg ttgtcagaag taagttggcc
     4501 gcagtgttat cactcatggt tatggcagca ctgcataatt ctcttactgt catgccatcc
     4561 gtaagatgct tttctgtgac tggtgagtac tcaaccaagt cattctgaga atagtgtatg
     4621 cggcgaccga gttgctcttg cccggcgtca atacgggata ataccgcgcc acatagcaga
     4681 actttaaaag tgctcatcat tggaaaacgt tcttcggggc gaaaactctc aaggatctta
     4741 ccgctgttga gatccagttc gatgtaaccc actcgtgcac ccaactgatc ttcagcatct
     4801 tttactttca ccagcgtttc tgggtgagca aaaacaggaa ggcaaaatgc cgcaaaaaag
     4861 ggaataaggg cgacacggaa atgttgaata ctcatactct tcctttttca atattattga
     4921 agcatttatc agggttattg tctcatgagc ggatacatat ttgaatgtat ttagaaaaat
     4981 aaacaaatag gggttccgcg cacatttccc cgaaaagtgc cacctgacgc gccctgtagc
     5041 ggcgcattaa gcgcggcggg tgtggtggtt acgcgcagcg tgaccgctac acttgccagc
     5101 gccctagcgc ccgctccttt cgctttcttc ccttcctttc tcgccacgtt cgccggcttt
     5161 ccccgtcaag ctctaaatcg ggggctccct ttagggttcc gatttagtgc tttacggcac
     5221 ctcgacccca aaaaacttga ttagggtgat ggttcacgta gtgggccatc gccctgatag
     5281 acggtttttc gccctttgac gttggagtcc acgttcttta atagtggact cttgttccaa
     5341 actggaacaa cactcaaccc tatctcggtc tattcttttg atttataagg gattttgccg
     5401 atttcggcct attggttaaa aaatgagctg atttaacaaa aatttaacgc gaattttaac
     5461 aaaatattaa cgcttacaat ttccattcgc cattcaggct gcgcaactgt tgggaagggc
     5521 gatcggtgcg ggcctcttcg ctattacgcc agctggcgaa agggggatgt gctgcaaggc
     5581 gattaagttg ggtaacgcca gggttttccc agtcacgacg ttgtaaaacg acggccagtg
     5641 ccaagctgat ctatacattg aatcaatatt ggcaattagc catat
//
