LOCUS       sequence-260945-        6063 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Expresses human NFL containing the Charcot-Marie-Tooth disease type 
            2E P22S 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-P22S
FEATURES             Location/Qualifiers
     source          1..6063
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     enhancer        101..404
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
     promoter        405..608
                     /label=CMV promoter
                     /note="human cytomegalovirus (CMV) immediate early 
                     promoter"
     primer_bind     559..579
                     /label=CMV-F
                     /note="Human CMV immediate early promoter, forward primer"
     misc_feature    649..705
                     /label=MCS
                     /note="multiple cloning site"
     polyA_signal    2891..3012
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(2928..2947)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     2982..3001
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     rep_origin      complement(3019..3474)
                     /direction=LEFT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(3156..3177)
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     3368..3387
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     promoter        3501..3605
                     /gene="bla"
                     /label=AmpR promoter
     promoter        3607..3964
                     /label=SV40 promoter
                     /note="SV40 enhancer and early promoter"
     enhancer        3607..3798
                     /label=SV40 enhancer
                     /note="enhancer for the SV40 early promoter (Herr, 1993)"
     primer_bind     complement(3630..3650)
                     /label=pBABE 3'
                     /note="SV40 enhancer, reverse primer for pBABE vectors"
     rep_origin      3815..3950
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     CDS             3999..4793
                     /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             3999..4793
                     /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(4053..4072)
                     /label=Neo-R
                     /note="Neomycin resistance gene, reverse primer"
     primer_bind     4663..4682
                     /label=Neo-F
                     /note="Neomycin resistance gene, forward primer"
     primer_bind     complement(4981..5000)
                     /label=TK-pA-R
                     /note="Thymidine kinase polyA, reverse primer"
     polyA_signal    5025..5072
                     /label=HSV TK poly(A) signal
                     /note="herpes simplex virus thymidine kinase 
                     polyadenylation signal (Cole and Stacy, 1985)"
     rep_origin      5401..5989
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     5890..5909
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
ORIGIN
        1 atcccctgat tctgtggata accgtattac cgccatgcat tagttattaa tagtaatcaa
       61 ttacggggtc attagttcat agcccatata tggagttccg cgttacataa cttacggtaa
      121 atggcccgcc tggctgaccg cccaacgacc cccgcccatt gacgtcaata atgacgtatg
      181 ttcccatagt aacgccaata gggactttcc attgacgtca atgggtggag tatttacggt
      241 aaactgccca cttggcagta catcaagtgt atcatatgcc aagtacgccc cctattgacg
      301 tcaatgacgg taaatggccc gcctggcatt atgcccagta catgacctta tgggactttc
      361 ctacttggca gtacatctac gtattagtca tcgctattac catggtgatg cggttttggc
      421 agtacatcaa tgggcgtgga tagcggtttg actcacgggg atttccaagt ctccacccca
      481 ttgacgtcaa tgggagtttg ttttggcacc aaaatcaacg ggactttcca aaatgtcgta
      541 acaactccgc cccattgacg caaatgggcg gtaggcgtgt acggtgggag gtctatataa
      601 gcagagctgg tttagtgaac cgtcagatcc gctagcgcta ccggactcag atctcgagct
      661 caagcttcga attctgcagt cgacggtacc gcgggcccgg gatccgggtg gcagaacggc
      721 gccgagccgc acacagccat ccatcctccc ccttccctct ctcccctgtc ctctctctcc
      781 gggctcccac cgccgccgcg ggccggggag ccaccggccg ccaccatgag ttccttcagc
      841 tacgagccgt actactcgac ctcctacaag cggcgctacg tggagacgag ccgggtgcac
      901 atctccagcg tgcgcagcgg ctacagcacc gcacgctcag cttactccag ctactcggcg
      961 ccggtgtctt cctcgctgtc cgtgcgccgc agctactcct ccagctctgg atcgttgatg
     1021 cccagtctgg agaacctcga cctgagccag gtagccgcca tcagcaacga cctcaagtcc
     1081 atccgcacgc aggagaaggc gcagctccag gacctcaatg accgcttcgc cagcttcatc
     1141 gagcgcgtgc acgagctgga gcagcagaac aaggtcctgg aagccgagct gctggtgctg
     1201 cgccagaagc actccgagcc atcccgcttc cgggcgctgt acgagcagga gatccgcgac
     1261 ctgcgcctgg cggcggaaga tgccaccaac gagaagcagg cgctccaggg cgagcgcgaa
     1321 gggctggagg agaccctgcg caacctgcag gcgcgctatg aagaggaggt gctgagccgc
     1381 gaggacgccg agggccggct gatggaagcg cgcaaaggcg ccgacgaggc ggcgctcgct
     1441 cgcgccgagc tcgagaagcg catcgacagc ttgatggacg aaatctcttt tctgaagaaa
     1501 gtgcacgaag aggagatcgc cgaactgcag gcgcagatcc agtacgcgca gatctccgtg
     1561 gagatggacg tgaccaagcc cgacctttcc gccgcgctca aggacatccg cgcgcagtac
     1621 gagaagctgg ccgccaagaa catgcagaac gctgaggaat ggttcaagag ccgcttcacc
     1681 gtgctgaccg agagcgccgc caagaacacc gacgccgtgc gcgccgccaa ggacgaggtg
     1741 tccgagagcc gtcgtctgct caaggccaag accctggaaa tcgaagcatg ccggggcatg
     1801 aatgaagcgc tggagaagca gctgcaggag ctggaggaca agcagaacgc cgacatcagc
     1861 gctatgcagg acacgatcaa caaattagaa aatgaattga ggaccacaaa gagtgaaatg
     1921 gcacgatacc taaaagaata ccaagacctc ctcaacgtga agatggcttt ggatattgag
     1981 attgcagctt acaggaaact cttggaaggc gaggagaccc gactcagttt caccagcgtg
     2041 ggaagcataa ccagtggcta ctcccagagc tcccaggtct ttggccgatc tgcctacggc
     2101 ggtttacaga ccagctccta tctgatgtcc acccgctcct tcccgtccta ctacaccagc
     2161 catgtccaag aggagcagat cgaagtggag gaaaccattg aggctgccaa ggctgaggaa
     2221 gccaaggatg agcccccctc tgaaggagaa gccgaggagg aggagaagga caaggaagag
     2281 gccgaggaag aggaggcagc tgaagaggaa gaagctgcca aggaagagtc tgaagaagca
     2341 aaagaagaag aagaaggagg tgaaggtgaa gaaggagagg aaaccaaaga agctgaagag
     2401 gaggagaaga aagttgaagg tgctggggag gaacaagcag ctaagaagaa agattgaacc
     2461 cccatttcct taattatttc aggaataatt ctcccgaaat caggtcaacc ccatcaccaa
     2521 ccaaccaacc agttgagttc cagattctat gtgaattaaa aagtcaatat atgtataatt
     2581 ctgagatgac ttaggttgga cattcaatgt tgtgctatga atttcctctt tatgcagagt
     2641 atctgtttgc ttgcagagtg gctttctggc ttgctgccag cctgtgcatg gtccacaagc
     2701 cgaattcaga tctggtacca cgcgtatcga taagcttgca tgcctgcagg tcgactctag
     2761 aggatccacc ggatctagat aactgatcat aatcagccat accacatttg tagaggtttt
     2821 acttgcttta aaaaacctcc cacacctccc cctgaacctg aaacataaaa tgaatgcaat
     2881 tgttgttgtt aacttgttta ttgcagctta taatggttac aaataaagca atagcatcac
     2941 aaatttcaca aataaagcat ttttttcact gcattctagt tgtggtttgt ccaaactcat
     3001 caatgtatct taacgcgtaa attgtaagcg ttaatatttt gttaaaattc gcgttaaatt
     3061 tttgttaaat cagctcattt tttaaccaat aggccgaaat cggcaaaatc ccttataaat
     3121 caaaagaata gaccgagata gggttgagtg ttgttccagt ttggaacaag agtccactat
     3181 taaagaacgt ggactccaac gtcaaagggc gaaaaaccgt ctatcagggc gatggcccac
     3241 tacgtgaacc atcaccctaa tcaagttttt tggggtcgag gtgccgtaaa gcactaaatc
     3301 ggaaccctaa agggagcccc cgatttagag cttgacgggg aaagccggcg aacgtggcga
     3361 gaaaggaagg gaagaaagcg aaaggagcgg gcgctagggc gctggcaagt gtagcggtca
     3421 cgctgcgcgt aaccaccaca cccgccgcgc ttaatgcgcc gctacagggc gcgtcaggtg
     3481 gcacttttcg gggaaatgtg cgcggaaccc ctatttgttt atttttctaa atacattcaa
     3541 atatgtatcc gctcatgaga caataaccct gataaatgct tcaataatat tgaaaaagga
     3601 agagtcctga ggcggaaaga accagctgtg gaatgtgtgt cagttagggt gtggaaagtc
     3661 cccaggctcc ccagcaggca gaagtatgca aagcatgcat ctcaattagt cagcaaccag
     3721 gtgtggaaag tccccaggct ccccagcagg cagaagtatg caaagcatgc atctcaatta
     3781 gtcagcaacc atagtcccgc ccctaactcc gcccatcccg cccctaactc cgcccagttc
     3841 cgcccattct ccgccccatg gctgactaat tttttttatt tatgcagagg ccgaggccgc
     3901 ctcggcctct gagctattcc agaagtagtg aggaggcttt tttggaggcc taggcttttg
     3961 caaagatcga tcaagagaca ggatgaggat cgtttcgcat gattgaacaa gatggattgc
     4021 acgcaggttc tccggccgct tgggtggaga ggctattcgg ctatgactgg gcacaacaga
     4081 caatcggctg ctctgatgcc gccgtgttcc ggctgtcagc gcaggggcgc ccggttcttt
     4141 ttgtcaagac cgacctgtcc ggtgccctga atgaactgca agacgaggca gcgcggctat
     4201 cgtggctggc cacgacgggc gttccttgcg cagctgtgct cgacgttgtc actgaagcgg
     4261 gaagggactg gctgctattg ggcgaagtgc cggggcagga tctcctgtca tctcaccttg
     4321 ctcctgccga gaaagtatcc atcatggctg atgcaatgcg gcggctgcat acgcttgatc
     4381 cggctacctg cccattcgac caccaagcga aacatcgcat cgagcgagca cgtactcgga
     4441 tggaagccgg tcttgtcgat caggatgatc tggacgaaga gcatcagggg ctcgcgccag
     4501 ccgaactgtt cgccaggctc aaggcgagca tgcccgacgg cgaggatctc gtcgtgaccc
     4561 atggcgatgc ctgcttgccg aatatcatgg tggaaaatgg ccgcttttct ggattcatcg
     4621 actgtggccg gctgggtgtg gcggaccgct atcaggacat agcgttggct acccgtgata
     4681 ttgctgaaga gcttggcggc gaatgggctg accgcttcct cgtgctttac ggtatcgccg
     4741 ctcccgattc gcagcgcatc gccttctatc gccttcttga cgagttcttc tgagcgggac
     4801 tctggggttc gaaatgaccg accaagcgac gcccaacctg ccatcacgag atttcgattc
     4861 caccgccgcc ttctatgaaa ggttgggctt cggaatcgtt ttccgggacg ccggctggat
     4921 gatcctccag cgcggggatc tcatgctgga gttcttcgcc caccctaggg ggaggctaac
     4981 tgaaacacgg aaggagacaa taccggaagg aacccgcgct atgacggcaa taaaaagaca
     5041 gaataaaacg cacggtgttg ggtcgtttgt tcataaacgc ggggttcggt cccagggctg
     5101 gcactctgtc gataccccac cgagacccca ttggggccaa tacgcccgcg tttcttcctt
     5161 ttccccaccc caccccccaa gttcgggtga aggcccaggg ctcgcagcca acgtcggggc
     5221 ggcaggccct gccatagcct caggttactc atatatactt tagattgatt taaaacttca
     5281 tttttaattt aaaaggatct aggtgaagat cctttttgat aatctcatga ccaaaatccc
     5341 ttaacgtgag ttttcgttcc actgagcgtc agaccccgta gaaaagatca aaggatcttc
     5401 ttgagatcct ttttttctgc gcgtaatctg ctgcttgcaa acaaaaaaac caccgctacc
     5461 agcggtggtt tgtttgccgg atcaagagct accaactctt tttccgaagg taactggctt
     5521 cagcagagcg cagataccaa atactgttct tctagtgtag ccgtagttag gccaccactt
     5581 caagaactct gtagcaccgc ctacatacct cgctctgcta atcctgttac cagtggctgc
     5641 tgccagtggc gataagtcgt gtcttaccgg gttggactca agacgatagt taccggataa
     5701 ggcgcagcgg tcgggctgaa cggggggttc gtgcacacag cccagcttgg agcgaacgac
     5761 ctacaccgaa ctgagatacc tacagcgtga gctatgagaa agcgccacgc ttcccgaagg
     5821 gagaaaggcg gacaggtatc cggtaagcgg cagggtcgga acaggagagc gcacgaggga
     5881 gcttccaggg ggaaacgcct ggtatcttta tagtcctgtc gggtttcgcc acctctgact
     5941 tgagcgtcga tttttgtgat gctcgtcagg ggggcggagc ctatggaaaa acgccagcaa
     6001 cgcggccttt ttacggttcc tggccttttg ctggcctttt gctcacatgt tctttcctgc
     6061 gtt
//
