LOCUS       sequence-260943-        6063 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Expresses human 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 6063)
  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 6063)
  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: phNFL-P8R
FEATURES             Location/Qualifiers
     source          1..6063
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     enhancer        73..376
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
     promoter        377..580
                     /label=CMV promoter
                     /note="human cytomegalovirus (CMV) immediate early 
                     promoter"
     primer_bind     531..551
                     /label=CMV-F
                     /note="Human CMV immediate early promoter, forward primer"
     misc_feature    621..677
                     /label=MCS
                     /note="multiple cloning site"
     polyA_signal    2863..2984
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(2900..2919)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     2954..2973
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     rep_origin      complement(2991..3446)
                     /direction=LEFT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(3128..3149)
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     3340..3359
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     promoter        3473..3577
                     /gene="bla"
                     /label=AmpR promoter
     promoter        3579..3936
                     /label=SV40 promoter
                     /note="SV40 enhancer and early promoter"
     enhancer        3579..3770
                     /label=SV40 enhancer
                     /note="enhancer for the SV40 early promoter (Herr, 1993)"
     primer_bind     complement(3602..3622)
                     /label=pBABE 3'
                     /note="SV40 enhancer, reverse primer for pBABE vectors"
     rep_origin      3787..3922
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     CDS             3971..4765
                     /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             3971..4765
                     /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(4025..4044)
                     /label=Neo-R
                     /note="Neomycin resistance gene, reverse primer"
     primer_bind     4635..4654
                     /label=Neo-F
                     /note="Neomycin resistance gene, forward primer"
     primer_bind     complement(4953..4972)
                     /label=TK-pA-R
                     /note="Thymidine kinase polyA, reverse primer"
     polyA_signal    4997..5044
                     /label=HSV TK poly(A) signal
                     /note="herpes simplex virus thymidine kinase 
                     polyadenylation signal (Cole and Stacy, 1985)"
     rep_origin      5373..5961
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     5862..5881
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
ORIGIN
        1 accgccatgc attagttatt aatagtaatc aattacgggg tcattagttc atagcccata
       61 tatggagttc cgcgttacat aacttacggt aaatggcccg cctggctgac cgcccaacga
      121 cccccgccca ttgacgtcaa taatgacgta tgttcccata gtaacgccaa tagggacttt
      181 ccattgacgt caatgggtgg agtatttacg gtaaactgcc cacttggcag tacatcaagt
      241 gtatcatatg ccaagtacgc cccctattga cgtcaatgac ggtaaatggc ccgcctggca
      301 ttatgcccag tacatgacct tatgggactt tcctacttgg cagtacatct acgtattagt
      361 catcgctatt accatggtga tgcggttttg gcagtacatc aatgggcgtg gatagcggtt
      421 tgactcacgg ggatttccaa gtctccaccc cattgacgtc aatgggagtt tgttttggca
      481 ccaaaatcaa cgggactttc caaaatgtcg taacaactcc gccccattga cgcaaatggg
      541 cggtaggcgt gtacggtggg aggtctatat aagcagagct ggtttagtga accgtcagat
      601 ccgctagcgc taccggactc agatctcgag ctcaagcttc gaattctgca gtcgacggta
      661 ccgcgggccc gggatccggg tggcagaacg gcgccgagcc gcacacagcc atccatcctc
      721 ccccttccct ctctcccctg tcctctctct ccgggctccc accgccgccg cgggccgggg
      781 agccaccggc cgccaccatg agttccttca gctacgagcg gtactactcg acctcctaca
      841 agcggcgcta cgtggagacg ccccgggtgc acatctccag cgtgcgcagc ggctacagca
      901 ccgcacgctc agcttactcc agctactcgg cgccggtgtc ttcctcgctg tccgtgcgcc
      961 gcagctactc ctccagctct ggatcgttga tgcccagtct ggagaacctc gacctgagcc
     1021 aggtagccgc catcagcaac gacctcaagt ccatccgcac gcaggagaag gcgcagctcc
     1081 aggacctcaa tgaccgcttc gccagcttca tcgagcgcgt gcacgagctg gagcagcaga
     1141 acaaggtcct ggaagccgag ctgctggtgc tgcgccagaa gcactccgag ccatcccgct
     1201 tccgggcgct gtacgagcag gagatccgcg acctgcgcct ggcggcggaa gatgccacca
     1261 acgagaagca ggcgctccag ggcgagcgcg aagggctgga ggagaccctg cgcaacctgc
     1321 aggcgcgcta tgaagaggag gtgctgagcc gcgaggacgc cgagggccgg ctgatggaag
     1381 cgcgcaaagg cgccgacgag gcggcgctcg ctcgcgccga gctcgagaag cgcatcgaca
     1441 gcttgatgga cgaaatctct tttctgaaga aagtgcacga agaggagatc gccgaactgc
     1501 aggcgcagat ccagtacgcg cagatctccg tggagatgga cgtgaccaag cccgaccttt
     1561 ccgccgcgct caaggacatc cgcgcgcagt acgagaagct ggccgccaag aacatgcaga
     1621 acgctgagga atggttcaag agccgcttca ccgtgctgac cgagagcgcc gccaagaaca
     1681 ccgacgccgt gcgcgccgcc aaggacgagg tgtccgagag ccgtcgtctg ctcaaggcca
     1741 agaccctgga aatcgaagca tgccggggca tgaatgaagc gctggagaag cagctgcagg
     1801 agctggagga caagcagaac gccgacatca gcgctatgca ggacacgatc aacaaattag
     1861 aaaatgaatt gaggaccaca aagagtgaaa tggcacgata cctaaaagaa taccaagacc
     1921 tcctcaacgt gaagatggct ttggatattg agattgcagc ttacaggaaa ctcttggaag
     1981 gcgaggagac ccgactcagt ttcaccagcg tgggaagcat aaccagtggc tactcccaga
     2041 gctcccaggt ctttggccga tctgcctacg gcggtttaca gaccagctcc tatctgatgt
     2101 ccacccgctc cttcccgtcc tactacacca gccatgtcca agaggagcag atcgaagtgg
     2161 aggaaaccat tgaggctgcc aaggctgagg aagccaagga tgagcccccc tctgaaggag
     2221 aagccgagga ggaggagaag gacaaggaag aggccgagga agaggaggca gctgaagagg
     2281 aagaagctgc caaggaagag tctgaagaag caaaagaaga agaagaagga ggtgaaggtg
     2341 aagaaggaga ggaaaccaaa gaagctgaag aggaggagaa gaaagttgaa ggtgctgggg
     2401 aggaacaagc agctaagaag aaagattgaa cccccatttc cttaattatt tcaggaataa
     2461 ttctcccgaa atcaggtcaa ccccatcacc aaccaaccaa ccagttgagt tccagattct
     2521 atgtgaatta aaaagtcaat atatgtataa ttctgagatg acttaggttg gacattcaat
     2581 gttgtgctat gaatttcctc tttatgcaga gtatctgttt gcttgcagag tggctttctg
     2641 gcttgctgcc agcctgtgca tggtccacaa gccgaattca gatctggtac cacgcgtatc
     2701 gataagcttg catgcctgca ggtcgactct agaggatcca ccggatctag ataactgatc
     2761 ataatcagcc ataccacatt tgtagaggtt ttacttgctt taaaaaacct cccacacctc
     2821 cccctgaacc tgaaacataa aatgaatgca attgttgttg ttaacttgtt tattgcagct
     2881 tataatggtt acaaataaag caatagcatc acaaatttca caaataaagc atttttttca
     2941 ctgcattcta gttgtggttt gtccaaactc atcaatgtat cttaacgcgt aaattgtaag
     3001 cgttaatatt ttgttaaaat tcgcgttaaa tttttgttaa atcagctcat tttttaacca
     3061 ataggccgaa atcggcaaaa tcccttataa atcaaaagaa tagaccgaga tagggttgag
     3121 tgttgttcca gtttggaaca agagtccact attaaagaac gtggactcca acgtcaaagg
     3181 gcgaaaaacc gtctatcagg gcgatggccc actacgtgaa ccatcaccct aatcaagttt
     3241 tttggggtcg aggtgccgta aagcactaaa tcggaaccct aaagggagcc cccgatttag
     3301 agcttgacgg ggaaagccgg cgaacgtggc gagaaaggaa gggaagaaag cgaaaggagc
     3361 gggcgctagg gcgctggcaa gtgtagcggt cacgctgcgc gtaaccacca cacccgccgc
     3421 gcttaatgcg ccgctacagg gcgcgtcagg tggcactttt cggggaaatg tgcgcggaac
     3481 ccctatttgt ttatttttct aaatacattc aaatatgtat ccgctcatga gacaataacc
     3541 ctgataaatg cttcaataat attgaaaaag gaagagtcct gaggcggaaa gaaccagctg
     3601 tggaatgtgt gtcagttagg gtgtggaaag tccccaggct ccccagcagg cagaagtatg
     3661 caaagcatgc atctcaatta gtcagcaacc aggtgtggaa agtccccagg ctccccagca
     3721 ggcagaagta tgcaaagcat gcatctcaat tagtcagcaa ccatagtccc gcccctaact
     3781 ccgcccatcc cgcccctaac tccgcccagt tccgcccatt ctccgcccca tggctgacta
     3841 atttttttta tttatgcaga ggccgaggcc gcctcggcct ctgagctatt ccagaagtag
     3901 tgaggaggct tttttggagg cctaggcttt tgcaaagatc gatcaagaga caggatgagg
     3961 atcgtttcgc atgattgaac aagatggatt gcacgcaggt tctccggccg cttgggtgga
     4021 gaggctattc ggctatgact gggcacaaca gacaatcggc tgctctgatg ccgccgtgtt
     4081 ccggctgtca gcgcaggggc gcccggttct ttttgtcaag accgacctgt ccggtgccct
     4141 gaatgaactg caagacgagg cagcgcggct atcgtggctg gccacgacgg gcgttccttg
     4201 cgcagctgtg ctcgacgttg tcactgaagc gggaagggac tggctgctat tgggcgaagt
     4261 gccggggcag gatctcctgt catctcacct tgctcctgcc gagaaagtat ccatcatggc
     4321 tgatgcaatg cggcggctgc atacgcttga tccggctacc tgcccattcg accaccaagc
     4381 gaaacatcgc atcgagcgag cacgtactcg gatggaagcc ggtcttgtcg atcaggatga
     4441 tctggacgaa gagcatcagg ggctcgcgcc agccgaactg ttcgccaggc tcaaggcgag
     4501 catgcccgac ggcgaggatc tcgtcgtgac ccatggcgat gcctgcttgc cgaatatcat
     4561 ggtggaaaat ggccgctttt ctggattcat cgactgtggc cggctgggtg tggcggaccg
     4621 ctatcaggac atagcgttgg ctacccgtga tattgctgaa gagcttggcg gcgaatgggc
     4681 tgaccgcttc ctcgtgcttt acggtatcgc cgctcccgat tcgcagcgca tcgccttcta
     4741 tcgccttctt gacgagttct tctgagcggg actctggggt tcgaaatgac cgaccaagcg
     4801 acgcccaacc tgccatcacg agatttcgat tccaccgccg ccttctatga aaggttgggc
     4861 ttcggaatcg ttttccggga cgccggctgg atgatcctcc agcgcgggga tctcatgctg
     4921 gagttcttcg cccaccctag ggggaggcta actgaaacac ggaaggagac aataccggaa
     4981 ggaacccgcg ctatgacggc aataaaaaga cagaataaaa cgcacggtgt tgggtcgttt
     5041 gttcataaac gcggggttcg gtcccagggc tggcactctg tcgatacccc accgagaccc
     5101 cattggggcc aatacgcccg cgtttcttcc ttttccccac cccacccccc aagttcgggt
     5161 gaaggcccag ggctcgcagc caacgtcggg gcggcaggcc ctgccatagc ctcaggttac
     5221 tcatatatac tttagattga tttaaaactt catttttaat ttaaaaggat ctaggtgaag
     5281 atcctttttg ataatctcat gaccaaaatc ccttaacgtg agttttcgtt ccactgagcg
     5341 tcagaccccg tagaaaagat caaaggatct tcttgagatc ctttttttct gcgcgtaatc
     5401 tgctgcttgc aaacaaaaaa accaccgcta ccagcggtgg tttgtttgcc ggatcaagag
     5461 ctaccaactc tttttccgaa ggtaactggc ttcagcagag cgcagatacc aaatactgtt
     5521 cttctagtgt agccgtagtt aggccaccac ttcaagaact ctgtagcacc gcctacatac
     5581 ctcgctctgc taatcctgtt accagtggct gctgccagtg gcgataagtc gtgtcttacc
     5641 gggttggact caagacgata gttaccggat aaggcgcagc ggtcgggctg aacggggggt
     5701 tcgtgcacac agcccagctt ggagcgaacg acctacaccg aactgagata cctacagcgt
     5761 gagctatgag aaagcgccac gcttcccgaa gggagaaagg cggacaggta tccggtaagc
     5821 ggcagggtcg gaacaggaga gcgcacgagg gagcttccag ggggaaacgc ctggtatctt
     5881 tatagtcctg tcgggtttcg ccacctctga cttgagcgtc gatttttgtg atgctcgtca
     5941 ggggggcgga gcctatggaa aaacgccagc aacgcggcct ttttacggtt cctggccttt
     6001 tgctggcctt ttgctcacat gttctttcct gcgttatccc ctgattctgt ggataaccgt
     6061 att
//
