LOCUS       sequence-322480-        5460 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Expression in mammalian cells.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 5460)
  AUTHORS   Maejima I, Takahashi A, Omori H, Kimura T, Takabatake Y, Saitoh T, 
            Yamamoto A, Hamasaki M, Noda T, Isaka Y, Yoshimori T
  TITLE     Autophagy sequesters damaged lysosomes to control lysosomal 
            biogenesis and kidney injury.
  JOURNAL   EMBO J. 2013 Aug 28;32(17):2336-47. doi: 10.1038/emboj.2013.171. 
            Epub 2013 Aug 6.
  PUBMED    23921551
REFERENCE   2  (bases 1 to 5460)
  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: 
            "10.1038/emboj.2013.171"; journalName: "EMBO J"; date: "2013-08-28- 
            28"; volume: "32"; issue: "17"; pages: "2336-47"
COMMENT     Sequence Label: pEGFP-hGal3
FEATURES             Location/Qualifiers
     source          1..5460
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     enhancer        115..418
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
     promoter        419..622
                     /label=CMV promoter
                     /note="human cytomegalovirus (CMV) immediate early 
                     promoter"
     primer_bind     573..593
                     /label=CMV-F
                     /note="Human CMV immediate early promoter, forward primer"
     regulatory      661..670
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     CDS             join(667..669,670..672,673..1383)
                     /codon_start=1
                     /product="enhanced GFP"
                     /label=EGFP
                     /translation="MVSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTL
                     KFICTTGKLPVPWPTLVTTLTYGVQCFSRYPDHMKQHDFFKSAMPEGYVQERTIFFKDD
                     GNYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIK
                     VNFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSALSKDPNEKRDHMVLL
                     EFVTAAGITLGMDELYK"
     CDS             join(667..669,670..672,673..1383)
                     /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(712..733)
                     /label=EGFP-N
                     /note="EGFP, reverse primer"
     primer_bind     complement(973..992)
                     /label=EXFP-R
                     /note="For distinguishing EGFP variants, reverse primer"
     primer_bind     1320..1341
                     /label=EGFP-C
                     /note="EGFP, forward primer"
     polyA_signal    2302..2423
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(2339..2358)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     2393..2412
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     rep_origin      complement(2430..2885)
                     /direction=LEFT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(2567..2588)
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     2779..2798
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     promoter        2912..3016
                     /gene="bla"
                     /label=AmpR promoter
     promoter        3018..3375
                     /label=SV40 promoter
                     /note="SV40 enhancer and early promoter"
     enhancer        3018..3209
                     /label=SV40 enhancer
                     /note="enhancer for the SV40 early promoter (Herr, 1993)"
     primer_bind     complement(3041..3061)
                     /label=pBABE 3'
                     /note="SV40 enhancer, reverse primer for pBABE vectors"
     rep_origin      3226..3361
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     CDS             3410..4204
                     /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             3410..4204
                     /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(3464..3483)
                     /label=Neo-R
                     /note="Neomycin resistance gene, reverse primer"
     primer_bind     4074..4093
                     /label=Neo-F
                     /note="Neomycin resistance gene, forward primer"
     primer_bind     complement(4392..4411)
                     /label=TK-pA-R
                     /note="Thymidine kinase polyA, reverse primer"
     polyA_signal    4436..4483
                     /label=HSV TK poly(A) signal
                     /note="herpes simplex virus thymidine kinase 
                     polyadenylation signal (Cole and Stacy, 1985)"
     rep_origin      4812..5400
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     5301..5320
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
ORIGIN
        1 ttctttcctg cgttatcccc tgattctgtg gataaccgta ttaccgccat gcattagtta
       61 ttaatagtaa tcaattacgg ggtcattagt tcatagccca tatatggagt tccgcgttac
      121 ataacttacg gtaaatggcc cgcctggctg accgcccaac gacccccgcc cattgacgtc
      181 aataatgacg tatgttccca tagtaacgcc aatagggact ttccattgac gtcaatgggt
      241 ggagtattta cggtaaactg cccacttggc agtacatcaa gtgtatcata tgccaagtac
      301 gccccctatt gacgtcaatg acggtaaatg gcccgcctgg cattatgccc agtacatgac
      361 cttatgggac tttcctactt ggcagtacat ctacgtatta gtcatcgcta ttaccatggt
      421 gatgcggttt tggcagtaca tcaatgggcg tggatagcgg tttgactcac ggggatttcc
      481 aagtctccac cccattgacg tcaatgggag tttgttttgg caccaaaatc aacgggactt
      541 tccaaaatgt cgtaacaact ccgccccatt gacgcaaatg ggcggtaggc gtgtacggtg
      601 ggaggtctat ataagcagag ctggtttagt gaaccgtcag atccgctagc gctaccggtc
      661 gccaccatgg tgagcaaggg cgaggagctg ttcaccgggg tggtgcccat cctggtcgag
      721 ctggacggcg acgtaaacgg ccacaagttc agcgtgtccg gcgagggcga gggcgatgcc
      781 acctacggca agctgaccct gaagttcatc tgcaccaccg gcaagctgcc cgtgccctgg
      841 cccaccctcg tgaccaccct gacctacggc gtgcagtgct tcagccgcta ccccgaccac
      901 atgaagcagc acgacttctt caagtccgcc atgcccgaag gctacgtcca ggagcgcacc
      961 atcttcttca aggacgacgg caactacaag acccgcgccg aggtgaagtt cgagggcgac
     1021 accctggtga accgcatcga gctgaagggc atcgacttca aggaggacgg caacatcctg
     1081 gggcacaagc tggagtacaa ctacaacagc cacaacgtct atatcatggc cgacaagcag
     1141 aagaacggca tcaaggtgaa cttcaagatc cgccacaaca tcgaggacgg cagcgtgcag
     1201 ctcgccgacc actaccagca gaacaccccc atcggcgacg gccccgtgct gctgcccgac
     1261 aaccactacc tgagcaccca gtccgccctg agcaaagacc ccaacgagaa gcgcgatcac
     1321 atggtcctgc tggagttcgt gaccgccgcc gggatcactc tcggcatgga cgagctgtac
     1381 aagtccggac tcagatctat ggcagacaat ttttcgctcc atgatgcgtt atctgggtct
     1441 ggaaacccaa accctcaagg atggcctggc gcatggggga accagcctgc tggggcaggg
     1501 ggctacccag gggcttccta tcctggggcc taccccgggc aggcaccccc aggggcttat
     1561 cctggacagg cacctccagg cgcctaccct ggagcacctg gagcttatcc cggagcacct
     1621 gcacctggag tctacccagg gccacccagc ggccctgggg cctacccatc ttctggacag
     1681 ccaagtgccc ccggagccta ccctgccact ggcccctatg gcgcccctgc tgggccactg
     1741 attgtgcctt ataacctgcc tttgcctggg ggagtggtgc ctcgcatgct gataacaatt
     1801 ctgggcacgg tgaagcccaa tgcaaacaga attgctttag atttccaaag agggaatgat
     1861 gttgccttcc actttaaccc acgcttcaat gagaacaaca ggagagtcat tgtttgcaat
     1921 acaaagctgg ataataactg gggaagggaa gaaagacagt cagttttccc atttgaaagt
     1981 gggaaaccat tcaaaataca agtactggtt gaacctgacc acttcaaggt tgcagtgaat
     2041 gatgctcact tgttgcagta caatcatcgg gttaaaaaac tcaatgaaat cagcaaactg
     2101 ggaatttctg gtgacataga cctcaccagt gcttcatata ccatgatata agtcgacggt
     2161 accgcgggcc cgggatccac cggatctaga taactgatca taatcagcca taccacattt
     2221 gtagaggttt tacttgcttt aaaaaacctc ccacacctcc ccctgaacct gaaacataaa
     2281 atgaatgcaa ttgttgttgt taacttgttt attgcagctt ataatggtta caaataaagc
     2341 aatagcatca caaatttcac aaataaagca tttttttcac tgcattctag ttgtggtttg
     2401 tccaaactca tcaatgtatc ttaacgcgta aattgtaagc gttaatattt tgttaaaatt
     2461 cgcgttaaat ttttgttaaa tcagctcatt ttttaaccaa taggccgaaa tcggcaaaat
     2521 cccttataaa tcaaaagaat agaccgagat agggttgagt gttgttccag tttggaacaa
     2581 gagtccacta ttaaagaacg tggactccaa cgtcaaaggg cgaaaaaccg tctatcaggg
     2641 cgatggccca ctacgtgaac catcacccta atcaagtttt ttggggtcga ggtgccgtaa
     2701 agcactaaat cggaacccta aagggagccc ccgatttaga gcttgacggg gaaagccggc
     2761 gaacgtggcg agaaaggaag ggaagaaagc gaaaggagcg ggcgctaggg cgctggcaag
     2821 tgtagcggtc acgctgcgcg taaccaccac acccgccgcg cttaatgcgc cgctacaggg
     2881 cgcgtcaggt ggcacttttc ggggaaatgt gcgcggaacc cctatttgtt tatttttcta
     2941 aatacattca aatatgtatc cgctcatgag acaataaccc tgataaatgc ttcaataata
     3001 ttgaaaaagg aagagtcctg aggcggaaag aaccagctgt ggaatgtgtg tcagttaggg
     3061 tgtggaaagt ccccaggctc cccagcaggc agaagtatgc aaagcatgca tctcaattag
     3121 tcagcaacca ggtgtggaaa gtccccaggc tccccagcag gcagaagtat gcaaagcatg
     3181 catctcaatt agtcagcaac catagtcccg cccctaactc cgcccatccc gcccctaact
     3241 ccgcccagtt ccgcccattc tccgccccat ggctgactaa ttttttttat ttatgcagag
     3301 gccgaggccg cctcggcctc tgagctattc cagaagtagt gaggaggctt ttttggaggc
     3361 ctaggctttt gcaaagatcg atcaagagac aggatgagga tcgtttcgca tgattgaaca
     3421 agatggattg cacgcaggtt ctccggccgc ttgggtggag aggctattcg gctatgactg
     3481 ggcacaacag acaatcggct gctctgatgc cgccgtgttc cggctgtcag cgcaggggcg
     3541 cccggttctt tttgtcaaga ccgacctgtc cggtgccctg aatgaactgc aagacgaggc
     3601 agcgcggcta tcgtggctgg ccacgacggg cgttccttgc gcagctgtgc tcgacgttgt
     3661 cactgaagcg ggaagggact ggctgctatt gggcgaagtg ccggggcagg atctcctgtc
     3721 atctcacctt gctcctgccg agaaagtatc catcatggct gatgcaatgc ggcggctgca
     3781 tacgcttgat ccggctacct gcccattcga ccaccaagcg aaacatcgca tcgagcgagc
     3841 acgtactcgg atggaagccg gtcttgtcga tcaggatgat ctggacgaag agcatcaggg
     3901 gctcgcgcca gccgaactgt tcgccaggct caaggcgagc atgcccgacg gcgaggatct
     3961 cgtcgtgacc catggcgatg cctgcttgcc gaatatcatg gtggaaaatg gccgcttttc
     4021 tggattcatc gactgtggcc ggctgggtgt ggcggaccgc tatcaggaca tagcgttggc
     4081 tacccgtgat attgctgaag agcttggcgg cgaatgggct gaccgcttcc tcgtgcttta
     4141 cggtatcgcc gctcccgatt cgcagcgcat cgccttctat cgccttcttg acgagttctt
     4201 ctgagcggga ctctggggtt cgaaatgacc gaccaagcga cgcccaacct gccatcacga
     4261 gatttcgatt ccaccgccgc cttctatgaa aggttgggct tcggaatcgt tttccgggac
     4321 gccggctgga tgatcctcca gcgcggggat ctcatgctgg agttcttcgc ccaccctagg
     4381 gggaggctaa ctgaaacacg gaaggagaca ataccggaag gaacccgcgc tatgacggca
     4441 ataaaaagac agaataaaac gcacggtgtt gggtcgtttg ttcataaacg cggggttcgg
     4501 tcccagggct ggcactctgt cgatacccca ccgagacccc attggggcca atacgcccgc
     4561 gtttcttcct tttccccacc ccacccccca agttcgggtg aaggcccagg gctcgcagcc
     4621 aacgtcgggg cggcaggccc tgccatagcc tcaggttact catatatact ttagattgat
     4681 ttaaaacttc atttttaatt taaaaggatc taggtgaaga tcctttttga taatctcatg
     4741 accaaaatcc cttaacgtga gttttcgttc cactgagcgt cagaccccgt agaaaagatc
     4801 aaaggatctt cttgagatcc tttttttctg cgcgtaatct gctgcttgca aacaaaaaaa
     4861 ccaccgctac cagcggtggt ttgtttgccg gatcaagagc taccaactct ttttccgaag
     4921 gtaactggct tcagcagagc gcagatacca aatactgttc ttctagtgta gccgtagtta
     4981 ggccaccact tcaagaactc tgtagcaccg cctacatacc tcgctctgct aatcctgtta
     5041 ccagtggctg ctgccagtgg cgataagtcg tgtcttaccg ggttggactc aagacgatag
     5101 ttaccggata aggcgcagcg gtcgggctga acggggggtt cgtgcacaca gcccagcttg
     5161 gagcgaacga cctacaccga actgagatac ctacagcgtg agctatgaga aagcgccacg
     5221 cttcccgaag ggagaaaggc ggacaggtat ccggtaagcg gcagggtcgg aacaggagag
     5281 cgcacgaggg agcttccagg gggaaacgcc tggtatcttt atagtcctgt cgggtttcgc
     5341 cacctctgac ttgagcgtcg atttttgtga tgctcgtcag gggggcggag cctatggaaa
     5401 aacgccagca acgcggcctt tttacggttc ctggcctttt gctggccttt tgctcacatg
//
