LOCUS       sequence-254756-        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        61..364
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
     promoter        365..568
                     /label=CMV promoter
                     /note="human cytomegalovirus (CMV) immediate early 
                     promoter"
     primer_bind     519..539
                     /label=CMV-F
                     /note="Human CMV immediate early promoter, forward primer"
     misc_feature    609..665
                     /label=MCS
                     /note="multiple cloning site"
     polyA_signal    2851..2972
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(2888..2907)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     2942..2961
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     rep_origin      complement(2979..3434)
                     /direction=LEFT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(3116..3137)
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     3328..3347
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     promoter        3461..3565
                     /gene="bla"
                     /label=AmpR promoter
     promoter        3567..3924
                     /label=SV40 promoter
                     /note="SV40 enhancer and early promoter"
     enhancer        3567..3758
                     /label=SV40 enhancer
                     /note="enhancer for the SV40 early promoter (Herr, 1993)"
     primer_bind     complement(3590..3610)
                     /label=pBABE 3'
                     /note="SV40 enhancer, reverse primer for pBABE vectors"
     rep_origin      3775..3910
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     CDS             3959..4753
                     /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             3959..4753
                     /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(4013..4032)
                     /label=Neo-R
                     /note="Neomycin resistance gene, reverse primer"
     primer_bind     4623..4642
                     /label=Neo-F
                     /note="Neomycin resistance gene, forward primer"
     primer_bind     complement(4941..4960)
                     /label=TK-pA-R
                     /note="Thymidine kinase polyA, reverse primer"
     polyA_signal    4985..5032
                     /label=HSV TK poly(A) signal
                     /note="herpes simplex virus thymidine kinase 
                     polyadenylation signal (Cole and Stacy, 1985)"
     rep_origin      5361..5949
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     5850..5869
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
ORIGIN
        1 tagttattaa tagtaatcaa ttacggggtc attagttcat agcccatata tggagttccg
       61 cgttacataa cttacggtaa atggcccgcc tggctgaccg cccaacgacc cccgcccatt
      121 gacgtcaata atgacgtatg ttcccatagt aacgccaata gggactttcc attgacgtca
      181 atgggtggag tatttacggt aaactgccca cttggcagta catcaagtgt atcatatgcc
      241 aagtacgccc cctattgacg tcaatgacgg taaatggccc gcctggcatt atgcccagta
      301 catgacctta tgggactttc ctacttggca gtacatctac gtattagtca tcgctattac
      361 catggtgatg cggttttggc agtacatcaa tgggcgtgga tagcggtttg actcacgggg
      421 atttccaagt ctccacccca ttgacgtcaa tgggagtttg ttttggcacc aaaatcaacg
      481 ggactttcca aaatgtcgta acaactccgc cccattgacg caaatgggcg gtaggcgtgt
      541 acggtgggag gtctatataa gcagagctgg tttagtgaac cgtcagatcc gctagcgcta
      601 ccggactcag atctcgagct caagcttcga attctgcagt cgacggtacc gcgggcccgg
      661 gatccgggtg gcagaacggc gccgagccgc acacagccat ccatcctccc ccttccctct
      721 ctcccctgtc ctctctctcc gggctcccac cgccgccgcg ggccggggag ccaccggccg
      781 ccaccatgag ttccttcagc tacgagccgt actactcgac ctcctacaag cggcgctacg
      841 tggagacgcc ccgggtgcac atctccagcg tgcgcagcgg ctacagcacc gcacgctcag
      901 cttactccag ctactcggcg ccggtgtctt cctcgctgtc cgtgcgccgc agctactcct
      961 ccagctctgg atcgttgatg cccagtctgg agaacctcga cctgagccag gtagccgcca
     1021 tcagcaacga cctcaagtcc atccgcacgc aggagaaggc gcagctccag gacctcaatg
     1081 accgcttcgc cagcttcatc gagcgcgtgc acgagctgga gcagcagaac aaggtcctgg
     1141 aagccgagct gctggtgctg cgccagaagc actccgagcc atcccgcttc cgggcgctgt
     1201 acgagcagga gatccgcgac ctgcgcctgg cggcggaaga tgccaccaac gagaagcagg
     1261 cgctccaggg cgagcgcgaa gggctggagg agaccctgcg caacctgcag gcgcgctatg
     1321 aagaggaggt gctgagccgc gaggacgccg agggccggct gatggaagcg cgcaaaggcg
     1381 ccgacgaggc ggcgctcgct cgcgccgagc tcgagaagcg catcgacagc ttgatggacg
     1441 aaatctcttt tctgaagaaa gtgcacgaag aggagatcgc cgaactgcag gcgcagatcc
     1501 agtacgcgca gatctccgtg gagatggacg tgaccaagcc cgacctttcc gccgcgctca
     1561 aggacatccg cgcgcagtac gagaagctgg ccgccaagaa catgcagaac gctgaggaat
     1621 ggttcaagag ccgcttcacc gtgctgaccg agagcgccgc caagaacacc gacgccgtgc
     1681 gcgccgccaa ggacgaggtg tccgagagcc gtcgtctgct caaggccaag accctggaaa
     1741 tcgaagcatg ccggggcatg aatgaagcgc tggagaagca gctgcaggag ctggaggaca
     1801 agcagaacgc cgacatcagc gctatgcagg acacgatcaa caaattagaa aatgaattga
     1861 ggaccacaaa gagtgaaatg gcacgatacc taaaagaata ccaagacctc ctcaacgtga
     1921 agatggcttt ggatattgag attgcagctt acaggaaact cttggaaggc aaggagaccc
     1981 gactcagttt caccagcgtg ggaagcataa ccagtggcta ctcccagagc tcccaggtct
     2041 ttggccgatc tgcctacggc ggtttacaga ccagctccta tctgatgtcc acccgctcct
     2101 tcccgtccta ctacaccagc catgtccaag aggagcagat cgaagtggag gaaaccattg
     2161 aggctgccaa ggctgaggaa gccaaggatg agcccccctc tgaaggagaa gccgaggagg
     2221 aggagaagga caaggaagag gccgaggaag aggaggcagc tgaagaggaa gaagctgcca
     2281 aggaagagtc tgaagaagca aaagaagaag aagaaggagg tgaaggtgaa gaaggagagg
     2341 aaaccaaaga agctgaagag gaggagaaga aagttgaagg tgctggggag gaacaagcag
     2401 ctaagaagaa agattgaacc cccatttcct taattatttc aggaataatt ctcccgaaat
     2461 caggtcaacc ccatcaccaa ccaaccaacc agttgagttc cagattctat gtgaattaaa
     2521 aagtcaatat atgtataatt ctgagatgac ttaggttgga cattcaatgt tgtgctatga
     2581 atttcctctt tatgcagagt atctgtttgc ttgcagagtg gctttctggc ttgctgccag
     2641 cctgtgcatg gtccacaagc cgaattcaga tctggtacca cgcgtatcga taagcttgca
     2701 tgcctgcagg tcgactctag aggatccacc ggatctagat aactgatcat aatcagccat
     2761 accacatttg tagaggtttt acttgcttta aaaaacctcc cacacctccc cctgaacctg
     2821 aaacataaaa tgaatgcaat tgttgttgtt aacttgttta ttgcagctta taatggttac
     2881 aaataaagca atagcatcac aaatttcaca aataaagcat ttttttcact gcattctagt
     2941 tgtggtttgt ccaaactcat caatgtatct taacgcgtaa attgtaagcg ttaatatttt
     3001 gttaaaattc gcgttaaatt tttgttaaat cagctcattt tttaaccaat aggccgaaat
     3061 cggcaaaatc ccttataaat caaaagaata gaccgagata gggttgagtg ttgttccagt
     3121 ttggaacaag agtccactat taaagaacgt ggactccaac gtcaaagggc gaaaaaccgt
     3181 ctatcagggc gatggcccac tacgtgaacc atcaccctaa tcaagttttt tggggtcgag
     3241 gtgccgtaaa gcactaaatc ggaaccctaa agggagcccc cgatttagag cttgacgggg
     3301 aaagccggcg aacgtggcga gaaaggaagg gaagaaagcg aaaggagcgg gcgctagggc
     3361 gctggcaagt gtagcggtca cgctgcgcgt aaccaccaca cccgccgcgc ttaatgcgcc
     3421 gctacagggc gcgtcaggtg gcacttttcg gggaaatgtg cgcggaaccc ctatttgttt
     3481 atttttctaa atacattcaa atatgtatcc gctcatgaga caataaccct gataaatgct
     3541 tcaataatat tgaaaaagga agagtcctga ggcggaaaga accagctgtg gaatgtgtgt
     3601 cagttagggt gtggaaagtc cccaggctcc ccagcaggca gaagtatgca aagcatgcat
     3661 ctcaattagt cagcaaccag gtgtggaaag tccccaggct ccccagcagg cagaagtatg
     3721 caaagcatgc atctcaatta gtcagcaacc atagtcccgc ccctaactcc gcccatcccg
     3781 cccctaactc cgcccagttc cgcccattct ccgccccatg gctgactaat tttttttatt
     3841 tatgcagagg ccgaggccgc ctcggcctct gagctattcc agaagtagtg aggaggcttt
     3901 tttggaggcc taggcttttg caaagatcga tcaagagaca ggatgaggat cgtttcgcat
     3961 gattgaacaa gatggattgc acgcaggttc tccggccgct tgggtggaga ggctattcgg
     4021 ctatgactgg gcacaacaga caatcggctg ctctgatgcc gccgtgttcc ggctgtcagc
     4081 gcaggggcgc ccggttcttt ttgtcaagac cgacctgtcc ggtgccctga atgaactgca
     4141 agacgaggca gcgcggctat cgtggctggc cacgacgggc gttccttgcg cagctgtgct
     4201 cgacgttgtc actgaagcgg gaagggactg gctgctattg ggcgaagtgc cggggcagga
     4261 tctcctgtca tctcaccttg ctcctgccga gaaagtatcc atcatggctg atgcaatgcg
     4321 gcggctgcat acgcttgatc cggctacctg cccattcgac caccaagcga aacatcgcat
     4381 cgagcgagca cgtactcgga tggaagccgg tcttgtcgat caggatgatc tggacgaaga
     4441 gcatcagggg ctcgcgccag ccgaactgtt cgccaggctc aaggcgagca tgcccgacgg
     4501 cgaggatctc gtcgtgaccc atggcgatgc ctgcttgccg aatatcatgg tggaaaatgg
     4561 ccgcttttct ggattcatcg actgtggccg gctgggtgtg gcggaccgct atcaggacat
     4621 agcgttggct acccgtgata ttgctgaaga gcttggcggc gaatgggctg accgcttcct
     4681 cgtgctttac ggtatcgccg ctcccgattc gcagcgcatc gccttctatc gccttcttga
     4741 cgagttcttc tgagcgggac tctggggttc gaaatgaccg accaagcgac gcccaacctg
     4801 ccatcacgag atttcgattc caccgccgcc ttctatgaaa ggttgggctt cggaatcgtt
     4861 ttccgggacg ccggctggat gatcctccag cgcggggatc tcatgctgga gttcttcgcc
     4921 caccctaggg ggaggctaac tgaaacacgg aaggagacaa taccggaagg aacccgcgct
     4981 atgacggcaa taaaaagaca gaataaaacg cacggtgttg ggtcgtttgt tcataaacgc
     5041 ggggttcggt cccagggctg gcactctgtc gataccccac cgagacccca ttggggccaa
     5101 tacgcccgcg tttcttcctt ttccccaccc caccccccaa gttcgggtga aggcccaggg
     5161 ctcgcagcca acgtcggggc ggcaggccct gccatagcct caggttactc atatatactt
     5221 tagattgatt taaaacttca tttttaattt aaaaggatct aggtgaagat cctttttgat
     5281 aatctcatga ccaaaatccc ttaacgtgag ttttcgttcc actgagcgtc agaccccgta
     5341 gaaaagatca aaggatcttc ttgagatcct ttttttctgc gcgtaatctg ctgcttgcaa
     5401 acaaaaaaac caccgctacc agcggtggtt tgtttgccgg atcaagagct accaactctt
     5461 tttccgaagg taactggctt cagcagagcg cagataccaa atactgtcct tctagtgtag
     5521 ccgtagttag gccaccactt caagaactct gtagcaccgc ctacatacct cgctctgcta
     5581 atcctgttac cagtggctgc tgccagtggc gataagtcgt gtcttaccgg gttggactca
     5641 agacgatagt taccggataa ggcgcagcgg tcgggctgaa cggggggttc gtgcacacag
     5701 cccagcttgg agcgaacgac ctacaccgaa ctgagatacc tacagcgtga gctatgagaa
     5761 agcgccacgc ttcccgaagg gagaaaggcg gacaggtatc cggtaagcgg cagggtcgga
     5821 acaggagagc gcacgaggga gcttccaggg ggaaacgcct ggtatcttta tagtcctgtc
     5881 gggtttcgcc acctctgact tgagcgtcga tttttgtgat gctcgtcagg ggggcggagc
     5941 ctatggaaaa acgccagcaa cgcggccttt ttacggttcc tggccttttg ctggcctttt
     6001 gctcacatgt tctttcctgc gttatcccct gattctgtgg ataaccgtat taccgccatg
     6061 cat
//
