LOCUS       sequence-260971-        6354 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Expresses EGFP-tagged rat NFL containing the Charcot-Marie-Tooth 
            disease type 2E P8R mutation.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 6354)
  AUTHORS   Stone EJ, Uchida A, Brown A
  TITLE     Charcot-Marie-Tooth disease Type 2E/1F mutant neurofilament proteins
            assemble into neurofilaments.
  JOURNAL   Cytoskeleton (Hoboken). 2019 Jul;76(7-8):423-439. doi: 
            10.1002/cm.21566. Epub 2019 Nov 6.
  PUBMED    31574566
REFERENCE   2  (bases 1 to 6354)
  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: 
            "Cytoskeleton (Hoboken)."; date: "2019-07"; volume: 
            "76(7-8):423-439. doi"; pages: " 10.1002/cm.21566. Epub 2019 Nov 6"
COMMENT     Sequence Label: prNFL-P8R-EGFP
FEATURES             Location/Qualifiers
     source          1..6354
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     enhancer        42..345
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
     promoter        346..549
                     /label=CMV promoter
                     /note="human cytomegalovirus (CMV) immediate early 
                     promoter"
     primer_bind     500..520
                     /label=CMV-F
                     /note="Human CMV immediate early promoter, forward primer"
     regulatory      2275..2284
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     CDS             join(2281..2283,2284..2286,2287..3000)
                     /codon_start=1
                     /product="the original enhanced GFP (Yang et al., 1996)"
                     /label=EGFP
                     /note="mammalian codon-optimized"
                     /translation="MVSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTL
                     KFICTTGKLPVPWPTLVTTLTYGVQCFSRYPDHMKQHDFFKSAMPEGYVQERTIFFKDD
                     GNYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIK
                     VNFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSALSKDPNEKRDHMVLL
                     EFVTAAGITLGMDELYK"
     CDS             join(2281..2283,2284..2286,2287..2997)
                     /codon_start=1
                     /product="enhanced GFP"
                     /label=EGFP
                     /translation="MVSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTL
                     KFICTTGKLPVPWPTLVTTLTYGVQCFSRYPDHMKQHDFFKSAMPEGYVQERTIFFKDD
                     GNYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIK
                     VNFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSALSKDPNEKRDHMVLL
                     EFVTAAGITLGMDELYK"
     primer_bind     complement(2326..2347)
                     /label=EGFP-N
                     /note="EGFP, reverse primer"
     primer_bind     complement(2587..2606)
                     /label=EXFP-R
                     /note="For distinguishing EGFP variants, reverse primer"
     primer_bind     2934..2955
                     /label=EGFP-C
                     /note="EGFP, forward primer"
     polyA_signal    3123..3244
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(3160..3179)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     3214..3233
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     rep_origin      complement(3251..3706)
                     /direction=LEFT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(3388..3409)
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     3600..3619
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     promoter        3733..3837
                     /gene="bla"
                     /label=AmpR promoter
     promoter        3839..4196
                     /label=SV40 promoter
                     /note="SV40 enhancer and early promoter"
     enhancer        3839..4030
                     /label=SV40 enhancer
                     /note="enhancer for the SV40 early promoter (Herr, 1993)"
     primer_bind     complement(3862..3882)
                     /label=pBABE 3'
                     /note="SV40 enhancer, reverse primer for pBABE vectors"
     rep_origin      4047..4182
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     CDS             4231..5025
                     /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             4231..5025
                     /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(4285..4304)
                     /label=Neo-R
                     /note="Neomycin resistance gene, reverse primer"
     primer_bind     4895..4914
                     /label=Neo-F
                     /note="Neomycin resistance gene, forward primer"
     primer_bind     complement(5213..5232)
                     /label=TK-pA-R
                     /note="Thymidine kinase polyA, reverse primer"
     polyA_signal    5257..5304
                     /label=HSV TK poly(A) signal
                     /note="herpes simplex virus thymidine kinase 
                     polyadenylation signal (Cole and Stacy, 1985)"
     rep_origin      5633..6221
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     6122..6141
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
ORIGIN
        1 attacggggt cattagttca tagcccatat atggagttcc gcgttacata acttacggta
       61 aatggcccgc ctggctgacc gcccaacgac ccccgcccat tgacgtcaat aatgacgtat
      121 gttcccatag taacgccaat agggactttc cattgacgtc aatgggtgga gtatttacgg
      181 taaactgccc acttggcagt acatcaagtg tatcatatgc caagtacgcc ccctattgac
      241 gtcaatgacg gtaaatggcc cgcctggcat tatgcccagt acatgacctt atgggacttt
      301 cctacttggc agtacatcta cgtattagtc atcgctatta ccatggtgat gcggttttgg
      361 cagtacatca atgggcgtgg atagcggttt gactcacggg gatttccaag tctccacccc
      421 attgacgtca atgggagttt gttttggcac caaaatcaac gggactttcc aaaatgtcgt
      481 aacaactccg ccccattgac gcaaatgggc ggtaggcgtg tacggtggga ggtctatata
      541 agcagagctg gtttagtgaa ccgtcagatc cgctagcgct accggactca gatctcgagc
      601 tcaagcttcg aattcctgag gggcgcaccg accgccacca tgagttcgtt cagctacgag
      661 cggtactttt cgacctccta caagcggcgc tacgtggaga cgccccgggt gcacatctcc
      721 agcgtgcgca gcggctacag cacggcgcgc tctgcgtact ccagctactc cgcgcccgtc
      781 tcctcctctc tgtcggtgcg ccgcagctac tcatccagct ccggctcttt gatgcccagc
      841 ctggagaacc tcgatctgag ccaggtagcc gccatcagca acgacctcaa gtctatccgc
      901 acacaggaga aggcacagct gcaggacctc aacgatcgct tcgccagctt catcgagcgc
      961 gtgcacgagc tggagcagca gaacaaggtc ctggaagccg agctgttggt gctgcgccag
     1021 aagcactcag agccttcccg cttccgcgcc ctgtatgagc aggagatccg tgatctgcga
     1081 ctggcggccg aagacgccac taacgagaag caggcgctgc agggcgagcg cgaggggctg
     1141 gaggagactc tgcgcaacct gcaggctcgc tacgaggagg aggtgctgag ccgcgaggac
     1201 gccgagggcc ggctgatgga agcccgcaaa ggcgcggatg aggctgcgct cgcccgcgcc
     1261 gagctggaga agcgcatcga cagcctgatg gacgagatag ccttcctgaa aaaggtgcac
     1321 gaggaagaga tcgccgagct gcaggctcag atccagtatg ctcagatctc cgtggagatg
     1381 gacgtgtcct ccaagcccga cctctccgcc gctctcaagg acatccgcgc tcagtacgag
     1441 aagctggccg ccaagaatat gcagaatgcc gaagagtggt tcaagagccg cttcacggtg
     1501 ctaaccgaga gcgccgccaa gaacaccgac gcagtgcgcg ctgccaagga cgaggtgtcg
     1561 gaaagccgcc gcctacttaa ggctaagacc ctggagatcg aagcctgccg gggtatgaac
     1621 gaagctctag agaagcagct gcaggagctg gaggacaagc agaatgcaga catcagcgcc
     1681 atgcaggaca caatcaacaa actggagaat gagctgcgaa gcacgaagag cgagatggcc
     1741 aggtacctga aggagtacca ggacctcctc aatgtcaaga tggcattgga cattgagatt
     1801 gcagcttaca ggaaactctt ggaaggcgaa gaaaccaggc tcagtttcac cagcgtgggt
     1861 agcataacca gcggctactc tcagagctcg caggtctttg gccgttctgc ttacagtggc
     1921 ttgcagagca gctcctactt gatgtctgct cgagcattcc cagcctacta taccagccac
     1981 gtccaggaag agcagtcaga ggtggaggag accattgagg ctacgaaagc tgaggaggcc
     2041 aaggatgagc ccccctctga aggagaagca gaagaggagg agaaggagaa agaggagggg
     2101 gaggaagagg aaggtgctga ggaggaagaa gctgccaagg atgagtctga agatgccaaa
     2161 gaagaagaag gtggtgaggg tgaagaggaa gacaccaaag agtcagaaga ggaagagaag
     2221 aaagaggaaa gtgctgggga ggagcaagct gctaagaaga aagatggatc catcgccacc
     2281 atggtgagca agggcgagga gctgttcacc ggggtggtgc ccatcctggt cgagctggac
     2341 ggcgacgtaa acggccacaa gttcagcgtg tccggcgagg gcgagggcga tgccacctac
     2401 ggcaagctga ccctgaagtt catctgcacc accggcaagc tgcccgtgcc ctggcccacc
     2461 ctcgtgacca ccctgaccta cggcgtgcag tgcttcagcc gctaccccga ccacatgaag
     2521 cagcacgact tcttcaagtc cgccatgccc gaaggctacg tccaggagcg caccatcttc
     2581 ttcaaggacg acggcaacta caagacccgc gccgaggtga agttcgaggg cgacaccctg
     2641 gtgaaccgca tcgagctgaa gggcatcgac ttcaaggagg acggcaacat cctggggcac
     2701 aagctggagt acaactacaa cagccacaac gtctatatca tggccgacaa gcagaagaac
     2761 ggcatcaagg tgaacttcaa gatccgccac aacatcgagg acggcagcgt gcagctcgcc
     2821 gaccactacc agcagaacac ccccatcggc gacggccccg tgctgctgcc cgacaaccac
     2881 tacctgagca cccagtccgc cctgagcaaa gaccccaacg agaagcgcga tcacatggtc
     2941 ctgctggagt tcgtgaccgc cgccgggatc actctcggca tggacgagct gtacaagtaa
     3001 agcggccgcg actctagatc ataatcagcc ataccacatt tgtagaggtt ttacttgctt
     3061 taaaaaacct cccacacctc cccctgaacc tgaaacataa aatgaatgca attgttgttg
     3121 ttaacttgtt tattgcagct tataatggtt acaaataaag caatagcatc acaaatttca
     3181 caaataaagc atttttttca ctgcattcta gttgtggttt gtccaaactc atcaatgtat
     3241 cttaaggcgt aaattgtaag cgttaatatt ttgttaaaat tcgcgttaaa tttttgttaa
     3301 atcagctcat tttttaacca ataggccgaa atcggcaaaa tcccttataa atcaaaagaa
     3361 tagaccgaga tagggttgag tgttgttcca gtttggaaca agagtccact attaaagaac
     3421 gtggactcca acgtcaaagg gcgaaaaacc gtctatcagg gcgatggccc actacgtgaa
     3481 ccatcaccct aatcaagttt tttggggtcg aggtgccgta aagcactaaa tcggaaccct
     3541 aaagggagcc cccgatttag agcttgacgg ggaaagccgg cgaacgtggc gagaaaggaa
     3601 gggaagaaag cgaaaggagc gggcgctagg gcgctggcaa gtgtagcggt cacgctgcgc
     3661 gtaaccacca cacccgccgc gcttaatgcg ccgctacagg gcgcgtcagg tggcactttt
     3721 cggggaaatg tgcgcggaac ccctatttgt ttatttttct aaatacattc aaatatgtat
     3781 ccgctcatga gacaataacc ctgataaatg cttcaataat attgaaaaag gaagagtcct
     3841 gaggcggaaa gaaccagctg tggaatgtgt gtcagttagg gtgtggaaag tccccaggct
     3901 ccccagcagg cagaagtatg caaagcatgc atctcaatta gtcagcaacc aggtgtggaa
     3961 agtccccagg ctccccagca ggcagaagta tgcaaagcat gcatctcaat tagtcagcaa
     4021 ccatagtccc gcccctaact ccgcccatcc cgcccctaac tccgcccagt tccgcccatt
     4081 ctccgcccca tggctgacta atttttttta tttatgcaga ggccgaggcc gcctcggcct
     4141 ctgagctatt ccagaagtag tgaggaggct tttttggagg cctaggcttt tgcaaagatc
     4201 gatcaagaga caggatgagg atcgtttcgc atgattgaac aagatggatt gcacgcaggt
     4261 tctccggccg cttgggtgga gaggctattc ggctatgact gggcacaaca gacaatcggc
     4321 tgctctgatg ccgccgtgtt ccggctgtca gcgcaggggc gcccggttct ttttgtcaag
     4381 accgacctgt ccggtgccct gaatgaactg caagacgagg cagcgcggct atcgtggctg
     4441 gccacgacgg gcgttccttg cgcagctgtg ctcgacgttg tcactgaagc gggaagggac
     4501 tggctgctat tgggcgaagt gccggggcag gatctcctgt catctcacct tgctcctgcc
     4561 gagaaagtat ccatcatggc tgatgcaatg cggcggctgc atacgcttga tccggctacc
     4621 tgcccattcg accaccaagc gaaacatcgc atcgagcgag cacgtactcg gatggaagcc
     4681 ggtcttgtcg atcaggatga tctggacgaa gagcatcagg ggctcgcgcc agccgaactg
     4741 ttcgccaggc tcaaggcgag catgcccgac ggcgaggatc tcgtcgtgac ccatggcgat
     4801 gcctgcttgc cgaatatcat ggtggaaaat ggccgctttt ctggattcat cgactgtggc
     4861 cggctgggtg tggcggaccg ctatcaggac atagcgttgg ctacccgtga tattgctgaa
     4921 gagcttggcg gcgaatgggc tgaccgcttc ctcgtgcttt acggtatcgc cgctcccgat
     4981 tcgcagcgca tcgccttcta tcgccttctt gacgagttct tctgagcggg actctggggt
     5041 tcgaaatgac cgaccaagcg acgcccaacc tgccatcacg agatttcgat tccaccgccg
     5101 ccttctatga aaggttgggc ttcggaatcg ttttccggga cgccggctgg atgatcctcc
     5161 agcgcgggga tctcatgctg gagttcttcg cccaccctag ggggaggcta actgaaacac
     5221 ggaaggagac aataccggaa ggaacccgcg ctatgacggc aataaaaaga cagaataaaa
     5281 cgcacggtgt tgggtcgttt gttcataaac gcggggttcg gtcccagggc tggcactctg
     5341 tcgatacccc accgagaccc cattggggcc aatacgcccg cgtttcttcc ttttccccac
     5401 cccacccccc aagttcgggt gaaggcccag ggctcgcagc caacgtcggg gcggcaggcc
     5461 ctgccatagc ctcaggttac tcatatatac tttagattga tttaaaactt catttttaat
     5521 ttaaaaggat ctaggtgaag atcctttttg ataatctcat gaccaaaatc ccttaacgtg
     5581 agttttcgtt ccactgagcg tcagaccccg tagaaaagat caaaggatct tcttgagatc
     5641 ctttttttct gcgcgtaatc tgctgcttgc aaacaaaaaa accaccgcta ccagcggtgg
     5701 tttgtttgcc ggatcaagag ctaccaactc tttttccgaa ggtaactggc ttcagcagag
     5761 cgcagatacc aaatactgtt cttctagtgt agccgtagtt aggccaccac ttcaagaact
     5821 ctgtagcacc gcctacatac ctcgctctgc taatcctgtt accagtggct gctgccagtg
     5881 gcgataagtc gtgtcttacc gggttggact caagacgata gttaccggat aaggcgcagc
     5941 ggtcgggctg aacggggggt tcgtgcacac agcccagctt ggagcgaacg acctacaccg
     6001 aactgagata cctacagcgt gagctatgag aaagcgccac gcttcccgaa gggagaaagg
     6061 cggacaggta tccggtaagc ggcagggtcg gaacaggaga gcgcacgagg gagcttccag
     6121 ggggaaacgc ctggtatctt tatagtcctg tcgggtttcg ccacctctga cttgagcgtc
     6181 gatttttgtg atgctcgtca ggggggcgga gcctatggaa aaacgccagc aacgcggcct
     6241 ttttacggtt cctggccttt tgctggcctt ttgctcacat gttctttcct gcgttatccc
     6301 ctgattctgt ggataaccgt attaccgcca tgcattagtt attaatagta atca
//
