LOCUS       sequence-393645-        5019 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 5019)
  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 5019)
  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 EGFP-LC3B
FEATURES             Location/Qualifiers
     source          1..5019
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     promoter        119..629
                     /label=hPGK promoter
                     /note="human phosphoglycerate kinase 1 promoter"
     primer_bind     548..566
                     /label=hPGK-F
                     /note="Human PGK promoter, forward primer"
     regulatory      645..654
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     CDS             join(651..653,654..656,657..1367)
                     /codon_start=1
                     /product="enhanced GFP"
                     /label=EGFP
                     /translation="MVSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTL
                     KFICTTGKLPVPWPTLVTTLTYGVQCFSRYPDHMKQHDFFKSAMPEGYVQERTIFFKDD
                     GNYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIK
                     VNFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSALSKDPNEKRDHMVLL
                     EFVTAAGITLGMDELYK"
     CDS             join(651..653,654..656,657..1367)
                     /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(696..717)
                     /label=EGFP-N
                     /note="EGFP, reverse primer"
     primer_bind     complement(957..976)
                     /label=EXFP-R
                     /note="For distinguishing EGFP variants, reverse primer"
     primer_bind     1304..1325
                     /label=EGFP-C
                     /note="EGFP, forward primer"
     CDS             1398..1772
                     /codon_start=1
                     /gene="MAP1LC3B"
                     /product="human LC3 protein"
                     /label=LC3B
                     /note="Atg8 family member found on autophagosomes"
                     /translation="MPSEKTFKQRRTFEQRVEDVRLIREQHPTKIPVIIERYKGEKQLP
                     VLDKTKFLVPDHVNMSELIKIIRRRLQLNANQAFFLLVNGHSMVSVSTPISEVYESEKD
                     EDGFLYMVYASQETFGMKLSV"
     polyA_signal    1910..2031
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(1947..1966)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     2001..2020
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     rep_origin      complement(2038..2493)
                     /direction=LEFT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(2175..2196)
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     2387..2406
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     promoter        2520..2624
                     /gene="bla"
                     /label=AmpR promoter
     promoter        2626..2983
                     /label=SV40 promoter
                     /note="SV40 enhancer and early promoter"
     enhancer        2626..2817
                     /label=SV40 enhancer
                     /note="enhancer for the SV40 early promoter (Herr, 1993)"
     primer_bind     complement(2649..2669)
                     /label=pBABE 3'
                     /note="SV40 enhancer, reverse primer for pBABE vectors"
     rep_origin      2834..2969
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     CDS             3018..3812
                     /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             3018..3812
                     /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(3072..3091)
                     /label=Neo-R
                     /note="Neomycin resistance gene, reverse primer"
     primer_bind     3682..3701
                     /label=Neo-F
                     /note="Neomycin resistance gene, forward primer"
     primer_bind     complement(4000..4019)
                     /label=TK-pA-R
                     /note="Thymidine kinase polyA, reverse primer"
     polyA_signal    4044..4091
                     /label=HSV TK poly(A) signal
                     /note="herpes simplex virus thymidine kinase 
                     polyadenylation signal (Cole and Stacy, 1985)"
     rep_origin      4420..5008
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     4909..4928
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
ORIGIN
        1 cgcggccttt ttacggttcc tggccttttg ctggcctttt gctcacatgt tctttcctgc
       61 gttatcccct gattctgtgg ataaccgtat taccgccatg cattagttat taatccacgg
      121 ggttggggtt gcgccttttc caaggcagcc ctgggtttgc gcagggacgc ggctgctctg
      181 ggcgtggttc cgggaaacgc agcggcgccg accctgggtc tcgcacattc ttcacgtccg
      241 ttcgcagcgt cacccggatc ttcgccgcta cccttgtggg ccccccggcg acgcttcctg
      301 ctccgcccct aagtcgggaa ggttccttgc ggttcgcggc gtgccggacg tgacaaacgg
      361 aagccgcacg tctcactagt accctcgcag acggacagcg ccagggagca atggcagcgc
      421 gccgaccgcg atgggctgtg gccaatagcg gctgctcagc agggcgcgcc gagagcagcg
      481 gccgggaagg ggcggtgcgg gaggcggggt gtggggcggt agtgtgggcc ctgttcctgc
      541 ccgcgcggtg ttccgcattc tgcaagcctc cggagcgcac gtcggcagtc ggctccctcg
      601 ttgaccgaat caccgacctc tctccccagg tggatccacc ggtcgccacc atggtgagca
      661 agggcgagga gctgttcacc ggggtggtgc ccatcctggt cgagctggac ggcgacgtaa
      721 acggccacaa gttcagcgtg tccggcgagg gcgagggcga tgccacctac ggcaagctga
      781 ccctgaagtt catctgcacc accggcaagc tgcccgtgcc ctggcccacc ctcgtgacca
      841 ccctgaccta cggcgtgcag tgcttcagcc gctaccccga ccacatgaag cagcacgact
      901 tcttcaagtc cgccatgccc gaaggctacg tccaggagcg caccatcttc ttcaaggacg
      961 acggcaacta caagacccgc gccgaggtga agttcgaggg cgacaccctg gtgaaccgca
     1021 tcgagctgaa gggcatcgac ttcaaggagg acggcaacat cctggggcac aagctggagt
     1081 acaactacaa cagccacaac gtctatatca tggccgacaa gcagaagaac ggcatcaagg
     1141 tgaacttcaa gatccgccac aacatcgagg acggcagcgt gcagctcgcc gaccactacc
     1201 agcagaacac ccccatcggc gacggccccg tgctgctgcc cgacaaccac tacctgagca
     1261 cccagtccgc cctgagcaaa gaccccaacg agaagcgcga tcacatggtc ctgctggagt
     1321 tcgtgaccgc cgccgggatc actctcggca tggacgagct gtacaagtcc ggactcagat
     1381 ctggctccag cgcaggcatg ccgtcggaga agaccttcaa gcagcgccgc accttcgaac
     1441 aaagagtaga agatgtccga cttattcgag agcagcatcc aaccaaaatc ccggtgataa
     1501 tagaacgata caagggtgag aagcagcttc ctgttctgga taaaacaaag ttccttgtac
     1561 ctgaccatgt caacatgagt gagctcatca agataattag aaggcgctta cagctcaatg
     1621 ctaatcaggc cttcttcctg ttggtgaacg gacacagcat ggtcagcgtc tccacaccaa
     1681 tctcagaggt gtatgagagt gagaaagatg aagatggatt cctgtacatg gtctacgcct
     1741 cccaggagac gttcgggatg aaattgtcag tgtaacgcgg ggatccaccg gatctagata
     1801 actgatcata atcagccata ccacatttgt agaggtttta cttgctttaa aaaacctccc
     1861 acacctcccc ctgaacctga aacataaaat gaatgcaatt gttgttgtta acttgtttat
     1921 tgcagcttat aatggttaca aataaagcaa tagcatcaca aatttcacaa ataaagcatt
     1981 tttttcactg cattctagtt gtggtttgtc caaactcatc aatgtatctt aacgcgtaaa
     2041 ttgtaagcgt taatattttg ttaaaattcg cgttaaattt ttgttaaatc agctcatttt
     2101 ttaaccaata ggccgaaatc ggcaaaatcc cttataaatc aaaagaatag accgagatag
     2161 ggttgagtgt tgttccagtt tggaacaaga gtccactatt aaagaacgtg gactccaacg
     2221 tcaaagggcg aaaaaccgtc tatcagggcg atggcccact acgtgaacca tcaccctaat
     2281 caagtttttt ggggtcgagg tgccgtaaag cactaaatcg gaaccctaaa gggagccccc
     2341 gatttagagc ttgacgggga aagccggcga acgtggcgag aaaggaaggg aagaaagcga
     2401 aaggagcggg cgctagggcg ctggcaagtg tagcggtcac gctgcgcgta accaccacac
     2461 ccgccgcgct taatgcgccg ctacagggcg cgtcaggtgg cacttttcgg ggaaatgtgc
     2521 gcggaacccc tatttgttta tttttctaaa tacattcaaa tatgtatccg ctcatgagac
     2581 aataaccctg ataaatgctt caataatatt gaaaaaggaa gagtcctgag gcggaaagaa
     2641 ccagctgtgg aatgtgtgtc agttagggtg tggaaagtcc ccaggctccc cagcaggcag
     2701 aagtatgcaa agcatgcatc tcaattagtc agcaaccagg tgtggaaagt ccccaggctc
     2761 cccagcaggc agaagtatgc aaagcatgca tctcaattag tcagcaacca tagtcccgcc
     2821 cctaactccg cccatcccgc ccctaactcc gcccagttcc gcccattctc cgccccatgg
     2881 ctgactaatt ttttttattt atgcagaggc cgaggccgcc tcggcctctg agctattcca
     2941 gaagtagtga ggaggctttt ttggaggcct aggcttttgc aaagatcgat caagagacag
     3001 gatgaggatc gtttcgcatg attgaacaag atggattgca cgcaggttct ccggccgctt
     3061 gggtggagag gctattcggc tatgactggg cacaacagac aatcggctgc tctgatgccg
     3121 ccgtgttccg gctgtcagcg caggggcgcc cggttctttt tgtcaagacc gacctgtccg
     3181 gtgccctgaa tgaactgcaa gacgaggcag cgcggctatc gtggctggcc acgacgggcg
     3241 ttccttgcgc agctgtgctc gacgttgtca ctgaagcggg aagggactgg ctgctattgg
     3301 gcgaagtgcc ggggcaggat ctcctgtcat ctcaccttgc tcctgccgag aaagtatcca
     3361 tcatggctga tgcaatgcgg cggctgcata cgcttgatcc ggctacctgc ccattcgacc
     3421 accaagcgaa acatcgcatc gagcgagcac gtactcggat ggaagccggt cttgtcgatc
     3481 aggatgatct ggacgaagag catcaggggc tcgcgccagc cgaactgttc gccaggctca
     3541 aggcgagcat gcccgacggc gaggatctcg tcgtgaccca tggcgatgcc tgcttgccga
     3601 atatcatggt ggaaaatggc cgcttttctg gattcatcga ctgtggccgg ctgggtgtgg
     3661 cggaccgcta tcaggacata gcgttggcta cccgtgatat tgctgaagag cttggcggcg
     3721 aatgggctga ccgcttcctc gtgctttacg gtatcgccgc tcccgattcg cagcgcatcg
     3781 ccttctatcg ccttcttgac gagttcttct gagcgggact ctggggttcg aaatgaccga
     3841 ccaagcgacg cccaacctgc catcacgaga tttcgattcc accgccgcct tctatgaaag
     3901 gttgggcttc ggaatcgttt tccgggacgc cggctggatg atcctccagc gcggggatct
     3961 catgctggag ttcttcgccc accctagggg gaggctaact gaaacacgga aggagacaat
     4021 accggaagga acccgcgcta tgacggcaat aaaaagacag aataaaacgc acggtgttgg
     4081 gtcgtttgtt cataaacgcg gggttcggtc ccagggctgg cactctgtcg ataccccacc
     4141 gagaccccat tggggccaat acgcccgcgt ttcttccttt tccccacccc accccccaag
     4201 ttcgggtgaa ggcccagggc tcgcagccaa cgtcggggcg gcaggccctg ccatagcctc
     4261 aggttactca tatatacttt agattgattt aaaacttcat ttttaattta aaaggatcta
     4321 ggtgaagatc ctttttgata atctcatgac caaaatccct taacgtgagt tttcgttcca
     4381 ctgagcgtca gaccccgtag aaaagatcaa aggatcttct tgagatcctt tttttctgcg
     4441 cgtaatctgc tgcttgcaaa caaaaaaacc accgctacca gcggtggttt gtttgccgga
     4501 tcaagagcta ccaactcttt ttccgaaggt aactggcttc agcagagcgc agataccaaa
     4561 tactgttctt ctagtgtagc cgtagttagg ccaccacttc aagaactctg tagcaccgcc
     4621 tacatacctc gctctgctaa tcctgttacc agtggctgct gccagtggcg ataagtcgtg
     4681 tcttaccggg ttggactcaa gacgatagtt accggataag gcgcagcggt cgggctgaac
     4741 ggggggttcg tgcacacagc ccagcttgga gcgaacgacc tacaccgaac tgagatacct
     4801 acagcgtgag ctatgagaaa gcgccacgct tcccgaaggg agaaaggcgg acaggtatcc
     4861 ggtaagcggc agggtcggaa caggagagcg cacgagggag cttccagggg gaaacgcctg
     4921 gtatctttat agtcctgtcg ggtttcgcca cctctgactt gagcgtcgat ttttgtgatg
     4981 ctcgtcaggg gggcggagcc tatggaaaaa cgccagcaa
//
