LOCUS       sequence-244427-        5118 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Expresses 3xFLAG-LC3B wild-type in mammalian cells..
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 5118)
  AUTHORS   Agrotis A, Pengo N, Burden JJ, Ketteler R
  TITLE     Redundancy of human ATG4 protease isoforms in autophagy and 
            LC3/GABARAP processing revealed in cells.
  JOURNAL   Autophagy. 2019 Jan 21. doi: 10.1080/15548627.2019.1569925.
  PUBMED    30661429
REFERENCE   2  (bases 1 to 5118)
  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: "Autophagy. 
            2019 Jan 21. doi: 10.1080/15548627.2019.1569925."
COMMENT     Sequence Label: pCMV 3xFLAG-LC3B WT
FEATURES             Location/Qualifiers
     source          1..5118
                     /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..2227
                     /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     2353..2375
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     primer_bind     2367..2384
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     2368..2384
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     enhancer        2545..2924
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
     promoter        2925..3128
                     /label=CMV promoter
                     /note="human cytomegalovirus (CMV) immediate early 
                     promoter"
     primer_bind     3079..3099
                     /label=CMV-F
                     /note="Human CMV immediate early promoter, forward primer"
     CDS             3165..3230
                     /codon_start=1
                     /product="three tandem FLAG(R) epitope tags, followed by an
                     enterokinase cleavage site"
                     /label=3xFLAG
                     /translation="DYKDHDGDYKDHDIDYKDDDDK"
     protein_bind    3234..3258
                     /gene="mutant version of attB"
                     /label=attB1
                     /bound_moiety="BP Clonase(TM)"
                     /note="recombination site for the Gateway(R) BP reaction"
     CDS             3261..3635
                     /codon_start=1
                     /gene="MAP1LC3B"
                     /product="human LC3 protein"
                     /label=LC3B
                     /note="Atg8 family member found on autophagosomes"
                     /translation="MPSEKTFKQRRTFEQRVEDVRLIREQHPTKIPVIIERYKGEKQLP
                     VLDKTKFLVPDHVNMSELIKIIRRRLQLNANQAFFLLVNGHSMVSVSTPISEVYESEKD
                     EDGFLYMVYASQETFGMKLSV"
     protein_bind    complement(3640..3664)
                     /gene="mutant version of attB"
                     /label=attB2
                     /bound_moiety="BP Clonase(TM)"
                     /note="recombination site for the Gateway(R) BP reaction"
     polyA_signal    3682..4304
                     /label=hGH poly(A) signal
                     /note="human growth hormone polyadenylation signal"
     primer_bind     complement(3881..3900)
                     /label=hGH-PA-R
                     /note="Human growth hormone terminator, reverse primer"
     primer_bind     complement(4328..4348)
                     /label=pBABE 3'
                     /note="SV40 enhancer, reverse primer for pBABE vectors"
     promoter        4333..4662
                     /label=SV40 promoter
                     /note="SV40 enhancer and early promoter"
     rep_origin      4513..4648
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     primer_bind     complement(4704..4723)
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        complement(4705..4723)
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     primer_bind     complement(4737..4753)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(4737..4753)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(4750..4772)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    4761..4777
                     /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(4785..4791,4792..4809,4810..4815))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     primer_bind     complement(4968..4985)
                     /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 tctcatagct 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 gaacagtatt 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 aatcgggggc 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 attaacgctt
     2221 acaatttcca ttcgccattc aggctgcgca actgttggga agggcgatcg gtgcgggcct
     2281 cttcgctatt acgccagctg gcgaaagggg gatgtgctgc aaggcgatta agttgggtaa
     2341 cgccagggtt ttcccagtca cgacgttgta aaacgacggc cagtgccaag ctgatctata
     2401 cattgaatca atattggcaa ttagccatat tagtcattgg ttatatagca taaatcaata
     2461 ttggctattg gccattgcat acgttgtatc tatatcataa tatgtacatt tatattggct
     2521 catgtccaat atgaccgcca tgttgacatt gattattgac tagttattaa tagtaatcaa
     2581 ttacggggtc attagttcat agcccatata tggagttccg cgttacataa cttacggtaa
     2641 atggcccgcc tggctgaccg cccaacgacc cccgcccatt gacgtcaata atgacgtatg
     2701 ttcccatagt aacgccaata gggactttcc attgacgtca atgggtggag tatttacggt
     2761 aaactgccca cttggcagta catcaagtgt atcatatgcc aagtccgccc cctattgacg
     2821 tcaatgacgg taaatggccc gcctggcatt atgcccagta catgacctta cgggactttc
     2881 ctacttggca gtacatctac gtattagtca tcgctattac catggtgatg cggttttggc
     2941 agtacaccaa tgggcgtgga tagcggtttg actcacgggg atttccaagt ctccacccca
     3001 ttgacgtcaa tgggagtttg ttttggcacc aaaatcaacg ggactttcca aaatgtcgta
     3061 ataaccccgc cccgttgacg caaatgggcg gtaggcgtgt acggtgggag gtctatataa
     3121 gcagagctcg tttagtgaac cgtcagaatt gatctggtac catggactac aaagaccatg
     3181 acggtgatta taaagatcat gacatcgatt acaaggatga cgatgacaag cttacaagtt
     3241 tgtacaaaaa agcaggctta atgccgtcgg agaagacctt caagcagcgg cgcaccttcg
     3301 aacaaagagt agaagatgtc cgacttattc gagagcagca tccaaccaaa atcccggtga
     3361 taatagaacg atacaagggt gagaagcagc ttcctgttct ggataaaaca aagttccttg
     3421 tacctgacca tgtcaacatg agtgagctca tcaagataat tagaaggcgc ttacagctca
     3481 atgctaatca ggccttcttc ctgttggtga acggacacag catggtcagc gtctccacac
     3541 caatctcaga ggtgtatgag agtgagaaag atgaagatgg attcctgtac atggtctatg
     3601 cctcccagga gacgttcggg atgaaattgt cagtgtaata cccagctttc ttgtacaaag
     3661 tggtgtgatc tagaggatcc cgggtggcat ccctgtgacc cctccccagt gcctctcctg
     3721 gccctggaag ttgccactcc agtgcccacc agccttgtcc taataaaatt aagttgcatc
     3781 attttgtctg actaggtgtc cttctataat attatggggt ggaggggggt ggtatggagc
     3841 aaggggcaag ttgggaagac aacctgtagg gcctgcgggg tctattggga accaagctgg
     3901 agtgcagtgg cacaatcttg gctcactgca atctccgcct cctgggttca agcgattctc
     3961 ctgcctcagc ctcccgagtt gttgggattc caggcatgca tgaccaggct cagctaattt
     4021 ttgttttttt ggtagagacg gggtttcacc atattggcca ggctggtctc caactcctaa
     4081 tctcaggtga tctacccacc ttggcctccc aaattgctgg gattacaggc gtgaaccact
     4141 gctcccttcc ctgtccttct gattttaaaa taactatacc agcaggagga cgtccagaca
     4201 cagcataggc tacctggcca tgcccaaccg gtgggacatt tgagttgctt gcttggcact
     4261 gtcctctcat gcgttgggtc cactcagtag atgcctgttg aattgggtac gcggccagct
     4321 tggctgtgga atgtgtgtca gttagggtgt ggaaagtccc caggctcccc agcaggcaga
     4381 agtatgcaaa gcatgcatct caattagtca gcaaccaggt gtggaaagtc cccaggctcc
     4441 ccagcaggca gaagtatgca aagcatgcat ctcaattagt cagcaaccat agtcccgccc
     4501 ctaactccgc ccatcccgcc cctaactccg cccagttccg cccattctcc gccccatggc
     4561 tgactaattt tttttattta tgcagaggcc gaggccgcct cggcctctga gctattccag
     4621 aagtagtgag gaggcttttt tggaggccta ggcttttgca aaaagctcct cgatcgagga
     4681 actgaaaaac cagaaagtta attccctata gtgagtcgta ttaaattcgt aatcatgtca
     4741 tagctgtttc ctgtgtgaaa ttgttatccg ctcacaattc cacacaacat acgagccgga
     4801 agcataaagt gtaaagcctg gggtgcctaa tgagcgagct aactcacatt aattgcgttg
     4861 cgctcactgc ccgctttcca gtcgggaaac ctgtcgtgcc agctgcatta atgaatcggc
     4921 caacgcgcgg ggagaggcgg tttgcgtatt gggcgctctt ccgcttcctc gctcactgac
     4981 tcgctgcgct cggtcgttcg gctgcggcga gcggtatcag ctcactcaaa ggcggtaata
     5041 cggttatcca cagaatcagg ggataacgca ggaaagaaca tgtgagcaaa aggccagcaa
     5101 aaggccagga accgtaaa
//
