LOCUS       sequence-393629-        5431 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Mammalian expression of fluorescently-tagged marker for autophagic 
            vesicles.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 5431)
  AUTHORS   Dou D, Smith EM, Evans CS, Boecker CA, Holzbaur ELF
  TITLE     Regulatory imbalance between LRRK2 kinase, PPM1H phosphatase, and 
            ARF6 GTPase disrupts the axonal transport of autophagosomes.
  JOURNAL   Cell Rep. 2023 May 1;42(5):112448. doi: 
            10.1016/j.celrep.2023.112448.
  PUBMED    37133994
REFERENCE   2  (bases 1 to 5431)
  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: "Cell Rep.";
            date: "2023-05-1"; volume: "42(5):112448. doi"; pages: " 
            10.1016/j.celrep.2023.112448"
COMMENT     Sequence Label: CMV mScarlet-LC3B
FEATURES             Location/Qualifiers
     source          1..5431
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     enhancer        87..390
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
     promoter        391..594
                     /label=CMV promoter
                     /note="human cytomegalovirus (CMV) immediate early 
                     promoter"
     primer_bind     545..565
                     /label=CMV-F
                     /note="Human CMV immediate early promoter, forward primer"
     regulatory      633..642
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     CDS             639..1334
                     /codon_start=1
                     /product="bright monomeric red fluorescent protein evolved 
                     from a synthetic template (Bindels et al., 2016)"
                     /label=mScarlet
                     /translation="MVSKGEAVIKEFMRFKVHMEGSMNGHEFEIEGEGEGRPYEGTQTA
                     KLKVTKGGPLPFSWDILSPQFMYGSRAFTKHPADIPDYYKQSFPEGFKWERVMNFEDGG
                     AVTVTQDTSLEDGTLIYKVKLRGTNFPPDGPVMQKKTMGWEASTERLYPEDGVLKGDIK
                     MALRLKDGGRYLADFKTTYKAKKPVQMPGAYNVDRKLDITSHNEDYTVVEQYERSEGRH
                     STGGMDELYK"
     primer_bind     complement(772..790)
                     /label=mCherry-R
                     /note="mCherry, reverse primer"
     primer_bind     1040..1059
                     /label=mCherry-F
                     /note="mCherry, forward primer"
     CDS             1353..1775
                     /codon_start=1
                     /gene="Map1lc3b"
                     /product="rat LC3 protein"
                     /label=LC3
                     /note="Atg8 family member found on autophagosomes"
                     /translation="PSEKTFKQRRSFEQRVEDVRLIREQHPTKIPVIIERYKGEKQLPV
                     LDKTKFLVPDHVNMSELIKIIRRRLQLNANQAFFLLVNGHSMVSVSTPISEVYESERDE
                     DGFLYMVYASQETFGTALAVTYMSALKATATGREPCL"
     polyA_signal    2245..2366
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(2282..2301)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     2336..2355
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     rep_origin      complement(2373..2828)
                     /direction=LEFT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(2510..2531)
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     2722..2741
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     promoter        2855..2959
                     /gene="bla"
                     /label=AmpR promoter
     promoter        2961..3318
                     /label=SV40 promoter
                     /note="SV40 enhancer and early promoter"
     enhancer        2961..3152
                     /label=SV40 enhancer
                     /note="enhancer for the SV40 early promoter (Herr, 1993)"
     primer_bind     complement(2984..3004)
                     /label=pBABE 3'
                     /note="SV40 enhancer, reverse primer for pBABE vectors"
     rep_origin      3169..3304
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     CDS             3353..4147
                     /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             3353..4147
                     /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(3407..3426)
                     /label=Neo-R
                     /note="Neomycin resistance gene, reverse primer"
     primer_bind     4017..4036
                     /label=Neo-F
                     /note="Neomycin resistance gene, forward primer"
     primer_bind     complement(4335..4354)
                     /label=TK-pA-R
                     /note="Thymidine kinase polyA, reverse primer"
     polyA_signal    4379..4426
                     /label=HSV TK poly(A) signal
                     /note="herpes simplex virus thymidine kinase 
                     polyadenylation signal (Cole and Stacy, 1985)"
     rep_origin      4755..5343
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     5244..5263
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
ORIGIN
        1 tggataaccg tattaccgcc atgcattagt tattaatagt aatcaattac ggggtcatta
       61 gttcatagcc catatatgga gttccgcgtt acataactta cggtaaatgg cccgcctggc
      121 tgaccgccca acgacccccg cccattgacg tcaataatga cgtatgttcc catagtaacg
      181 ccaataggga ctttccattg acgtcaatgg gtggagtatt tacggtaaac tgcccacttg
      241 gcagtacatc aagtgtatca tatgccaagt acgcccccta ttgacgtcaa tgacggtaaa
      301 tggcccgcct ggcattatgc ccagtacatg accttatggg actttcctac ttggcagtac
      361 atctacgtat tagtcatcgc tattaccatg gtgatgcggt tttggcagta catcaatggg
      421 cgtggatagc ggtttgactc acggggattt ccaagtctcc accccattga cgtcaatggg
      481 agtttgtttt ggcaccaaaa tcaacgggac tttccaaaat gtcgtaacaa ctccgcccca
      541 ttgacgcaaa tgggcggtag gcgtgtacgg tgggaggtct atataagcag agctggttta
      601 gtgaaccgtc agatccgcta gcgctaccgg tcgccaccat ggtgagcaag ggcgaggcag
      661 tgatcaagga gttcatgcgg ttcaaggtgc acatggaggg ctccatgaac ggccacgagt
      721 tcgagatcga gggcgagggc gagggccgcc cctacgaggg cacccagacc gccaagctga
      781 aggtgaccaa gggtggcccc ctgcccttct cctgggacat cctgtcccct cagttcatgt
      841 acggctccag ggccttcacc aagcaccccg ccgacatccc cgactactat aagcagtcct
      901 tccccgaggg cttcaagtgg gagcgcgtga tgaacttcga ggacggcggc gccgtgaccg
      961 tgacccagga cacctccctg gaggacggca ccctgatcta caaggtgaag ctccgcggca
     1021 ccaacttccc tcctgacggc cccgtaatgc agaagaagac aatgggctgg gaagcgtcca
     1081 ccgagcggtt gtaccccgag gacggcgtgc tgaagggcga cattaagatg gccctgcgcc
     1141 tgaaggacgg cggccgctac ctggcggact tcaagaccac ctacaaggcc aagaagcccg
     1201 tgcagatgcc cggcgcctac aacgtcgacc gcaagttgga catcacctcc cacaacgagg
     1261 actacaccgt ggtggaacag tacgaacgct ccgagggccg ccactccacc ggcggcatgg
     1321 acgagctgta caagtccgga ctcagatcta gaccgtccga gaagaccttc aaacagcgcc
     1381 ggagcttcga acaaagagtg gaagatgtcc ggctcatccg ggagcagcac cccaccaaga
     1441 tcccagtgat tatagagcga tacaagggtg agaagcagct gcccgtcctg gacaagacca
     1501 agttccttgt acctgatcac gtgaatatga gcgaactcat caagataatt agaaggcgcc
     1561 tgcagctcaa tgctaaccaa gccttcttcc tcctggtgaa tgggcacagc atggtgagtg
     1621 tgtccacacc catctctgaa gtgtacgaga gcgagagaga tgaagacggc ttcctgtaca
     1681 tggtctatgc ctcccaggag acgttcggga cagcactggc tgttacatac atgtcagctc
     1741 tgaaggcaac agcaacagga agagagccat gcttgtgaca gacatacagc cacttccaac
     1801 taaagcaagc ctctgctccc tgctacctgc atggagccca ctgtgacact cagaccatcc
     1861 ccggtcactc actcactcgt gtctgagaat ctcagtgaga gctgcctctg tcacggacgg
     1921 aagccaacac agccacctct cgacctggct ccccacagca cccaccctcc ctgcatgcag
     1981 ctgtccctgc taacccccaa tgttatgtta cactgtgtaa gtccccactg ctgccgtgtg
     2041 tgggttgtgt acgtcgtcat gtccctggtt tataactatg gtgcgaattc tgcagtcgac
     2101 ggtaccgcgg gcccgggatc caccggatct agataactga tcataatcag ccataccaca
     2161 tttgtagagg ttttacttgc tttaaaaaac ctcccacacc tccccctgaa cctgaaacat
     2221 aaaatgaatg caattgttgt tgttaacttg tttattgcag cttataatgg ttacaaataa
     2281 agcaatagca tcacaaattt cacaaataaa gcattttttt cactgcattc tagttgtggt
     2341 ttgtccaaac tcatcaatgt atcttaacgc gtaaattgta agcgttaata ttttgttaaa
     2401 attcgcgtta aatttttgtt aaatcagctc attttttaac caataggccg aaatcggcaa
     2461 aatcccttat aaatcaaaag aatagaccga gatagggttg agtgttgttc cagtttggaa
     2521 caagagtcca ctattaaaga acgtggactc caacgtcaaa gggcgaaaaa ccgtctatca
     2581 gggcgatggc ccactacgtg aaccatcacc ctaatcaagt tttttggggt cgaggtgccg
     2641 taaagcacta aatcggaacc ctaaagggag cccccgattt agagcttgac ggggaaagcc
     2701 ggcgaacgtg gcgagaaagg aagggaagaa agcgaaagga gcgggcgcta gggcgctggc
     2761 aagtgtagcg gtcacgctgc gcgtaaccac cacacccgcc gcgcttaatg cgccgctaca
     2821 gggcgcgtca ggtggcactt ttcggggaaa tgtgcgcgga acccctattt gtttattttt
     2881 ctaaatacat tcaaatatgt atccgctcat gagacaataa ccctgataaa tgcttcaata
     2941 atattgaaaa aggaagagtc ctgaggcgga aagaaccagc tgtggaatgt gtgtcagtta
     3001 gggtgtggaa agtccccagg ctccccagca ggcagaagta tgcaaagcat gcatctcaat
     3061 tagtcagcaa ccaggtgtgg aaagtcccca ggctccccag caggcagaag tatgcaaagc
     3121 atgcatctca attagtcagc aaccatagtc ccgcccctaa ctccgcccat cccgccccta
     3181 actccgccca gttccgccca ttctccgccc catggctgac taattttttt tatttatgca
     3241 gaggccgagg ccgcctcggc ctctgagcta ttccagaagt agtgaggagg cttttttgga
     3301 ggcctaggct tttgcaaaga tcgatcaaga gacaggatga ggatcgtttc gcatgattga
     3361 acaagatgga ttgcacgcag gttctccggc cgcttgggtg gagaggctat tcggctatga
     3421 ctgggcacaa cagacaatcg gctgctctga tgccgccgtg ttccggctgt cagcgcaggg
     3481 gcgcccggtt ctttttgtca agaccgacct gtccggtgcc ctgaatgaac tgcaagacga
     3541 ggcagcgcgg ctatcgtggc tggccacgac gggcgttcct tgcgcagctg tgctcgacgt
     3601 tgtcactgaa gcgggaaggg actggctgct attgggcgaa gtgccggggc aggatctcct
     3661 gtcatctcac cttgctcctg ccgagaaagt atccatcatg gctgatgcaa tgcggcggct
     3721 gcatacgctt gatccggcta cctgcccatt cgaccaccaa gcgaaacatc gcatcgagcg
     3781 agcacgtact cggatggaag ccggtcttgt cgatcaggat gatctggacg aagagcatca
     3841 ggggctcgcg ccagccgaac tgttcgccag gctcaaggcg agcatgcccg acggcgagga
     3901 tctcgtcgtg acccatggcg atgcctgctt gccgaatatc atggtggaaa atggccgctt
     3961 ttctggattc atcgactgtg gccggctggg tgtggcggac cgctatcagg acatagcgtt
     4021 ggctacccgt gatattgctg aagagcttgg cggcgaatgg gctgaccgct tcctcgtgct
     4081 ttacggtatc gccgctcccg attcgcagcg catcgccttc tatcgccttc ttgacgagtt
     4141 cttctgagcg ggactctggg gttcgaaatg accgaccaag cgacgcccaa cctgccatca
     4201 cgagatttcg attccaccgc cgccttctat gaaaggttgg gcttcggaat cgttttccgg
     4261 gacgccggct ggatgatcct ccagcgcggg gatctcatgc tggagttctt cgcccaccct
     4321 agggggaggc taactgaaac acggaaggag acaataccgg aaggaacccg cgctatgacg
     4381 gcaataaaaa gacagaataa aacgcacggt gttgggtcgt ttgttcataa acgcggggtt
     4441 cggtcccagg gctggcactc tgtcgatacc ccaccgagac cccattgggg ccaatacgcc
     4501 cgcgtttctt ccttttcccc accccacccc ccaagttcgg gtgaaggccc agggctcgca
     4561 gccaacgtcg gggcggcagg ccctgccata gcctcaggtt actcatatat actttagatt
     4621 gatttaaaac ttcattttta atttaaaagg atctaggtga agatcctttt tgataatctc
     4681 atgaccaaaa tcccttaacg tgagttttcg ttccactgag cgtcagaccc cgtagaaaag
     4741 atcaaaggat cttcttgaga tccttttttt ctgcgcgtaa tctgctgctt gcaaacaaaa
     4801 aaaccaccgc taccagcggt ggtttgtttg ccggatcaag agctaccaac tctttttccg
     4861 aaggtaactg gcttcagcag agcgcagata ccaaatactg ttcttctagt gtagccgtag
     4921 ttaggccacc acttcaagaa ctctgtagca ccgcctacat acctcgctct gctaatcctg
     4981 ttaccagtgg ctgctgccag tggcgataag tcgtgtctta ccgggttgga ctcaagacga
     5041 tagttaccgg ataaggcgca gcggtcgggc tgaacggggg gttcgtgcac acagcccagc
     5101 ttggagcgaa cgacctacac cgaactgaga tacctacagc gtgagctatg agaaagcgcc
     5161 acgcttcccg aagggagaaa ggcggacagg tatccggtaa gcggcagggt cggaacagga
     5221 gagcgcacga gggagcttcc agggggaaac gcctggtatc tttatagtcc tgtcgggttt
     5281 cgccacctct gacttgagcg tcgatttttg tgatgctcgt caggggggcg gagcctatgg
     5341 aaaaacgcca gcaacgcggc ctttttacgg ttcctggcct tttgctggcc ttttgctcac
     5401 atgttctttc ctgcgttatc ccctgattct g
//
