LOCUS       sequence-252996-        5103 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Expresses 3xFLAG-tagged deconjugation-resistant LC3B in mammalian 
            cells.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 5103)
  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 5103)
  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-LC3B Q116P G120
FEATURES             Location/Qualifiers
     source          1..5103
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     67..89
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     primer_bind     81..98
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     82..98
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     enhancer        259..638
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
     promoter        639..842
                     /label=CMV promoter
                     /note="human cytomegalovirus (CMV) immediate early 
                     promoter"
     primer_bind     793..813
                     /label=CMV-F
                     /note="Human CMV immediate early promoter, forward primer"
     CDS             879..944
                     /codon_start=1
                     /product="three tandem FLAG(R) epitope tags, followed by an
                     enterokinase cleavage site"
                     /label=3xFLAG
                     /translation="DYKDHDGDYKDHDIDYKDDDDK"
     protein_bind    948..972
                     /gene="mutant version of attB"
                     /label=attB1
                     /bound_moiety="BP Clonase(TM)"
                     /note="recombination site for the Gateway(R) BP reaction"
     protein_bind    complement(1339..1363)
                     /gene="mutant version of attB"
                     /label=attB2
                     /bound_moiety="BP Clonase(TM)"
                     /note="recombination site for the Gateway(R) BP reaction"
     polyA_signal    1381..2003
                     /label=hGH poly(A) signal
                     /note="human growth hormone polyadenylation signal"
     primer_bind     complement(1580..1599)
                     /label=hGH-PA-R
                     /note="Human growth hormone terminator, reverse primer"
     primer_bind     complement(2027..2047)
                     /label=pBABE 3'
                     /note="SV40 enhancer, reverse primer for pBABE vectors"
     promoter        2032..2361
                     /label=SV40 promoter
                     /note="SV40 enhancer and early promoter"
     rep_origin      2212..2347
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     primer_bind     complement(2403..2422)
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        complement(2404..2422)
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     primer_bind     complement(2436..2452)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(2436..2452)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(2449..2471)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    2460..2476
                     /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(2484..2490,2491..2508,2509..2514))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     primer_bind     complement(2667..2684)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(2838..3426)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(2918..2937)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(3597..4388,4389..4457))
                     /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     4220..4239
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(4458..4562)
                     /gene="bla"
                     /label=AmpR promoter
     rep_origin      4589..5044
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(4676..4695)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     4886..4907
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
ORIGIN
        1 tattacgcca gctggcgaaa gggggatgtg ctgcaaggcg attaagttgg gtaacgccag
       61 ggttttccca gtcacgacgt tgtaaaacga cggccagtgc caagctgatc tatacattga
      121 atcaatattg gcaattagcc atattagtca ttggttatat agcataaatc aatattggct
      181 attggccatt gcatacgttg tatctatatc ataatatgta catttatatt ggctcatgtc
      241 caatatgacc gccatgttga cattgattat tgactagtta ttaatagtaa tcaattacgg
      301 ggtcattagt tcatagccca tatatggagt tccgcgttac ataacttacg gtaaatggcc
      361 cgcctggctg accgcccaac gacccccgcc cattgacgtc aataatgacg tatgttccca
      421 tagtaacgcc aatagggact ttccattgac gtcaatgggt ggagtattta cggtaaactg
      481 cccacttggc agtacatcaa gtgtatcata tgccaagtcc gccccctatt gacgtcaatg
      541 acggtaaatg gcccgcctgg cattatgccc agtacatgac cttacgggac tttcctactt
      601 ggcagtacat ctacgtatta gtcatcgcta ttaccatggt gatgcggttt tggcagtaca
      661 ccaatgggcg tggatagcgg tttgactcac ggggatttcc aagtctccac cccattgacg
      721 tcaatgggag tttgttttgg caccaaaatc aacgggactt tccaaaatgt cgtaataacc
      781 ccgccccgtt gacgcaaatg ggcggtaggc gtgtacggtg ggaggtctat ataagcagag
      841 ctcgtttagt gaaccgtcag aattgatctg gtaccatgga ctacaaagac catgacggtg
      901 attataaaga tcatgacatc gattacaagg atgacgatga caagcttaca agtttgtaca
      961 aaaaagcagg cttaatgccg tcggagaaga ccttcaagca gcggcgcacc ttcgaacaaa
     1021 gagtagaaga tgtccgactt attcgagagc agcatccaac caaaatcccg gtgataatag
     1081 aacgatacaa gggtgagaag cagcttcctg ttctggataa aacaaagttc cttgtacctg
     1141 accatgtcaa catgagtgag ctcatcaaga taattagaag gcgcttacag ctcaatgcta
     1201 atcaggcctt cttcctgttg gtgaacggac acagcatggt cagcgtctcc acaccaatct
     1261 cagaggtgta tgagagtgag aaagatgaag atggattcct gtacatggtc tatgcctccc
     1321 cggagacgtt cgggtaatac ccagctttct tgtacaaagt ggtgtgatct agaggatccc
     1381 gggtggcatc cctgtgaccc ctccccagtg cctctcctgg ccctggaagt tgccactcca
     1441 gtgcccacca gccttgtcct aataaaatta agttgcatca ttttgtctga ctaggtgtcc
     1501 ttctataata ttatggggtg gaggggggtg gtatggagca aggggcaagt tgggaagaca
     1561 acctgtaggg cctgcggggt ctattgggaa ccaagctgga gtgcagtggc acaatcttgg
     1621 ctcactgcaa tctccgcctc ctgggttcaa gcgattctcc tgcctcagcc tcccgagttg
     1681 ttgggattcc aggcatgcat gaccaggctc agctaatttt tgtttttttg gtagagacgg
     1741 ggtttcacca tattggccag gctggtctcc aactcctaat ctcaggtgat ctacccacct
     1801 tggcctccca aattgctggg attacaggcg tgaaccactg ctcccttccc tgtccttctg
     1861 attttaaaat aactatacca gcaggaggac gtccagacac agcataggct acctggccat
     1921 gcccaaccgg tgggacattt gagttgcttg cttggcactg tcctctcatg cgttgggtcc
     1981 actcagtaga tgcctgttga attgggtacg cggccagctt ggctgtggaa tgtgtgtcag
     2041 ttagggtgtg gaaagtcccc aggctcccca gcaggcagaa gtatgcaaag catgcatctc
     2101 aattagtcag caaccaggtg tggaaagtcc ccaggctccc cagcaggcag aagtatgcaa
     2161 agcatgcatc tcaattagtc agcaaccata gtcccgcccc taactccgcc catcccgccc
     2221 ctaactccgc ccagttccgc ccattctccg ccccatggct gactaatttt ttttatttat
     2281 gcagaggccg aggccgcctc ggcctctgag ctattccaga agtagtgagg aggctttttt
     2341 ggaggcctag gcttttgcaa aaagctcctc gatcgaggaa ctgaaaaacc agaaagttaa
     2401 ttccctatag tgagtcgtat taaattcgta atcatgtcat agctgtttcc tgtgtgaaat
     2461 tgttatccgc tcacaattcc acacaacata cgagccggaa gcataaagtg taaagcctgg
     2521 ggtgcctaat gagcgagcta actcacatta attgcgttgc gctcactgcc cgctttccag
     2581 tcgggaaacc tgtcgtgcca gctgcattaa tgaatcggcc aacgcgcggg gagaggcggt
     2641 ttgcgtattg ggcgctcttc cgcttcctcg ctcactgact cgctgcgctc ggtcgttcgg
     2701 ctgcggcgag cggtatcagc tcactcaaag gcggtaatac ggttatccac agaatcaggg
     2761 gataacgcag gaaagaacat gtgagcaaaa ggccagcaaa aggccaggaa ccgtaaaaag
     2821 gccgcgttgc tggcgttttt ccataggctc cgcccccctg acgagcatca caaaaatcga
     2881 cgctcaagtc agaggtggcg aaacccgaca ggactataaa gataccaggc gtttccccct
     2941 ggaagctccc tcgtgcgctc tcctgttccg accctgccgc ttaccggata cctgtccgcc
     3001 tttctccctt cgggaagcgt ggcgctttct catagctcac gctgtaggta tctcagttcg
     3061 gtgtaggtcg ttcgctccaa gctgggctgt gtgcacgaac cccccgttca gcccgaccgc
     3121 tgcgccttat ccggtaacta tcgtcttgag tccaacccgg taagacacga cttatcgcca
     3181 ctggcagcag ccactggtaa caggattagc agagcgaggt atgtaggcgg tgctacagag
     3241 ttcttgaagt ggtggcctaa ctacggctac actagaagaa cagtatttgg tatctgcgct
     3301 ctgctgaagc cagttacctt cggaaaaaga gttggtagct cttgatccgg caaacaaacc
     3361 accgctggta gcggtggttt ttttgtttgc aagcagcaga ttacgcgcag aaaaaaagga
     3421 tctcaagaag atcctttgat cttttctacg gggtctgacg ctcagtggaa cgaaaactca
     3481 cgttaaggga ttttggtcat gagattatca aaaaggatct tcacctagat ccttttaaat
     3541 taaaaatgaa gttttaaatc aatctaaagt atatatgagt aaacttggtc tgacagttac
     3601 caatgcttaa tcagtgaggc acctatctca gcgatctgtc tatttcgttc atccatagtt
     3661 gcctgactcc ccgtcgtgta gataactacg atacgggagg gcttaccatc tggccccagt
     3721 gctgcaatga taccgcgaga cccacgctca ccggctccag atttatcagc aataaaccag
     3781 ccagccggaa gggccgagcg cagaagtggt cctgcaactt tatccgcctc catccagtct
     3841 attaattgtt gccgggaagc tagagtaagt agttcgccag ttaatagttt gcgcaacgtt
     3901 gttgccattg ctacaggcat cgtggtgtca cgctcgtcgt ttggtatggc ttcattcagc
     3961 tccggttccc aacgatcaag gcgagttaca tgatccccca tgttgtgcaa aaaagcggtt
     4021 agctccttcg gtcctccgat cgttgtcaga agtaagttgg ccgcagtgtt atcactcatg
     4081 gttatggcag cactgcataa ttctcttact gtcatgccat ccgtaagatg cttttctgtg
     4141 actggtgagt actcaaccaa gtcattctga gaatagtgta tgcggcgacc gagttgctct
     4201 tgcccggcgt caatacggga taataccgcg ccacatagca gaactttaaa agtgctcatc
     4261 attggaaaac gttcttcggg gcgaaaactc tcaaggatct taccgctgtt gagatccagt
     4321 tcgatgtaac ccactcgtgc acccaactga tcttcagcat cttttacttt caccagcgtt
     4381 tctgggtgag caaaaacagg aaggcaaaat gccgcaaaaa agggaataag ggcgacacgg
     4441 aaatgttgaa tactcatact cttccttttt caatattatt gaagcattta tcagggttat
     4501 tgtctcatga gcggatacat atttgaatgt atttagaaaa ataaacaaat aggggttccg
     4561 cgcacatttc cccgaaaagt gccacctgac gcgccctgta gcggcgcatt aagcgcggcg
     4621 ggtgtggtgg ttacgcgcag cgtgaccgct acacttgcca gcgccctagc gcccgctcct
     4681 ttcgctttct tcccttcctt tctcgccacg ttcgccggct ttccccgtca agctctaaat
     4741 cgggggctcc ctttagggtt ccgatttagt gctttacggc acctcgaccc caaaaaactt
     4801 gattagggtg atggttcacg tagtgggcca tcgccctgat agacggtttt tcgccctttg
     4861 acgttggagt ccacgttctt taatagtgga ctcttgttcc aaactggaac aacactcaac
     4921 cctatctcgg tctattcttt tgatttataa gggattttgc cgatttcggc ctattggtta
     4981 aaaaatgagc tgatttaaca aaaatttaac gcgaatttta acaaaatatt aacgcttaca
     5041 atttccattc gccattcagg ctgcgcaact gttgggaagg gcgatcggtg cgggcctctt
     5101 cgc
//
