LOCUS       sequence-393635-        5366 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 5366)
  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 5366)
  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: PGK mScarlet-LC3B
FEATURES             Location/Qualifiers
     source          1..5366
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     promoter        129..639
                     /label=hPGK promoter
                     /note="human phosphoglycerate kinase 1 promoter"
     primer_bind     558..576
                     /label=hPGK-F
                     /note="Human PGK promoter, forward primer"
     regulatory      655..664
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     CDS             661..1356
                     /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(794..812)
                     /label=mCherry-R
                     /note="mCherry, reverse primer"
     primer_bind     1062..1081
                     /label=mCherry-F
                     /note="mCherry, forward primer"
     CDS             1375..1797
                     /codon_start=1
                     /gene="Map1lc3b"
                     /product="rat LC3 protein"
                     /label=LC3
                     /note="Atg8 family member found on autophagosomes"
                     /translation="PSEKTFKQRRSFEQRVEDVRLIREQHPTKIPVIIERYKGEKQLPV
                     LDKTKFLVPDHVNMSELIKIIRRRLQLNANQAFFLLVNGHSMVSVSTPISEVYESERDE
                     DGFLYMVYASQETFGTALAVTYMSALKATATGREPCL"
     polyA_signal    2267..2388
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(2304..2323)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     2358..2377
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     rep_origin      complement(2395..2850)
                     /direction=LEFT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(2532..2553)
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     2744..2763
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     promoter        2877..2981
                     /gene="bla"
                     /label=AmpR promoter
     promoter        2983..3340
                     /label=SV40 promoter
                     /note="SV40 enhancer and early promoter"
     enhancer        2983..3174
                     /label=SV40 enhancer
                     /note="enhancer for the SV40 early promoter (Herr, 1993)"
     primer_bind     complement(3006..3026)
                     /label=pBABE 3'
                     /note="SV40 enhancer, reverse primer for pBABE vectors"
     rep_origin      3191..3326
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     CDS             3375..4169
                     /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             3375..4169
                     /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(3429..3448)
                     /label=Neo-R
                     /note="Neomycin resistance gene, reverse primer"
     primer_bind     4039..4058
                     /label=Neo-F
                     /note="Neomycin resistance gene, forward primer"
     primer_bind     complement(4357..4376)
                     /label=TK-pA-R
                     /note="Thymidine kinase polyA, reverse primer"
     polyA_signal    4401..4448
                     /label=HSV TK poly(A) signal
                     /note="herpes simplex virus thymidine kinase 
                     polyadenylation signal (Cole and Stacy, 1985)"
     rep_origin      4777..5365
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     5266..5285
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
ORIGIN
        1 acgccagcaa cgcggccttt ttacggttcc tggccttttg ctggcctttt gctcacatgt
       61 tctttcctgc gttatcccct gattctgtgg ataaccgtat taccgccatg cattagttat
      121 taatccacgg ggttggggtt gcgccttttc caaggcagcc ctgggtttgc gcagggacgc
      181 ggctgctctg ggcgtggttc cgggaaacgc agcggcgccg accctgggtc tcgcacattc
      241 ttcacgtccg ttcgcagcgt cacccggatc ttcgccgcta cccttgtggg ccccccggcg
      301 acgcttcctg ctccgcccct aagtcgggaa ggttccttgc ggttcgcggc gtgccggacg
      361 tgacaaacgg aagccgcacg tctcactagt accctcgcag acggacagcg ccagggagca
      421 atggcagcgc gccgaccgcg atgggctgtg gccaatagcg gctgctcagc agggcgcgcc
      481 gagagcagcg gccgggaagg ggcggtgcgg gaggcggggt gtggggcggt agtgtgggcc
      541 ctgttcctgc ccgcgcggtg ttccgcattc tgcaagcctc cggagcgcac gtcggcagtc
      601 ggctccctcg ttgaccgaat caccgacctc tctccccagg tggatccacc ggtcgccacc
      661 atggtgagca agggcgaggc agtgatcaag gagttcatgc ggttcaaggt gcacatggag
      721 ggctccatga acggccacga gttcgagatc gagggcgagg gcgagggccg cccctacgag
      781 ggcacccaga ccgccaagct gaaggtgacc aagggtggcc ccctgccctt ctcctgggac
      841 atcctgtccc ctcagttcat gtacggctcc agggccttca ccaagcaccc cgccgacatc
      901 cccgactact ataagcagtc cttccccgag ggcttcaagt gggagcgcgt gatgaacttc
      961 gaggacggcg gcgccgtgac cgtgacccag gacacctccc tggaggacgg caccctgatc
     1021 tacaaggtga agctccgcgg caccaacttc cctcctgacg gccccgtaat gcagaagaag
     1081 acaatgggct gggaagcgtc caccgagcgg ttgtaccccg aggacggcgt gctgaagggc
     1141 gacattaaga tggccctgcg cctgaaggac ggcggccgct acctggcgga cttcaagacc
     1201 acctacaagg ccaagaagcc cgtgcagatg cccggcgcct acaacgtcga ccgcaagttg
     1261 gacatcacct cccacaacga ggactacacc gtggtggaac agtacgaacg ctccgagggc
     1321 cgccactcca ccggcggcat ggacgagctg tacaagtccg gactcagatc tagaccgtcc
     1381 gagaagacct tcaaacagcg ccggagcttc gaacaaagag tggaagatgt ccggctcatc
     1441 cgggagcagc accccaccaa gatcccagtg attatagagc gatacaaggg tgagaagcag
     1501 ctgcccgtcc tggacaagac caagttcctt gtacctgatc acgtgaatat gagcgaactc
     1561 atcaagataa ttagaaggcg cctgcagctc aatgctaacc aagccttctt cctcctggtg
     1621 aatgggcaca gcatggtgag tgtgtccaca cccatctctg aagtgtacga gagcgagaga
     1681 gatgaagacg gcttcctgta catggtctat gcctcccagg agacgttcgg gacagcactg
     1741 gctgttacat acatgtcagc tctgaaggca acagcaacag gaagagagcc atgcttgtga
     1801 cagacataca gccacttcca actaaagcaa gcctctgctc cctgctacct gcatggagcc
     1861 cactgtgaca ctcagaccat ccccggtcac tcactcactc gtgtctgaga atctcagtga
     1921 gagctgcctc tgtcacggac ggaagccaac acagccacct ctcgacctgg ctccccacag
     1981 cacccaccct ccctgcatgc agctgtccct gctaaccccc aatgttatgt tacactgtgt
     2041 aagtccccac tgctgccgtg tgtgggttgt gtacgtcgtc atgtccctgg tttataacta
     2101 tggtgcgaat tctgcagtcg acggtaccgc gggcccggga tccaccggat ctagataact
     2161 gatcataatc agccatacca catttgtaga ggttttactt gctttaaaaa acctcccaca
     2221 cctccccctg aacctgaaac ataaaatgaa tgcaattgtt gttgttaact tgtttattgc
     2281 agcttataat ggttacaaat aaagcaatag catcacaaat ttcacaaata aagcattttt
     2341 ttcactgcat tctagttgtg gtttgtccaa actcatcaat gtatcttaac gcgtaaattg
     2401 taagcgttaa tattttgtta aaattcgcgt taaatttttg ttaaatcagc tcatttttta
     2461 accaataggc cgaaatcggc aaaatccctt ataaatcaaa agaatagacc gagatagggt
     2521 tgagtgttgt tccagtttgg aacaagagtc cactattaaa gaacgtggac tccaacgtca
     2581 aagggcgaaa aaccgtctat cagggcgatg gcccactacg tgaaccatca ccctaatcaa
     2641 gttttttggg gtcgaggtgc cgtaaagcac taaatcggaa ccctaaaggg agcccccgat
     2701 ttagagcttg acggggaaag ccggcgaacg tggcgagaaa ggaagggaag aaagcgaaag
     2761 gagcgggcgc tagggcgctg gcaagtgtag cggtcacgct gcgcgtaacc accacacccg
     2821 ccgcgcttaa tgcgccgcta cagggcgcgt caggtggcac ttttcgggga aatgtgcgcg
     2881 gaacccctat ttgtttattt ttctaaatac attcaaatat gtatccgctc atgagacaat
     2941 aaccctgata aatgcttcaa taatattgaa aaaggaagag tcctgaggcg gaaagaacca
     3001 gctgtggaat gtgtgtcagt tagggtgtgg aaagtcccca ggctccccag caggcagaag
     3061 tatgcaaagc atgcatctca attagtcagc aaccaggtgt ggaaagtccc caggctcccc
     3121 agcaggcaga agtatgcaaa gcatgcatct caattagtca gcaaccatag tcccgcccct
     3181 aactccgccc atcccgcccc taactccgcc cagttccgcc cattctccgc cccatggctg
     3241 actaattttt tttatttatg cagaggccga ggccgcctcg gcctctgagc tattccagaa
     3301 gtagtgagga ggcttttttg gaggcctagg cttttgcaaa gatcgatcaa gagacaggat
     3361 gaggatcgtt tcgcatgatt gaacaagatg gattgcacgc aggttctccg gccgcttggg
     3421 tggagaggct attcggctat gactgggcac aacagacaat cggctgctct gatgccgccg
     3481 tgttccggct gtcagcgcag gggcgcccgg ttctttttgt caagaccgac ctgtccggtg
     3541 ccctgaatga actgcaagac gaggcagcgc ggctatcgtg gctggccacg acgggcgttc
     3601 cttgcgcagc tgtgctcgac gttgtcactg aagcgggaag ggactggctg ctattgggcg
     3661 aagtgccggg gcaggatctc ctgtcatctc accttgctcc tgccgagaaa gtatccatca
     3721 tggctgatgc aatgcggcgg ctgcatacgc ttgatccggc tacctgccca ttcgaccacc
     3781 aagcgaaaca tcgcatcgag cgagcacgta ctcggatgga agccggtctt gtcgatcagg
     3841 atgatctgga cgaagagcat caggggctcg cgccagccga actgttcgcc aggctcaagg
     3901 cgagcatgcc cgacggcgag gatctcgtcg tgacccatgg cgatgcctgc ttgccgaata
     3961 tcatggtgga aaatggccgc ttttctggat tcatcgactg tggccggctg ggtgtggcgg
     4021 accgctatca ggacatagcg ttggctaccc gtgatattgc tgaagagctt ggcggcgaat
     4081 gggctgaccg cttcctcgtg ctttacggta tcgccgctcc cgattcgcag cgcatcgcct
     4141 tctatcgcct tcttgacgag ttcttctgag cgggactctg gggttcgaaa tgaccgacca
     4201 agcgacgccc aacctgccat cacgagattt cgattccacc gccgccttct atgaaaggtt
     4261 gggcttcgga atcgttttcc gggacgccgg ctggatgatc ctccagcgcg gggatctcat
     4321 gctggagttc ttcgcccacc ctagggggag gctaactgaa acacggaagg agacaatacc
     4381 ggaaggaacc cgcgctatga cggcaataaa aagacagaat aaaacgcacg gtgttgggtc
     4441 gtttgttcat aaacgcgggg ttcggtccca gggctggcac tctgtcgata ccccaccgag
     4501 accccattgg ggccaatacg cccgcgtttc ttccttttcc ccaccccacc ccccaagttc
     4561 gggtgaaggc ccagggctcg cagccaacgt cggggcggca ggccctgcca tagcctcagg
     4621 ttactcatat atactttaga ttgatttaaa acttcatttt taatttaaaa ggatctaggt
     4681 gaagatcctt tttgataatc tcatgaccaa aatcccttaa cgtgagtttt cgttccactg
     4741 agcgtcagac cccgtagaaa agatcaaagg atcttcttga gatccttttt ttctgcgcgt
     4801 aatctgctgc ttgcaaacaa aaaaaccacc gctaccagcg gtggtttgtt tgccggatca
     4861 agagctacca actctttttc cgaaggtaac tggcttcagc agagcgcaga taccaaatac
     4921 tgttcttcta gtgtagccgt agttaggcca ccacttcaag aactctgtag caccgcctac
     4981 atacctcgct ctgctaatcc tgttaccagt ggctgctgcc agtggcgata agtcgtgtct
     5041 taccgggttg gactcaagac gatagttacc ggataaggcg cagcggtcgg gctgaacggg
     5101 gggttcgtgc acacagccca gcttggagcg aacgacctac accgaactga gatacctaca
     5161 gcgtgagcta tgagaaagcg ccacgcttcc cgaagggaga aaggcggaca ggtatccggt
     5221 aagcggcagg gtcggaacag gagagcgcac gagggagctt ccagggggaa acgcctggta
     5281 tctttatagt cctgtcgggt ttcgccacct ctgacttgag cgtcgatttt tgtgatgctc
     5341 gtcagggggg cggagcctat ggaaaa
//
