LOCUS       sequence-333140-        5452 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Mammalian expression of rat LC3 fused to EGFP.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 5452)
  AUTHORS   Puri C, Manni MM, Vicinanza M, Hilcenko C, Zhu Y, Runwal G, 
            Stamatakou E, Menzies FM, Mamchaoui K, Bitoun M, Rubinsztein DC
  TITLE     A DNM2 Centronuclear Myopathy Mutation Reveals a Link between 
            Recycling Endosome Scission and Autophagy.
  JOURNAL   Dev Cell. 2020 Apr 20;53(2):154-168.e6. doi: 
            10.1016/j.devcel.2020.03.018.
  PUBMED    32315611
REFERENCE   2  (bases 1 to 5452)
  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: 
            "10.1016/j.devcel.2020.03"; journalName: "Dev Cell"; date: 
            "2020-04-20- 20"; volume: "53"; issue: "2"; pages: "154-168.e6"
COMMENT     Sequence Label: pEGFP-LC3-R70A
FEATURES             Location/Qualifiers
     source          1..5452
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     151..172
                     /label=EGFP-C
                     /note="EGFP, forward primer"
     CDS             233..655
                     /codon_start=1
                     /gene="Map1lc3b"
                     /product="rat LC3 protein"
                     /label=LC3
                     /note="Atg8 family member found on autophagosomes"
                     /translation="PSEKTFKQRRSFEQRVEDVRLIREQHPTKIPVIIERYKGEKQLPV
                     LDKTKFLVPDHVNMSELIKIIRRALQLNANQAFFLLVNGHSMVSVSTPISEVYESERDE
                     DGFLYMVYASQETFGTALAVTYMSALKATATGREPCL"
     polyA_signal    1125..1246
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(1162..1181)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     1216..1235
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     rep_origin      complement(1253..1708)
                     /direction=LEFT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(1390..1411)
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     1602..1621
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     promoter        1735..1839
                     /gene="bla"
                     /label=AmpR promoter
     promoter        1841..2198
                     /label=SV40 promoter
                     /note="SV40 enhancer and early promoter"
     enhancer        1841..2032
                     /label=SV40 enhancer
                     /note="enhancer for the SV40 early promoter (Herr, 1993)"
     primer_bind     complement(1864..1884)
                     /label=pBABE 3'
                     /note="SV40 enhancer, reverse primer for pBABE vectors"
     rep_origin      2049..2184
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     CDS             2233..3027
                     /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             2233..3027
                     /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(2287..2306)
                     /label=Neo-R
                     /note="Neomycin resistance gene, reverse primer"
     primer_bind     2897..2916
                     /label=Neo-F
                     /note="Neomycin resistance gene, forward primer"
     primer_bind     complement(3215..3234)
                     /label=TK-pA-R
                     /note="Thymidine kinase polyA, reverse primer"
     polyA_signal    3259..3306
                     /label=HSV TK poly(A) signal
                     /note="herpes simplex virus thymidine kinase 
                     polyadenylation signal (Cole and Stacy, 1985)"
     rep_origin      3635..4223
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     4124..4143
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     enhancer        4398..4701
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
     promoter        4702..4905
                     /label=CMV promoter
                     /note="human cytomegalovirus (CMV) immediate early 
                     promoter"
     primer_bind     4856..4876
                     /label=CMV-F
                     /note="Human CMV immediate early promoter, forward primer"
     regulatory      4944..4953
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     CDS             join(4950..4952,4953..4955,4956..5452,1..214)
                     /codon_start=1
                     /product="enhanced GFP"
                     /label=EGFP
                     /translation="MVSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTL
                     KFICTTGKLPVPWPTLVTTLTYGVQCFSRYPDHMKQHDFFKSAMPEGYVQERTIFFKDD
                     GNYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIK
                     VNFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSALSKDPNEKRDHMVLL
                     EFVTAAGITLGMDELYK"
     CDS             join(4950..4952,4953..4955,4956..5452,1..214)
                     /codon_start=1
                     /product="the original enhanced GFP (Yang et al., 1996)"
                     /label=EGFP
                     /note="mammalian codon-optimized"
                     /translation="MVSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTL
                     KFICTTGKLPVPWPTLVTTLTYGVQCFSRYPDHMKQHDFFKSAMPEGYVQERTIFFKDD
                     GNYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIK
                     VNFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSALSKDPNEKRDHMVLL
                     EFVTAAGITLGMDELYK"
     primer_bind     complement(4995..5016)
                     /label=EGFP-N
                     /note="EGFP, reverse primer"
     primer_bind     complement(5256..5275)
                     /label=EXFP-R
                     /note="For distinguishing EGFP variants, reverse primer"
ORIGIN
        1 ccgccacaac atcgaggacg gcagcgtgca gctcgccgac cactaccagc agaacacccc
       61 catcggcgac ggccccgtgc tgctgcccga caaccactac ctgagcaccc agtccgccct
      121 gagcaaagac cccaacgaga agcgcgatca catggtcctg ctggagttcg tgaccgccgc
      181 cgggatcact ctcggcatgg acgagctgta caagtccgga ctcagatcta gaccgtccga
      241 gaagaccttc aaacagcgcc ggagcttcga acaaagagtg gaagatgtcc ggctcatccg
      301 ggagcagcac cccaccaaga tcccagtgat tatagagcga tacaagggtg agaagcagct
      361 gcccgtcctg gacaagacca agttccttgt acctgatcac gtgaatatga gcgaactcat
      421 caagataatt agaagggccc tgcagctcaa tgctaaccaa gccttcttcc tcctggtgaa
      481 tgggcacagc atggtgagtg tgtccacacc catctctgaa gtgtacgaga gcgagagaga
      541 tgaagacggc ttcctgtaca tggtctatgc ctcccaggag acgttcggga cagcactggc
      601 tgttacatac atgtcagctc tgaaggcaac agcaacagga agagagccat gcttgtgaca
      661 gacatacagc cacttccaac taaagcaagc ctctgctccc tgctacctgc atggagccca
      721 ctgtgacact cagaccatcc ccggtcactc actcactcgt gtctgagaat ctcagtgaga
      781 gctgcctctg tcacggacgg aagccaacac agccacctct cgacctggct ccccacagca
      841 cccaccctcc ctgcatgcag ctgtccctgc taacccccaa tgttatgtta cactgtgtaa
      901 gtccccactg ctgccgtgtg tgggttgtgt acgtcgtcat gtccctggtt tataactatg
      961 gtgcgaattc tgcagtcgac ggtaccgcgg gcccgggatc caccggatct agataactga
     1021 tcataatcag ccataccaca tttgtagagg ttttacttgc tttaaaaaac ctcccacacc
     1081 tccccctgaa cctgaaacat aaaatgaatg caattgttgt tgttaacttg tttattgcag
     1141 cttataatgg ttacaaataa agcaatagca tcacaaattt cacaaataaa gcattttttt
     1201 cactgcattc tagttgtggt ttgtccaaac tcatcaatgt atcttaacgc gtaaattgta
     1261 agcgttaata ttttgttaaa attcgcgtta aatttttgtt aaatcagctc attttttaac
     1321 caataggccg aaatcggcaa aatcccttat aaatcaaaag aatagaccga gatagggttg
     1381 agtgttgttc cagtttggaa caagagtcca ctattaaaga acgtggactc caacgtcaaa
     1441 gggcgaaaaa ccgtctatca gggcgatggc ccactacgtg aaccatcacc ctaatcaagt
     1501 tttttggggt cgaggtgccg taaagcacta aatcggaacc ctaaagggag cccccgattt
     1561 agagcttgac ggggaaagcc ggcgaacgtg gcgagaaagg aagggaagaa agcgaaagga
     1621 gcgggcgcta gggcgctggc aagtgtagcg gtcacgctgc gcgtaaccac cacacccgcc
     1681 gcgcttaatg cgccgctaca gggcgcgtca ggtggcactt ttcggggaaa tgtgcgcgga
     1741 acccctattt gtttattttt ctaaatacat tcaaatatgt atccgctcat gagacaataa
     1801 ccctgataaa tgcttcaata atattgaaaa aggaagagtc ctgaggcgga aagaaccagc
     1861 tgtggaatgt gtgtcagtta gggtgtggaa agtccccagg ctccccagca ggcagaagta
     1921 tgcaaagcat gcatctcaat tagtcagcaa ccaggtgtgg aaagtcccca ggctccccag
     1981 caggcagaag tatgcaaagc atgcatctca attagtcagc aaccatagtc ccgcccctaa
     2041 ctccgcccat cccgccccta actccgccca gttccgccca ttctccgccc catggctgac
     2101 taattttttt tatttatgca gaggccgagg ccgcctcggc ctctgagcta ttccagaagt
     2161 agtgaggagg cttttttgga ggcctaggct tttgcaaaga tcgatcaaga gacaggatga
     2221 ggatcgtttc gcatgattga acaagatgga ttgcacgcag gttctccggc cgcttgggtg
     2281 gagaggctat tcggctatga ctgggcacaa cagacaatcg gctgctctga tgccgccgtg
     2341 ttccggctgt cagcgcaggg gcgcccggtt ctttttgtca agaccgacct gtccggtgcc
     2401 ctgaatgaac tgcaagacga ggcagcgcgg ctatcgtggc tggccacgac gggcgttcct
     2461 tgcgcagctg tgctcgacgt tgtcactgaa gcgggaaggg actggctgct attgggcgaa
     2521 gtgccggggc aggatctcct gtcatctcac cttgctcctg ccgagaaagt atccatcatg
     2581 gctgatgcaa tgcggcggct gcatacgctt gatccggcta cctgcccatt cgaccaccaa
     2641 gcgaaacatc gcatcgagcg agcacgtact cggatggaag ccggtcttgt cgatcaggat
     2701 gatctggacg aagagcatca ggggctcgcg ccagccgaac tgttcgccag gctcaaggcg
     2761 agcatgcccg acggcgagga tctcgtcgtg acccatggcg atgcctgctt gccgaatatc
     2821 atggtggaaa atggccgctt ttctggattc atcgactgtg gccggctggg tgtggcggac
     2881 cgctatcagg acatagcgtt ggctacccgt gatattgctg aagagcttgg cggcgaatgg
     2941 gctgaccgct tcctcgtgct ttacggtatc gccgctcccg attcgcagcg catcgccttc
     3001 tatcgccttc ttgacgagtt cttctgagcg ggactctggg gttcgaaatg accgaccaag
     3061 cgacgcccaa cctgccatca cgagatttcg attccaccgc cgccttctat gaaaggttgg
     3121 gcttcggaat cgttttccgg gacgccggct ggatgatcct ccagcgcggg gatctcatgc
     3181 tggagttctt cgcccaccct agggggaggc taactgaaac acggaaggag acaataccgg
     3241 aaggaacccg cgctatgacg gcaataaaaa gacagaataa aacgcacggt gttgggtcgt
     3301 ttgttcataa acgcggggtt cggtcccagg gctggcactc tgtcgatacc ccaccgagac
     3361 cccattgggg ccaatacgcc cgcgtttctt ccttttcccc accccacccc ccaagttcgg
     3421 gtgaaggccc agggctcgca gccaacgtcg gggcggcagg ccctgccata gcctcaggtt
     3481 actcatatat actttagatt gatttaaaac ttcattttta atttaaaagg atctaggtga
     3541 agatcctttt tgataatctc atgaccaaaa tcccttaacg tgagttttcg ttccactgag
     3601 cgtcagaccc cgtagaaaag atcaaaggat cttcttgaga tccttttttt ctgcgcgtaa
     3661 tctgctgctt gcaaacaaaa aaaccaccgc taccagcggt ggtttgtttg ccggatcaag
     3721 agctaccaac tctttttccg aaggtaactg gcttcagcag agcgcagata ccaaatactg
     3781 ttcttctagt gtagccgtag ttaggccacc acttcaagaa ctctgtagca ccgcctacat
     3841 acctcgctct gctaatcctg ttaccagtgg ctgctgccag tggcgataag tcgtgtctta
     3901 ccgggttgga ctcaagacga tagttaccgg ataaggcgca gcggtcgggc tgaacggggg
     3961 gttcgtgcac acagcccagc ttggagcgaa cgacctacac cgaactgaga tacctacagc
     4021 gtgagctatg agaaagcgcc acgcttcccg aagggagaaa ggcggacagg tatccggtaa
     4081 gcggcagggt cggaacagga gagcgcacga gggagcttcc agggggaaac gcctggtatc
     4141 tttatagtcc tgtcgggttt cgccacctct gacttgagcg tcgatttttg tgatgctcgt
     4201 caggggggcg gagcctatgg aaaaacgcca gcaacgcggc ctttttacgg ttcctggcct
     4261 tttgctggcc ttttgctcac atgttctttc ctgcgttatc ccctgattct gtggataacc
     4321 gtattaccgc catgcattag ttattaatag taatcaatta cggggtcatt agttcatagc
     4381 ccatatatgg agttccgcgt tacataactt acggtaaatg gcccgcctgg ctgaccgccc
     4441 aacgaccccc gcccattgac gtcaataatg acgtatgttc ccatagtaac gccaataggg
     4501 actttccatt gacgtcaatg ggtggagtat ttacggtaaa ctgcccactt ggcagtacat
     4561 caagtgtatc atatgccaag tacgccccct attgacgtca atgacggtaa atggcccgcc
     4621 tggcattatg cccagtacat gaccttatgg gactttccta cttggcagta catctacgta
     4681 ttagtcatcg ctattaccat ggtgatgcgg ttttggcagt acatcaatgg gcgtggatag
     4741 cggtttgact cacggggatt tccaagtctc caccccattg acgtcaatgg gagtttgttt
     4801 tggcaccaaa atcaacggga ctttccaaaa tgtcgtaaca actccgcccc attgacgcaa
     4861 atgggcggta ggcgtgtacg gtgggaggtc tatataagca gagctggttt agtgaaccgt
     4921 cagatccgct agcgctaccg gtcgccacca tggtgagcaa gggcgaggag ctgttcaccg
     4981 gggtggtgcc catcctggtc gagctggacg gcgacgtaaa cggccacaag ttcagcgtgt
     5041 ccggcgaggg cgagggcgat gccacctacg gcaagctgac cctgaagttc atctgcacca
     5101 ccggcaagct gcccgtgccc tggcccaccc tcgtgaccac cctgacctac ggcgtgcagt
     5161 gcttcagccg ctaccccgac cacatgaagc agcacgactt cttcaagtcc gccatgcccg
     5221 aaggctacgt ccaggagcgc accatcttct tcaaggacga cggcaactac aagacccgcg
     5281 ccgaggtgaa gttcgagggc gacaccctgg tgaaccgcat cgagctgaag ggcatcgact
     5341 tcaaggagga cggcaacatc ctggggcaca agctggagta caactacaac agccacaacg
     5401 tctatatcat ggccgacaag cagaagaacg gcatcaaggt gaacttcaag at
//
