LOCUS       sequence-260981-        6354 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Expresses hEGFP-tagged rat 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 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-P22S-EGFP
FEATURES             Location/Qualifiers
     source          1..6354
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     enhancer        72..263
                     /label=SV40 enhancer
                     /note="enhancer for the SV40 early promoter (Herr, 1993)"
     primer_bind     220..240
                     /label=pBABE 3'
                     /note="SV40 enhancer, reverse primer for pBABE vectors"
     promoter        complement(265..369)
                     /gene="bla"
                     /label=AmpR promoter
     rep_origin      396..851
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(483..502)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     693..714
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     polyA_signal    858..979
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(869..888)
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     primer_bind     923..942
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     CDS             complement(join(1102..1815,1816..1818,1819..1821))
                     /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             complement(join(1105..1815,1816..1818,1819..1821))
                     /codon_start=1
                     /product="enhanced GFP"
                     /label=EGFP
                     /translation="MVSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTL
                     KFICTTGKLPVPWPTLVTTLTYGVQCFSRYPDHMKQHDFFKSAMPEGYVQERTIFFKDD
                     GNYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIK
                     VNFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSALSKDPNEKRDHMVLL
                     EFVTAAGITLGMDELYK"
     primer_bind     complement(1147..1168)
                     /label=EGFP-C
                     /note="EGFP, forward primer"
     primer_bind     1496..1515
                     /label=EXFP-R
                     /note="For distinguishing EGFP variants, reverse primer"
     primer_bind     1755..1776
                     /label=EGFP-N
                     /note="EGFP, reverse primer"
     regulatory      1818..1827
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     promoter        complement(3553..3756)
                     /label=CMV promoter
                     /note="human cytomegalovirus (CMV) immediate early 
                     promoter"
     primer_bind     complement(3582..3602)
                     /label=CMV-F
                     /note="Human CMV immediate early promoter, forward primer"
     enhancer        3757..4060
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
     rep_origin      complement(4235..4823)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(4315..4334)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     polyA_signal    5152..5199
                     /label=HSV TK poly(A) signal
                     /note="herpes simplex virus thymidine kinase 
                     polyadenylation signal (Cole and Stacy, 1985)"
     primer_bind     5224..5243
                     /label=TK-pA-R
                     /note="Thymidine kinase polyA, reverse primer"
     CDS             complement(5431..6225)
                     /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             complement(5431..6225)
                     /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(5542..5561)
                     /label=Neo-F
                     /note="Neomycin resistance gene, forward primer"
     primer_bind     6152..6171
                     /label=Neo-R
                     /note="Neomycin resistance gene, reverse primer"
     promoter        complement(join(6260..6354,1..263))
                     /label=SV40 promoter
                     /note="SV40 enhancer and early promoter"
     rep_origin      join(6274..6354,1..55)
                     /label=SV40 ori
                     /note="SV40 origin of replication"
ORIGIN
        1 ttagtcagcc atggggcgga gaatgggcgg aactgggcgg agttaggggc gggatgggcg
       61 gagttagggg cgggactatg gttgctgact aattgagatg catgctttgc atacttctgc
      121 ctgctgggga gcctggggac tttccacacc tggttgctga ctaattgaga tgcatgcttt
      181 gcatacttct gcctgctggg gagcctgggg actttccaca ccctaactga cacacattcc
      241 acagctggtt ctttccgcct caggactctt cctttttcaa tattattgaa gcatttatca
      301 gggttattgt ctcatgagcg gatacatatt tgaatgtatt tagaaaaata aacaaatagg
      361 ggttccgcgc acatttcccc gaaaagtgcc acctgacgcg ccctgtagcg gcgcattaag
      421 cgcggcgggt gtggtggtta cgcgcagcgt gaccgctaca cttgccagcg ccctagcgcc
      481 cgctcctttc gctttcttcc cttcctttct cgccacgttc gccggctttc cccgtcaagc
      541 tctaaatcgg gggctccctt tagggttccg atttagtgct ttacggcacc tcgaccccaa
      601 aaaacttgat tagggtgatg gttcacgtag tgggccatcg ccctgataga cggtttttcg
      661 ccctttgacg ttggagtcca cgttctttaa tagtggactc ttgttccaaa ctggaacaac
      721 actcaaccct atctcggtct attcttttga tttataaggg attttgccga tttcggccta
      781 ttggttaaaa aatgagctga tttaacaaaa atttaacgcg aattttaaca aaatattaac
      841 gcttacaatt tacgccttaa gatacattga tgagtttgga caaaccacaa ctagaatgca
      901 gtgaaaaaaa tgctttattt gtgaaatttg tgatgctatt gctttatttg taaccattat
      961 aagctgcaat aaacaagtta acaacaacaa ttgcattcat tttatgtttc aggttcaggg
     1021 ggaggtgtgg gaggtttttt aaagcaagta aaacctctac aaatgtggta tggctgatta
     1081 tgatctagag tcgcggccgc tttacttgta cagctcgtcc atgccgagag tgatcccggc
     1141 ggcggtcacg aactccagca ggaccatgtg atcgcgcttc tcgttggggt ctttgctcag
     1201 ggcggactgg gtgctcaggt agtggttgtc gggcagcagc acggggccgt cgccgatggg
     1261 ggtgttctgc tggtagtggt cggcgagctg cacgctgccg tcctcgatgt tgtggcggat
     1321 cttgaagttc accttgatgc cgttcttctg cttgtcggcc atgatataga cgttgtggct
     1381 gttgtagttg tactccagct tgtgccccag gatgttgccg tcctccttga agtcgatgcc
     1441 cttcagctcg atgcggttca ccagggtgtc gccctcgaac ttcacctcgg cgcgggtctt
     1501 gtagttgccg tcgtccttga agaagatggt gcgctcctgg acgtagcctt cgggcatggc
     1561 ggacttgaag aagtcgtgct gcttcatgtg gtcggggtag cggctgaagc actgcacgcc
     1621 gtaggtcagg gtggtcacga gggtgggcca gggcacgggc agcttgccgg tggtgcagat
     1681 gaacttcagg gtcagcttgc cgtaggtggc atcgccctcg ccctcgccgg acacgctgaa
     1741 cttgtggccg tttacgtcgc cgtccagctc gaccaggatg ggcaccaccc cggtgaacag
     1801 ctcctcgccc ttgctcacca tggtggcgat ggatccatct ttcttcttag cagcttgctc
     1861 ctccccagca ctttcctctt tcttctcttc ctcttctgac tctttggtgt cttcctcttc
     1921 accctcacca ccttcttctt ctttggcatc ttcagactca tccttggcag cttcttcctc
     1981 ctcagcacct tcctcttcct ccccctcctc tttctccttc tcctcctctt ctgcttctcc
     2041 ttcagagggg ggctcatcct tggcctcctc agctttcgta gcctcaatgg tctcctccac
     2101 ctctgactgc tcttcctgga cgtggctggt atagtaggct gggaatgctc gagcagacat
     2161 caagtaggag ctgctctgca agccactgta agcagaacgg ccaaagacct gcgagctctg
     2221 agagtagccg ctggttatgc tacccacgct ggtgaaactg agcctggttt cttcgccttc
     2281 caagagtttc ctgtaagctg caatctcaat gtccaatgcc atcttgacat tgaggaggtc
     2341 ctggtactcc ttcaggtacc tggccatctc gctcttcgtg cttcgcagct cattctccag
     2401 tttgttgatt gtgtcctgca tggcgctgat gtctgcattc tgcttgtcct ccagctcctg
     2461 cagctgcttc tctagagctt cgttcatacc ccggcaggct tcgatctcca gggtcttagc
     2521 cttaagtagg cggcggcttt ccgacacctc gtccttggca gcgcgcactg cgtcggtgtt
     2581 cttggcggcg ctctcggtta gcaccgtgaa gcggctcttg aaccactctt cggcattctg
     2641 catattcttg gcggccagct tctcgtactg agcgcggatg tccttgagag cggcggagag
     2701 gtcgggcttg gaggacacgt ccatctccac ggagatctga gcatactgga tctgagcctg
     2761 cagctcggcg atctcttcct cgtgcacctt tttcaggaag gctatctcgt ccatcaggct
     2821 gtcgatgcgc ttctccagct cggcgcgggc gagcgcagcc tcatccgcgc ctttgcgggc
     2881 ttccatcagc cggccctcgg cgtcctcgcg gctcagcacc tcctcctcgt agcgagcctg
     2941 caggttgcgc agagtctcct ccagcccctc gcgctcgccc tgcagcgcct gcttctcgtt
     3001 agtggcgtct tcggccgcca gtcgcagatc acggatctcc tgctcataca gggcgcggaa
     3061 gcgggaaggc tctgagtgct tctggcgcag caccaacagc tcggcttcca ggaccttgtt
     3121 ctgctgctcc agctcgtgca cgcgctcgat gaagctggcg aagcgatcgt tgaggtcctg
     3181 cagctgtgcc ttctcctgtg tgcggataga cttgaggtcg ttgctgatgg cggctacctg
     3241 gctcagatcg aggttctcca ggctgggcat caaagagccg gagctggatg agtagctgcg
     3301 gcgcaccgac agagaggagg agacgggcgc ggagtagctg gagtacgcag agcgcgccgt
     3361 gctgtagccg ctgcgcacgc tggagatgtg cacccggctc gtctccacgt agcgccgctt
     3421 gtaggaggtc gaaaagtacg gctcgtagct gaacgaactc atggtggcgg tcggtgcgcc
     3481 cctcaggaat tcgaagcttg agctcgagat ctgagtccgg tagcgctagc ggatctgacg
     3541 gttcactaaa ccagctctgc ttatatagac ctcccaccgt acacgcctac cgcccatttg
     3601 cgtcaatggg gcggagttgt tacgacattt tggaaagtcc cgttgatttt ggtgccaaaa
     3661 caaactccca ttgacgtcaa tggggtggag acttggaaat ccccgtgagt caaaccgcta
     3721 tccacgccca ttgatgtact gccaaaaccg catcaccatg gtaatagcga tgactaatac
     3781 gtagatgtac tgccaagtag gaaagtccca taaggtcatg tactgggcat aatgccaggc
     3841 gggccattta ccgtcattga cgtcaatagg gggcgtactt ggcatatgat acacttgatg
     3901 tactgccaag tgggcagttt accgtaaata ctccacccat tgacgtcaat ggaaagtccc
     3961 tattggcgtt actatgggaa catacgtcat tattgacgtc aatgggcggg ggtcgttggg
     4021 cggtcagcca ggcgggccat ttaccgtaag ttatgtaacg cggaactcca tatatgggct
     4081 atgaactaat gaccccgtaa ttgattacta ttaataacta atgcatggcg gtaatacggt
     4141 tatccacaga atcaggggat aacgcaggaa agaacatgtg agcaaaaggc cagcaaaagg
     4201 ccaggaaccg taaaaaggcc gcgttgctgg cgtttttcca taggctccgc ccccctgacg
     4261 agcatcacaa aaatcgacgc tcaagtcaga ggtggcgaaa cccgacagga ctataaagat
     4321 accaggcgtt tccccctgga agctccctcg tgcgctctcc tgttccgacc ctgccgctta
     4381 ccggatacct gtccgccttt ctcccttcgg gaagcgtggc gctttctcat agctcacgct
     4441 gtaggtatct cagttcggtg taggtcgttc gctccaagct gggctgtgtg cacgaacccc
     4501 ccgttcagcc cgaccgctgc gccttatccg gtaactatcg tcttgagtcc aacccggtaa
     4561 gacacgactt atcgccactg gcagcagcca ctggtaacag gattagcaga gcgaggtatg
     4621 taggcggtgc tacagagttc ttgaagtggt ggcctaacta cggctacact agaagaacag
     4681 tatttggtat ctgcgctctg ctgaagccag ttaccttcgg aaaaagagtt ggtagctctt
     4741 gatccggcaa acaaaccacc gctggtagcg gtggtttttt tgtttgcaag cagcagatta
     4801 cgcgcagaaa aaaaggatct caagaagatc ctttgatctt ttctacgggg tctgacgctc
     4861 agtggaacga aaactcacgt taagggattt tggtcatgag attatcaaaa aggatcttca
     4921 cctagatcct tttaaattaa aaatgaagtt ttaaatcaat ctaaagtata tatgagtaac
     4981 ctgaggctat ggcagggcct gccgccccga cgttggctgc gagccctggg ccttcacccg
     5041 aacttggggg gtggggtggg gaaaaggaag aaacgcgggc gtattggccc caatggggtc
     5101 tcggtggggt atcgacagag tgccagccct gggaccgaac cccgcgttta tgaacaaacg
     5161 acccaacacc gtgcgtttta ttctgtcttt ttattgccgt catagcgcgg gttccttccg
     5221 gtattgtctc cttccgtgtt tcagttagcc tccccctagg gtgggcgaag aactccagca
     5281 tgagatcccc gcgctggagg atcatccagc cggcgtcccg gaaaacgatt ccgaagccca
     5341 acctttcata gaaggcggcg gtggaatcga aatctcgtga tggcaggttg ggcgtcgctt
     5401 ggtcggtcat ttcgaacccc agagtcccgc tcagaagaac tcgtcaagaa ggcgatagaa
     5461 ggcgatgcgc tgcgaatcgg gagcggcgat accgtaaagc acgaggaagc ggtcagccca
     5521 ttcgccgcca agctcttcag caatatcacg ggtagccaac gctatgtcct gatagcggtc
     5581 cgccacaccc agccggccac agtcgatgaa tccagaaaag cggccatttt ccaccatgat
     5641 attcggcaag caggcatcgc catgggtcac gacgagatcc tcgccgtcgg gcatgctcgc
     5701 cttgagcctg gcgaacagtt cggctggcgc gagcccctga tgctcttcgt ccagatcatc
     5761 ctgatcgaca agaccggctt ccatccgagt acgtgctcgc tcgatgcgat gtttcgcttg
     5821 gtggtcgaat gggcaggtag ccggatcaag cgtatgcagc cgccgcattg catcagccat
     5881 gatggatact ttctcggcag gagcaaggtg agatgacagg agatcctgcc ccggcacttc
     5941 gcccaatagc agccagtccc ttcccgcttc agtgacaacg tcgagcacag ctgcgcaagg
     6001 aacgcccgtc gtggccagcc acgatagccg cgctgcctcg tcttgcagtt cattcagggc
     6061 accggacagg tcggtcttga caaaaagaac cgggcgcccc tgcgctgaca gccggaacac
     6121 ggcggcatca gagcagccga ttgtctgttg tgcccagtca tagccgaata gcctctccac
     6181 ccaagcggcc ggagaacctg cgtgcaatcc atcttgttca atcatgcgaa acgatcctca
     6241 tcctgtctct tgatcgatct ttgcaaaagc ctaggcctcc aaaaaagcct cctcactact
     6301 tctggaatag ctcagaggcc gaggcggcct cggcctctgc ataaataaaa aaaa
//
