LOCUS       sequence-457349-        5095 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Encodes a donor-only, Aquamarine-tagged version of the inactive LC3B
            biosensor under the control of the CMV promoter.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 5095)
  AUTHORS   Gokerkucuk EB, Cheron A, Tramier M, Bertolin G
  TITLE     The LC3B FRET biosensor monitors the modes of action of ATG4B during
            autophagy in living cells.
  JOURNAL   Autophagy. 2023 Aug;19(8):2275-2295. doi: 
            10.1080/15548627.2023.2179845. Epub 2023 Feb 22.
  PUBMED    36814061
REFERENCE   2  (bases 1 to 5095)
  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"; doi: 
            "10.1080/15548627.2023.2179845"; journalName: "Autophagy"; date: 
            "2023-08"; volume: "19"; issue: "8"; pages: "2275-2295"
COMMENT     Sequence Label: Aquamarine-LC3B G120A
FEATURES             Location/Qualifiers
     source          1..5095
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     rep_origin      313..901
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     802..821
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     enhancer        1076..1379
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
     promoter        1380..1583
                     /label=CMV promoter
                     /note="human cytomegalovirus (CMV) immediate early 
                     promoter"
     primer_bind     1534..1554
                     /label=CMV-F
                     /note="Human CMV immediate early promoter, forward primer"
     regulatory      1622..1631
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     CDS             join(1628..1630,1631..1633,1634..2344)
                     /codon_start=1
                     /product="enhanced CFP"
                     /label=ECFP
                     /note="mammalian codon-optimized"
                     /translation="MVSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTL
                     KFICTTGKLPVPWPTLVTTLSWGVQCFSRYPDHMKQHDFFKSAMPEGYVQERTIFFKDD
                     GNYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYISGNVYITADKQKNGIK
                     ANFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSALSKDPNEKRDHMVLL
                     EFVTAAGITLGMDELYK"
     primer_bind     complement(1673..1694)
                     /label=EGFP-N
                     /note="EGFP, reverse primer"
     primer_bind     complement(1934..1953)
                     /label=EXFP-R
                     /note="For distinguishing EGFP variants, reverse primer"
     primer_bind     2281..2302
                     /label=EGFP-C
                     /note="EGFP, forward primer"
     CDS             2360..2734
                     /codon_start=1
                     /gene="MAP1LC3B"
                     /product="human LC3 protein"
                     /label=LC3B
                     /note="Atg8 family member found on autophagosomes"
                     /translation="MPSEKTFKQRRTFEQRVEDVRLIREQHPTKIPVIIERYKGEKQLP
                     VLDKTKFLVPDHVNMSELIKIIRRRLQLNANQAFFLLVNGHSMVSVSTPISEVYESEKD
                     EDGFLYMVYASQETFAMKLSV"
     polyA_signal    2898..3019
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(2935..2954)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     2989..3008
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     rep_origin      complement(3026..3481)
                     /direction=LEFT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(3163..3184)
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     3375..3394
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     promoter        3508..3612
                     /gene="bla"
                     /label=AmpR promoter
     promoter        3614..3971
                     /label=SV40 promoter
                     /note="SV40 enhancer and early promoter"
     enhancer        3614..3805
                     /label=SV40 enhancer
                     /note="enhancer for the SV40 early promoter (Herr, 1993)"
     primer_bind     complement(3637..3657)
                     /label=pBABE 3'
                     /note="SV40 enhancer, reverse primer for pBABE vectors"
     rep_origin      3822..3957
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     CDS             4006..4800
                     /codon_start=1
                     /product="aminoglycoside phosphotransferase"
                     /label=NeoR/KanR
                     /note="confers resistance to neomycin, kanamycin, and G418 
                     (Geneticin(R))"
                     /translation="MIEQDGLHAGSPAAWVERLFGYDWAQQTIGCSDAAVFRLSAQGRP
                     VLFVKTDLSGALNELQDEAARLSWLATTGVPCAAVLDVVTEAGRDWLLLGEVPGQDLLS
                     SHLAPAEKVSIMADAMRRLHTLDPATCPFDHQAKHRIERARTRMEAGLVDQDDLDEEHQ
                     GLAPAELFARLKASMPDGEDLVVTHGDACLPNIMVENGRFSGFIDCGRLGVADRYQDIA
                     LATRDIAEELGGEWADRFLVLYGIAAPDSQRIAFYRLLDEFF"
     CDS             4006..4800
                     /codon_start=1
                     /gene="aph(3')-II (or nptII)"
                     /product="aminoglycoside phosphotransferase from Tn5"
                     /label=NeoR/KanR
                     /note="confers resistance to neomycin, kanamycin, and G418 
                     (Geneticin(R))"
                     /translation="MIEQDGLHAGSPAAWVERLFGYDWAQQTIGCSDAAVFRLSAQGRP
                     VLFVKTDLSGALNELQDEAARLSWLATTGVPCAAVLDVVTEAGRDWLLLGEVPGQDLLS
                     SHLAPAEKVSIMADAMRRLHTLDPATCPFDHQAKHRIERARTRMEAGLVDQDDLDEEHQ
                     GLAPAELFARLKASMPDGEDLVVTHGDACLPNIMVENGRFSGFIDCGRLGVADRYQDIA
                     LATRDIAEELGGEWADRFLVLYGIAAPDSQRIAFYRLLDEFF"
     primer_bind     complement(4060..4079)
                     /label=Neo-R
                     /note="Neomycin resistance gene, reverse primer"
     primer_bind     4670..4689
                     /label=Neo-F
                     /note="Neomycin resistance gene, forward primer"
     primer_bind     complement(4988..5007)
                     /label=TK-pA-R
                     /note="Thymidine kinase polyA, reverse primer"
     polyA_signal    5032..5079
                     /label=HSV TK poly(A) signal
                     /note="herpes simplex virus thymidine kinase 
                     polyadenylation signal (Cole and Stacy, 1985)"
ORIGIN
        1 gtcccagggc tggcactctg tcgatacccc accgagaccc cattggggcc aatacgcccg
       61 cgtttcttcc ttttccccac cccacccccc aagttcgggt gaaggcccag ggctcgcagc
      121 caacgtcggg gcggcaggcc ctgccatagc ctcaggttac tcatatatac tttagattga
      181 tttaaaactt catttttaat ttaaaaggat ctaggtgaag atcctttttg ataatctcat
      241 gaccaaaatc ccttaacgtg agttttcgtt ccactgagcg tcagaccccg tagaaaagat
      301 caaaggatct tcttgagatc ctttttttct gcgcgtaatc tgctgcttgc aaacaaaaaa
      361 accaccgcta ccagcggtgg tttgtttgcc ggatcaagag ctaccaactc tttttccgaa
      421 ggtaactggc ttcagcagag cgcagatacc aaatactgtt cttctagtgt agccgtagtt
      481 aggccaccac ttcaagaact ctgtagcacc gcctacatac ctcgctctgc taatcctgtt
      541 accagtggct gctgccagtg gcgataagtc gtgtcttacc gggttggact caagacgata
      601 gttaccggat aaggcgcagc ggtcgggctg aacggggggt tcgtgcacac agcccagctt
      661 ggagcgaacg acctacaccg aactgagata cctacagcgt gagctatgag aaagcgccac
      721 gcttcccgaa gggagaaagg cggacaggta tccggtaagc ggcagggtcg gaacaggaga
      781 gcgcacgagg gagcttccag ggggaaacgc ctggtatctt tatagtcctg tcgggtttcg
      841 ccacctctga cttgagcgtc gatttttgtg atgctcgtca ggggggcgga gcctatggaa
      901 aaacgccagc aacgcggcct ttttacggtt cctggccttt tgctggcctt ttgctcacat
      961 gttctttcct gcgttatccc ctgattctgt ggataaccgt attaccgcca tgcattagtt
     1021 attaatagta atcaattacg gggtcattag ttcatagccc atatatggag ttccgcgtta
     1081 cataacttac ggtaaatggc ccgcctggct gaccgcccaa cgacccccgc ccattgacgt
     1141 caataatgac gtatgttccc atagtaacgc caatagggac tttccattga cgtcaatggg
     1201 tggagtattt acggtaaact gcccacttgg cagtacatca agtgtatcat atgccaagta
     1261 cgccccctat tgacgtcaat gacggtaaat ggcccgcctg gcattatgcc cagtacatga
     1321 ccttatggga ctttcctact tggcagtaca tctacgtatt agtcatcgct attaccatgg
     1381 tgatgcggtt ttggcagtac atcaatgggc gtggatagcg gtttgactca cggggatttc
     1441 caagtctcca ccccattgac gtcaatggga gtttgttttg gcaccaaaat caacgggact
     1501 ttccaaaatg tcgtaacaac tccgccccat tgacgcaaat gggcggtagg cgtgtacggt
     1561 gggaggtcta tataagcaga gctggtttag tgaaccgtca gatccgctag cgctaccggt
     1621 cgccaccatg gtgagcaagg gcgaggagct gttcaccggg gtggtgccca tcctggtcga
     1681 gctggacggc gacgtaaacg gccacaagtt cagcgtgtcc ggcgagggcg agggcgatgc
     1741 cacctacggc aagctgaccc tgaagttcat ctgcaccacc ggcaagctgc ccgtgccctg
     1801 gcccaccctc gtgaccaccc tgagctgggg cgtgcagtgc ttcagccgct accccgacca
     1861 catgaagcag cacgacttct tcaagtccgc catgcccgaa ggctacgtcc aggagcgcac
     1921 catcttcttc aaggacgacg gcaactacaa gacccgcgcc gaggtgaagt tcgagggcga
     1981 caccctggtg aaccgcatcg agctgaaggg catcgacttc aaggaggacg gcaacatcct
     2041 ggggcacaag ctggagtaca actacatcag cggcaacgtc tatatcaccg ccgacaagca
     2101 gaagaacggc atcaaggcca acttcaagat ccgccacaac atcgaggacg gcagcgtgca
     2161 gctcgccgac cactaccagc agaacacccc catcggcgac ggccccgtgc tgctgcccga
     2221 caaccactac ctgagcaccc agtccgccct gagcaaagac cccaacgaga agcgcgatca
     2281 catggtcctg ctggagttcg tgaccgccgc cgggatcact ctcggcatgg acgagctgta
     2341 caagtccgga ctcagatcta tgccgtcgga gaagaccttc aagcagcgcc gcaccttcga
     2401 acaaagagta gaagatgtcc gacttattcg agagcagcat ccaaccaaaa tcccggtgat
     2461 aatagaacga tacaagggtg agaagcagct tcctgttctg gataaaacaa agttccttgt
     2521 acctgaccat gtcaacatga gtgagctcat caagataatt agaaggcgct tacagctcaa
     2581 tgctaatcag gccttcttcc tgttggtgaa cggacacagc atggtcagcg tctccacacc
     2641 aatctcagag gtgtatgaga gtgagaaaga tgaagatgga ttcctgtaca tggtctacgc
     2701 ctcccaggag acgttcgcga tgaaattgtc agtgtaagaa ttctgcagtc gacggtaccg
     2761 cgggcccggg atccaccgga tctagataac tgatcataat cagccatacc acatttgtag
     2821 aggttttact tgctttaaaa aacctcccac acctccccct gaacctgaaa cataaaatga
     2881 atgcaattgt tgttgttaac ttgtttattg cagcttataa tggttacaaa taaagcaata
     2941 gcatcacaaa tttcacaaat aaagcatttt tttcactgca ttctagttgt ggtttgtcca
     3001 aactcatcaa tgtatcttaa ggcgtaaatt gtaagcgtta atattttgtt aaaattcgcg
     3061 ttaaattttt gttaaatcag ctcatttttt aaccaatagg ccgaaatcgg caaaatccct
     3121 tataaatcaa aagaatagac cgagataggg ttgagtgttg ttccagtttg gaacaagagt
     3181 ccactattaa agaacgtgga ctccaacgtc aaagggcgaa aaaccgtcta tcagggcgat
     3241 ggcccactac gtgaaccatc accctaatca agttttttgg ggtcgaggtg ccgtaaagca
     3301 ctaaatcgga accctaaagg gagcccccga tttagagctt gacggggaaa gccggcgaac
     3361 gtggcgagaa aggaagggaa gaaagcgaaa ggagcgggcg ctagggcgct ggcaagtgta
     3421 gcggtcacgc tgcgcgtaac caccacaccc gccgcgctta atgcgccgct acagggcgcg
     3481 tcaggtggca cttttcgggg aaatgtgcgc ggaaccccta tttgtttatt tttctaaata
     3541 cattcaaata tgtatccgct catgagacaa taaccctgat aaatgcttca ataatattga
     3601 aaaaggaaga gtcctgaggc ggaaagaacc agctgtggaa tgtgtgtcag ttagggtgtg
     3661 gaaagtcccc aggctcccca gcaggcagaa gtatgcaaag catgcatctc aattagtcag
     3721 caaccaggtg tggaaagtcc ccaggctccc cagcaggcag aagtatgcaa agcatgcatc
     3781 tcaattagtc agcaaccata gtcccgcccc taactccgcc catcccgccc ctaactccgc
     3841 ccagttccgc ccattctccg ccccatggct gactaatttt ttttatttat gcagaggccg
     3901 aggccgcctc ggcctctgag ctattccaga agtagtgagg aggctttttt ggaggcctag
     3961 gcttttgcaa agatcgatca agagacagga tgaggatcgt ttcgcatgat tgaacaagat
     4021 ggattgcacg caggttctcc ggccgcttgg gtggagaggc tattcggcta tgactgggca
     4081 caacagacaa tcggctgctc tgatgccgcc gtgttccggc tgtcagcgca ggggcgcccg
     4141 gttctttttg tcaagaccga cctgtccggt gccctgaatg aactgcaaga cgaggcagcg
     4201 cggctatcgt ggctggccac gacgggcgtt ccttgcgcag ctgtgctcga cgttgtcact
     4261 gaagcgggaa gggactggct gctattgggc gaagtgccgg ggcaggatct cctgtcatct
     4321 caccttgctc ctgccgagaa agtatccatc atggctgatg caatgcggcg gctgcatacg
     4381 cttgatccgg ctacctgccc attcgaccac caagcgaaac atcgcatcga gcgagcacgt
     4441 actcggatgg aagccggtct tgtcgatcag gatgatctgg acgaagagca tcaggggctc
     4501 gcgccagccg aactgttcgc caggctcaag gcgagcatgc ccgacggcga ggatctcgtc
     4561 gtgacccatg gcgatgcctg cttgccgaat atcatggtgg aaaatggccg cttttctgga
     4621 ttcatcgact gtggccggct gggtgtggcg gaccgctatc aggacatagc gttggctacc
     4681 cgtgatattg ctgaagagct tggcggcgaa tgggctgacc gcttcctcgt gctttacggt
     4741 atcgccgctc ccgattcgca gcgcatcgcc ttctatcgcc ttcttgacga gttcttctga
     4801 gcgggactct ggggttcgaa atgaccgacc aagcgacgcc caacctgcca tcacgagatt
     4861 tcgattccac cgccgccttc tatgaaaggt tgggcttcgg aatcgttttc cgggacgccg
     4921 gctggatgat cctccagcgc ggggatctca tgctggagtt cttcgcccac cctaggggga
     4981 ggctaactga aacacggaag gagacaatac cggaaggaac ccgcgctatg acggcaataa
     5041 aaagacagaa taaaacgcac ggtgttgggt cgtttgttca taaacgcggg gttcg
//
