LOCUS       sequence-457344-        6516 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Encodes an uncleavable version of the LC3B biosensor. Mutated LC3B 
            is flanked by the Aquamarine/TdLanYFP donor/accpetor FRET pair and 
            under the control of the CMV promoter.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 6516)
  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 6516)
  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-TdLanYFP
FEATURES             Location/Qualifiers
     source          1..6516
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     286..305
                     /label=Neo-F
                     /note="Neomycin resistance gene, forward primer"
     primer_bind     complement(604..623)
                     /label=TK-pA-R
                     /note="Thymidine kinase polyA, reverse primer"
     polyA_signal    648..695
                     /label=HSV TK poly(A) signal
                     /note="herpes simplex virus thymidine kinase 
                     polyadenylation signal (Cole and Stacy, 1985)"
     rep_origin      1024..1612
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     1513..1532
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     enhancer        1787..2090
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
     promoter        2091..2294
                     /label=CMV promoter
                     /note="human cytomegalovirus (CMV) immediate early 
                     promoter"
     primer_bind     2245..2265
                     /label=CMV-F
                     /note="Human CMV immediate early promoter, forward primer"
     regulatory      2333..2342
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     CDS             join(2339..2341,2342..2344,2345..3055)
                     /codon_start=1
                     /product="enhanced CFP"
                     /label=ECFP
                     /note="mammalian codon-optimized"
                     /translation="MVSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTL
                     KFICTTGKLPVPWPTLVTTLSWGVQCFSRYPDHMKQHDFFKSAMPEGYVQERTIFFKDD
                     GNYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYISGNVYITADKQKNGIK
                     ANFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSALSKDPNEKRDHMVLL
                     EFVTAAGITLGMDELYK"
     primer_bind     complement(2384..2405)
                     /label=EGFP-N
                     /note="EGFP, reverse primer"
     primer_bind     complement(2645..2664)
                     /label=EXFP-R
                     /note="For distinguishing EGFP variants, reverse primer"
     primer_bind     2992..3013
                     /label=EGFP-C
                     /note="EGFP, forward primer"
     CDS             3071..3445
                     /codon_start=1
                     /gene="MAP1LC3B"
                     /product="human LC3 protein"
                     /label=LC3B
                     /note="Atg8 family member found on autophagosomes"
                     /translation="MPSEKTFKQRRTFEQRVEDVRLIREQHPTKIPVIIERYKGEKQLP
                     VLDKTKFLVPDHVNMSELIKIIRRRLQLNANQAFFLLVNGHSMVSVSTPISEVYESEKD
                     EDGFLYMVYASQETFAMKLSV"
     CDS             3479..4138
                     /codon_start=1
                     /product="tetrameric yellow fluorescent protein from 
                     Branchiostoma lanceolatum, also known as blFP-Y3"
                     /label=LanYFP
                     /translation="MASLPATHELHIFGSFNGVDFDMVGRGTGNPNDGYEELNLKSTKG
                     DLQFSPWILVPQIGYGFHQYLPFPDGMSPFQAAMKDGSGYQVHRTMQFEDGASLTSNYR
                     YTYEGSHIKGEFQVKGTGFPADGPVMTNSLTAADWCVTKMLYPNDKTIISTFDWTYTTG
                     NGKRYQSTARTTYTFAKPMAANILKNQPMFVFRKTELKHSKTELNFKEWQKAFTDVM"
     CDS             4190..4849
                     /codon_start=1
                     /product="tetrameric yellow fluorescent protein from 
                     Branchiostoma lanceolatum, also known as blFP-Y3"
                     /label=LanYFP
                     /translation="MASLPATHELHIFGSFNGVDFDMVGRGTGNPNDGYEELNLKSTKG
                     DLQFSPWILVPQIGYGFHQYLPFPDGMSPFQAAMKDGSGYQVHRTMQFEDGASLTSNYR
                     YTYEGSHIKGEFQVKGTGFPADGPVMTNSLTAADWCVTKMLYPNDKTIISTFDWTYTTG
                     NGKRYQSTARTTYTFAKPMAANILKNQPMFVFRKTELKHSKTELNFKEWQKAFTDVM"
     polyA_signal    5030..5151
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(5067..5086)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     5121..5140
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     rep_origin      complement(5158..5613)
                     /direction=LEFT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(5295..5316)
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     5507..5526
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     promoter        5640..5744
                     /gene="bla"
                     /label=AmpR promoter
     promoter        5746..6103
                     /label=SV40 promoter
                     /note="SV40 enhancer and early promoter"
     enhancer        5746..5937
                     /label=SV40 enhancer
                     /note="enhancer for the SV40 early promoter (Herr, 1993)"
     primer_bind     complement(5769..5789)
                     /label=pBABE 3'
                     /note="SV40 enhancer, reverse primer for pBABE vectors"
     rep_origin      5954..6089
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     CDS             join(6138..6516,1..416)
                     /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             join(6138..6516,1..416)
                     /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(6192..6211)
                     /label=Neo-R
                     /note="Neomycin resistance gene, reverse primer"
ORIGIN
        1 atccggctac ctgcccattc gaccaccaag cgaaacatcg catcgagcga gcacgtactc
       61 ggatggaagc cggtcttgtc gatcaggatg atctggacga agagcatcag gggctcgcgc
      121 cagccgaact gttcgccagg ctcaaggcga gcatgcccga cggcgaggat ctcgtcgtga
      181 cccatggcga tgcctgcttg ccgaatatca tggtggaaaa tggccgcttt tctggattca
      241 tcgactgtgg ccggctgggt gtggcggacc gctatcagga catagcgttg gctacccgtg
      301 atattgctga agagcttggc ggcgaatggg ctgaccgctt cctcgtgctt tacggtatcg
      361 ccgctcccga ttcgcagcgc atcgccttct atcgccttct tgacgagttc ttctgagcgg
      421 gactctgggg ttcgaaatga ccgaccaagc gacgcccaac ctgccatcac gagatttcga
      481 ttccaccgcc gccttctatg aaaggttggg cttcggaatc gttttccggg acgccggctg
      541 gatgatcctc cagcgcgggg atctcatgct ggagttcttc gcccacccta gggggaggct
      601 aactgaaaca cggaaggaga caataccgga aggaacccgc gctatgacgg caataaaaag
      661 acagaataaa acgcacggtg ttgggtcgtt tgttcataaa cgcggggttc ggtcccaggg
      721 ctggcactct gtcgataccc caccgagacc ccattggggc caatacgccc gcgtttcttc
      781 cttttcccca ccccaccccc caagttcggg tgaaggccca gggctcgcag ccaacgtcgg
      841 ggcggcaggc cctgccatag cctcaggtta ctcatatata ctttagattg atttaaaact
      901 tcatttttaa tttaaaagga tctaggtgaa gatccttttt gataatctca tgaccaaaat
      961 cccttaacgt gagttttcgt tccactgagc gtcagacccc gtagaaaaga tcaaaggatc
     1021 ttcttgagat cctttttttc tgcgcgtaat ctgctgcttg caaacaaaaa aaccaccgct
     1081 accagcggtg gtttgtttgc cggatcaaga gctaccaact ctttttccga aggtaactgg
     1141 cttcagcaga gcgcagatac caaatactgt tcttctagtg tagccgtagt taggccacca
     1201 cttcaagaac tctgtagcac cgcctacata cctcgctctg ctaatcctgt taccagtggc
     1261 tgctgccagt ggcgataagt cgtgtcttac cgggttggac tcaagacgat agttaccgga
     1321 taaggcgcag cggtcgggct gaacgggggg ttcgtgcaca cagcccagct tggagcgaac
     1381 gacctacacc gaactgagat acctacagcg tgagctatga gaaagcgcca cgcttcccga
     1441 agggagaaag gcggacaggt atccggtaag cggcagggtc ggaacaggag agcgcacgag
     1501 ggagcttcca gggggaaacg cctggtatct ttatagtcct gtcgggtttc gccacctctg
     1561 acttgagcgt cgatttttgt gatgctcgtc aggggggcgg agcctatgga aaaacgccag
     1621 caacgcggcc tttttacggt tcctggcctt ttgctggcct tttgctcaca tgttctttcc
     1681 tgcgttatcc cctgattctg tggataaccg tattaccgcc atgcattagt tattaatagt
     1741 aatcaattac ggggtcatta gttcatagcc catatatgga gttccgcgtt acataactta
     1801 cggtaaatgg cccgcctggc tgaccgccca acgacccccg cccattgacg tcaataatga
     1861 cgtatgttcc catagtaacg ccaataggga ctttccattg acgtcaatgg gtggagtatt
     1921 tacggtaaac tgcccacttg gcagtacatc aagtgtatca tatgccaagt acgcccccta
     1981 ttgacgtcaa tgacggtaaa tggcccgcct ggcattatgc ccagtacatg accttatggg
     2041 actttcctac ttggcagtac atctacgtat tagtcatcgc tattaccatg gtgatgcggt
     2101 tttggcagta catcaatggg cgtggatagc ggtttgactc acggggattt ccaagtctcc
     2161 accccattga cgtcaatggg agtttgtttt ggcaccaaaa tcaacgggac tttccaaaat
     2221 gtcgtaacaa ctccgcccca ttgacgcaaa tgggcggtag gcgtgtacgg tgggaggtct
     2281 atataagcag agctggttta gtgaaccgtc agatccgcta gcgctaccgg tcgccaccat
     2341 ggtgagcaag ggcgaggagc tgttcaccgg ggtggtgccc atcctggtcg agctggacgg
     2401 cgacgtaaac ggccacaagt tcagcgtgtc cggcgagggc gagggcgatg ccacctacgg
     2461 caagctgacc ctgaagttca tctgcaccac cggcaagctg cccgtgccct ggcccaccct
     2521 cgtgaccacc ctgagctggg gcgtgcagtg cttcagccgc taccccgacc acatgaagca
     2581 gcacgacttc ttcaagtccg ccatgcccga aggctacgtc caggagcgca ccatcttctt
     2641 caaggacgac ggcaactaca agacccgcgc cgaggtgaag ttcgagggcg acaccctggt
     2701 gaaccgcatc gagctgaagg gcatcgactt caaggaggac ggcaacatcc tggggcacaa
     2761 gctggagtac aactacatca gcggcaacgt ctatatcacc gccgacaagc agaagaacgg
     2821 catcaaggcc aacttcaaga tccgccacaa catcgaggac ggcagcgtgc agctcgccga
     2881 ccactaccag cagaacaccc ccatcggcga cggccccgtg ctgctgcccg acaaccacta
     2941 cctgagcacc cagtccgccc tgagcaaaga ccccaacgag aagcgcgatc acatggtcct
     3001 gctggagttc gtgaccgccg ccgggatcac tctcggcatg gacgagctgt acaagtccgg
     3061 actcagatct atgccgtcgg agaagacctt caagcagcgc cgcaccttcg aacaaagagt
     3121 agaagatgtc cgacttattc gagagcagca tccaaccaaa atcccggtga taatagaacg
     3181 atacaagggt gagaagcagc ttcctgttct ggataaaaca aagttccttg tacctgacca
     3241 tgtcaacatg agtgagctca tcaagataat tagaaggcgc ttacagctca atgctaatca
     3301 ggccttcttc ctgttggtga acggacacag catggtcagc gtctccacac caatctcaga
     3361 ggtgtatgag agtgagaaag atgaagatgg attcctgtac atggtctacg cctcccagga
     3421 gacgttcgcg atgaaattgt cagtggaatt catggtctcc aaaggagagg aggataacat
     3481 ggcctctctc ccagcgacac atgagttaca catctttggc tccttcaacg gtgtggactt
     3541 tgacatggtg ggtcgtggca ccggcaatcc aaatgatggt tatgaggagt taaacctgaa
     3601 gtccaccaag ggtgacctcc agttctcccc ctggattctg gtccctcaaa tcgggtatgg
     3661 attccatcag tacctgccct tccccgacgg gatgtcgcct ttccaggccg ccatgaaaga
     3721 tggctccgga taccaagtcc atcgcacaat gcagtttgaa gacggtgcct ccctgacttc
     3781 caactaccgc tacacctacg agggaagcca catcaaagga gagtttcagg tgaaggggac
     3841 tggtttccct gctgacggtc ctgtgatgac caactcgctg accgctgcgg actggtgcgt
     3901 gaccaagatg ctgtacccca acgacaaaac catcatcagc acctttgact ggacttacac
     3961 cactggaaat ggcaagcgct accagagcac tgcgcggacc acctacacct ttgccaagcc
     4021 aatggcggcc aacatcctga agaaccagcc gatgttcgtg ttccgtaaga cggaactcaa
     4081 gcactccaag accgaactca acttcaagga gtggcaaaag gcatttaccg atgtgatgtc
     4141 gactggcact ggttctacgg gctcgggctc ctcaggagag gaggataaca tggcctctct
     4201 cccagcgaca catgagttac acatctttgg ctccttcaac ggtgtggact ttgacatggt
     4261 gggtcgtggc accggcaatc caaatgatgg ttatgaggag ttaaacctga agtccaccaa
     4321 gggtgacctc cagttctccc cctggattct ggtccctcaa atcgggtatg gcttccatca
     4381 gtacctgccc ttccccgacg ggatgtcgcc tttccaggcc gccatgaaag atggctccgg
     4441 ataccaagtc catcgcacaa tgcagtttga agacggtgcc tccctgactt ccaactaccg
     4501 ctacacctac gagggaagcc acatcaaagg agagtttcag gtgaagggga ctggtttccc
     4561 tgctgacggt cctgtgatga ccaactcgct gaccgctgcg gactggtgcg tgaccaagat
     4621 gctgtacccc aacgacaaaa ccatcatcag cacctttgac tggacttaca ccactggaaa
     4681 tggcaagcgc taccagagca ctgcgcggac cacctacacc tttgccaagc caatggcggc
     4741 caacatcctg aagaaccagc cgatgttcgt gttccgtaag acggaactca agcactccaa
     4801 gaccgaactc aacttcaagg agtggcaaaa ggcatttacc gatgtgatgg gcatggacga
     4861 gctgtacaag taaagcggcc gcgacggtac cgcgggcccg ggatccaccg gatctagata
     4921 actgatcata atcagccata ccacatttgt agaggtttta cttgctttaa aaaacctccc
     4981 acacctcccc ctgaacctga aacataaaat gaatgcaatt gttgttgtta acttgtttat
     5041 tgcagcttat aatggttaca aataaagcaa tagcatcaca aatttcacaa ataaagcatt
     5101 tttttcactg cattctagtt gtggtttgtc caaactcatc aatgtatctt aacgcgtaaa
     5161 ttgtaagcgt taatattttg ttaaaattcg cgttaaattt ttgttaaatc agctcatttt
     5221 ttaaccaata ggccgaaatc ggcaaaatcc cttataaatc aaaagaatag accgagatag
     5281 ggttgagtgt tgttccagtt tggaacaaga gtccactatt aaagaacgtg gactccaacg
     5341 tcaaagggcg aaaaaccgtc tatcagggcg atggcccact acgtgaacca tcaccctaat
     5401 caagtttttt ggggtcgagg tgccgtaaag cactaaatcg gaaccctaaa gggagccccc
     5461 gatttagagc ttgacgggga aagccggcga acgtggcgag aaaggaaggg aagaaagcga
     5521 aaggagcggg cgctagggcg ctggcaagtg tagcggtcac gctgcgcgta accaccacac
     5581 ccgccgcgct taatgcgccg ctacagggcg cgtcaggtgg cacttttcgg ggaaatgtgc
     5641 gcggaacccc tatttgttta tttttctaaa tacattcaaa tatgtatccg ctcatgagac
     5701 aataaccctg ataaatgctt caataatatt gaaaaaggaa gagtcctgag gcggaaagaa
     5761 ccagctgtgg aatgtgtgtc agttagggtg tggaaagtcc ccaggctccc cagcaggcag
     5821 aagtatgcaa agcatgcatc tcaattagtc agcaaccagg tgtggaaagt ccccaggctc
     5881 cccagcaggc agaagtatgc aaagcatgca tctcaattag tcagcaacca tagtcccgcc
     5941 cctaactccg cccatcccgc ccctaactcc gcccagttcc gcccattctc cgccccatgg
     6001 ctgactaatt ttttttattt atgcagaggc cgaggccgcc tcggcctctg agctattcca
     6061 gaagtagtga ggaggctttt ttggaggcct aggcttttgc aaagatcgat caagagacag
     6121 gatgaggatc gtttcgcatg attgaacaag atggattgca cgcaggttct ccggccgctt
     6181 gggtggagag gctattcggc tatgactggg cacaacagac aatcggctgc tctgatgccg
     6241 ccgtgttccg gctgtcagcg caggggcgcc cggttctttt tgtcaagacc gacctgtccg
     6301 gtgccctgaa tgaactgcaa gacgaggcag cgcggctatc gtggctggcc acgacgggcg
     6361 ttccttgcgc agctgtgctc gacgttgtca ctgaagcggg aagggactgg ctgctattgg
     6421 gcgaagtgcc ggggcaggat ctcctgtcat ctcaccttgc tcctgccgag aaagtatcca
     6481 tcatggctga tgcaatgcgg cggctgcata cgcttg
//
