LOCUS       sequence-260028-        6063 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Expresses human NFL containing the Charcot-Marie-Tooth disease type 
            2E E396K 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-E396K
FEATURES             Location/Qualifiers
     source          1..6063
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     enhancer        75..378
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
     promoter        379..582
                     /label=CMV promoter
                     /note="human cytomegalovirus (CMV) immediate early 
                     promoter"
     primer_bind     533..553
                     /label=CMV-F
                     /note="Human CMV immediate early promoter, forward primer"
     misc_feature    623..679
                     /label=MCS
                     /note="multiple cloning site"
     polyA_signal    2865..2986
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(2902..2921)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     2956..2975
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     rep_origin      complement(2993..3448)
                     /direction=LEFT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(3130..3151)
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     3342..3361
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     promoter        3475..3579
                     /gene="bla"
                     /label=AmpR promoter
     promoter        3581..3938
                     /label=SV40 promoter
                     /note="SV40 enhancer and early promoter"
     enhancer        3581..3772
                     /label=SV40 enhancer
                     /note="enhancer for the SV40 early promoter (Herr, 1993)"
     primer_bind     complement(3604..3624)
                     /label=pBABE 3'
                     /note="SV40 enhancer, reverse primer for pBABE vectors"
     rep_origin      3789..3924
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     CDS             3973..4767
                     /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             3973..4767
                     /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(4027..4046)
                     /label=Neo-R
                     /note="Neomycin resistance gene, reverse primer"
     primer_bind     4637..4656
                     /label=Neo-F
                     /note="Neomycin resistance gene, forward primer"
     primer_bind     complement(4955..4974)
                     /label=TK-pA-R
                     /note="Thymidine kinase polyA, reverse primer"
     polyA_signal    4999..5046
                     /label=HSV TK poly(A) signal
                     /note="herpes simplex virus thymidine kinase 
                     polyadenylation signal (Cole and Stacy, 1985)"
     rep_origin      5375..5963
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     5864..5883
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
ORIGIN
        1 ttaccgccat gcattagtta ttaatagtaa tcaattacgg ggtcattagt tcatagccca
       61 tatatggagt tccgcgttac ataacttacg gtaaatggcc cgcctggctg accgcccaac
      121 gacccccgcc cattgacgtc aataatgacg tatgttccca tagtaacgcc aatagggact
      181 ttccattgac gtcaatgggt ggagtattta cggtaaactg cccacttggc agtacatcaa
      241 gtgtatcata tgccaagtac gccccctatt gacgtcaatg acggtaaatg gcccgcctgg
      301 cattatgccc agtacatgac cttatgggac tttcctactt ggcagtacat ctacgtatta
      361 gtcatcgcta ttaccatggt gatgcggttt tggcagtaca tcaatgggcg tggatagcgg
      421 tttgactcac ggggatttcc aagtctccac cccattgacg tcaatgggag tttgttttgg
      481 caccaaaatc aacgggactt tccaaaatgt cgtaacaact ccgccccatt gacgcaaatg
      541 ggcggtaggc gtgtacggtg ggaggtctat ataagcagag ctggtttagt gaaccgtcag
      601 atccgctagc gctaccggac tcagatctcg agctcaagct tcgaattctg cagtcgacgg
      661 taccgcgggc ccgggatccg ggtggcagaa cggcgccgag ccgcacacag ccatccatcc
      721 tcccccttcc ctctctcccc tgtcctctct ctccgggctc ccaccgccgc cgcgggccgg
      781 ggagccaccg gccgccacca tgagttcctt cagctacgag ccgtactact cgacctccta
      841 caagcggcgc tacgtggaga cgccccgggt gcacatctcc agcgtgcgca gcggctacag
      901 caccgcacgc tcagcttact ccagctactc ggcgccggtg tcttcctcgc tgtccgtgcg
      961 ccgcagctac tcctccagct ctggatcgtt gatgcccagt ctggagaacc tcgacctgag
     1021 ccaggtagcc gccatcagca acgacctcaa gtccatccgc acgcaggaga aggcgcagct
     1081 ccaggacctc aatgaccgct tcgccagctt catcgagcgc gtgcacgagc tggagcagca
     1141 gaacaaggtc ctggaagccg agctgctggt gctgcgccag aagcactccg agccatcccg
     1201 cttccgggcg ctgtacgagc aggagatccg cgacctgcgc ctggcggcgg aagatgccac
     1261 caacgagaag caggcgctcc agggcgagcg cgaagggctg gaggagaccc tgcgcaacct
     1321 gcaggcgcgc tatgaagagg aggtgctgag ccgcgaggac gccgagggcc ggctgatgga
     1381 agcgcgcaaa ggcgccgacg aggcggcgct cgctcgcgcc gagctcgaga agcgcatcga
     1441 cagcttgatg gacgaaatct cttttctgaa gaaagtgcac gaagaggaga tcgccgaact
     1501 gcaggcgcag atccagtacg cgcagatctc cgtggagatg gacgtgacca agcccgacct
     1561 ttccgccgcg ctcaaggaca tccgcgcgca gtacgagaag ctggccgcca agaacatgca
     1621 gaacgctgag gaatggttca agagccgctt caccgtgctg accgagagcg ccgccaagaa
     1681 caccgacgcc gtgcgcgccg ccaaggacga ggtgtccgag agccgtcgtc tgctcaaggc
     1741 caagaccctg gaaatcgaag catgccgggg catgaatgaa gcgctggaga agcagctgca
     1801 ggagctggag gacaagcaga acgccgacat cagcgctatg caggacacga tcaacaaatt
     1861 agaaaatgaa ttgaggacca caaagagtga aatggcacga tacctaaaag aataccaaga
     1921 cctcctcaac gtgaagatgg ctttggatat tgagattgca gcttacagga aactcttgga
     1981 aggcaaggag acccgactca gtttcaccag cgtgggaagc ataaccagtg gctactccca
     2041 gagctcccag gtctttggcc gatctgccta cggcggttta cagaccagct cctatctgat
     2101 gtccacccgc tccttcccgt cctactacac cagccatgtc caagaggagc agatcgaagt
     2161 ggaggaaacc attgaggctg ccaaggctga ggaagccaag gatgagcccc cctctgaagg
     2221 agaagccgag gaggaggaga aggacaagga agaggccgag gaagaggagg cagctgaaga
     2281 ggaagaagct gccaaggaag agtctgaaga agcaaaagaa gaagaagaag gaggtgaagg
     2341 tgaagaagga gaggaaacca aagaagctga agaggaggag aagaaagttg aaggtgctgg
     2401 ggaggaacaa gcagctaaga agaaagattg aacccccatt tccttaatta tttcaggaat
     2461 aattctcccg aaatcaggtc aaccccatca ccaaccaacc aaccagttga gttccagatt
     2521 ctatgtgaat taaaaagtca atatatgtat aattctgaga tgacttaggt tggacattca
     2581 atgttgtgct atgaatttcc tctttatgca gagtatctgt ttgcttgcag agtggctttc
     2641 tggcttgctg ccagcctgtg catggtccac aagccgaatt cagatctggt accacgcgta
     2701 tcgataagct tgcatgcctg caggtcgact ctagaggatc caccggatct agataactga
     2761 tcataatcag ccataccaca tttgtagagg ttttacttgc tttaaaaaac ctcccacacc
     2821 tccccctgaa cctgaaacat aaaatgaatg caattgttgt tgttaacttg tttattgcag
     2881 cttataatgg ttacaaataa agcaatagca tcacaaattt cacaaataaa gcattttttt
     2941 cactgcattc tagttgtggt ttgtccaaac tcatcaatgt atcttaacgc gtaaattgta
     3001 agcgttaata ttttgttaaa attcgcgtta aatttttgtt aaatcagctc attttttaac
     3061 caataggccg aaatcggcaa aatcccttat aaatcaaaag aatagaccga gatagggttg
     3121 agtgttgttc cagtttggaa caagagtcca ctattaaaga acgtggactc caacgtcaaa
     3181 gggcgaaaaa ccgtctatca gggcgatggc ccactacgtg aaccatcacc ctaatcaagt
     3241 tttttggggt cgaggtgccg taaagcacta aatcggaacc ctaaagggag cccccgattt
     3301 agagcttgac ggggaaagcc ggcgaacgtg gcgagaaagg aagggaagaa agcgaaagga
     3361 gcgggcgcta gggcgctggc aagtgtagcg gtcacgctgc gcgtaaccac cacacccgcc
     3421 gcgcttaatg cgccgctaca gggcgcgtca ggtggcactt ttcggggaaa tgtgcgcgga
     3481 acccctattt gtttattttt ctaaatacat tcaaatatgt atccgctcat gagacaataa
     3541 ccctgataaa tgcttcaata atattgaaaa aggaagagtc ctgaggcgga aagaaccagc
     3601 tgtggaatgt gtgtcagtta gggtgtggaa agtccccagg ctccccagca ggcagaagta
     3661 tgcaaagcat gcatctcaat tagtcagcaa ccaggtgtgg aaagtcccca ggctccccag
     3721 caggcagaag tatgcaaagc atgcatctca attagtcagc aaccatagtc ccgcccctaa
     3781 ctccgcccat cccgccccta actccgccca gttccgccca ttctccgccc catggctgac
     3841 taattttttt tatttatgca gaggccgagg ccgcctcggc ctctgagcta ttccagaagt
     3901 agtgaggagg cttttttgga ggcctaggct tttgcaaaga tcgatcaaga gacaggatga
     3961 ggatcgtttc gcatgattga acaagatgga ttgcacgcag gttctccggc cgcttgggtg
     4021 gagaggctat tcggctatga ctgggcacaa cagacaatcg gctgctctga tgccgccgtg
     4081 ttccggctgt cagcgcaggg gcgcccggtt ctttttgtca agaccgacct gtccggtgcc
     4141 ctgaatgaac tgcaagacga ggcagcgcgg ctatcgtggc tggccacgac gggcgttcct
     4201 tgcgcagctg tgctcgacgt tgtcactgaa gcgggaaggg actggctgct attgggcgaa
     4261 gtgccggggc aggatctcct gtcatctcac cttgctcctg ccgagaaagt atccatcatg
     4321 gctgatgcaa tgcggcggct gcatacgctt gatccggcta cctgcccatt cgaccaccaa
     4381 gcgaaacatc gcatcgagcg agcacgtact cggatggaag ccggtcttgt cgatcaggat
     4441 gatctggacg aagagcatca ggggctcgcg ccagccgaac tgttcgccag gctcaaggcg
     4501 agcatgcccg acggcgagga tctcgtcgtg acccatggcg atgcctgctt gccgaatatc
     4561 atggtggaaa atggccgctt ttctggattc atcgactgtg gccggctggg tgtggcggac
     4621 cgctatcagg acatagcgtt ggctacccgt gatattgctg aagagcttgg cggcgaatgg
     4681 gctgaccgct tcctcgtgct ttacggtatc gccgctcccg attcgcagcg catcgccttc
     4741 tatcgccttc ttgacgagtt cttctgagcg ggactctggg gttcgaaatg accgaccaag
     4801 cgacgcccaa cctgccatca cgagatttcg attccaccgc cgccttctat gaaaggttgg
     4861 gcttcggaat cgttttccgg gacgccggct ggatgatcct ccagcgcggg gatctcatgc
     4921 tggagttctt cgcccaccct agggggaggc taactgaaac acggaaggag acaataccgg
     4981 aaggaacccg cgctatgacg gcaataaaaa gacagaataa aacgcacggt gttgggtcgt
     5041 ttgttcataa acgcggggtt cggtcccagg gctggcactc tgtcgatacc ccaccgagac
     5101 cccattgggg ccaatacgcc cgcgtttctt ccttttcccc accccacccc ccaagttcgg
     5161 gtgaaggccc agggctcgca gccaacgtcg gggcggcagg ccctgccata gcctcaggtt
     5221 actcatatat actttagatt gatttaaaac ttcattttta atttaaaagg atctaggtga
     5281 agatcctttt tgataatctc atgaccaaaa tcccttaacg tgagttttcg ttccactgag
     5341 cgtcagaccc cgtagaaaag atcaaaggat cttcttgaga tccttttttt ctgcgcgtaa
     5401 tctgctgctt gcaaacaaaa aaaccaccgc taccagcggt ggtttgtttg ccggatcaag
     5461 agctaccaac tctttttccg aaggtaactg gcttcagcag agcgcagata ccaaatactg
     5521 ttcttctagt gtagccgtag ttaggccacc acttcaagaa ctctgtagca ccgcctacat
     5581 acctcgctct gctaatcctg ttaccagtgg ctgctgccag tggcgataag tcgtgtctta
     5641 ccgggttgga ctcaagacga tagttaccgg ataaggcgca gcggtcgggc tgaacggggg
     5701 gttcgtgcac acagcccagc ttggagcgaa cgacctacac cgaactgaga tacctacagc
     5761 gtgagctatg agaaagcgcc acgcttcccg aagggagaaa ggcggacagg tatccggtaa
     5821 gcggcagggt cggaacagga gagcgcacga gggagcttcc agggggaaac gcctggtatc
     5881 tttatagtcc tgtcgggttt cgccacctct gacttgagcg tcgatttttg tgatgctcgt
     5941 caggggggcg gagcctatgg aaaaacgcca gcaacgcggc ctttttacgg ttcctggcct
     6001 tttgctggcc ttttgctcac atgttctttc ctgcgttatc ccctgattct gtggataacc
     6061 gta
//
