LOCUS       sequence-260177-        6354 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Expresses EGFP-tagged rat NFL containing the Charcot-Marie-Tooth 
            disease type 2E Q333P 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-Q333P-EGFP
FEATURES             Location/Qualifiers
     source          1..6354
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     enhancer        127..430
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
     promoter        431..634
                     /label=CMV promoter
                     /note="human cytomegalovirus (CMV) immediate early 
                     promoter"
     primer_bind     585..605
                     /label=CMV-F
                     /note="Human CMV immediate early promoter, forward primer"
     regulatory      2360..2369
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     CDS             join(2366..2368,2369..2371,2372..3085)
                     /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             join(2366..2368,2369..2371,2372..3082)
                     /codon_start=1
                     /product="enhanced GFP"
                     /label=EGFP
                     /translation="MVSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTL
                     KFICTTGKLPVPWPTLVTTLTYGVQCFSRYPDHMKQHDFFKSAMPEGYVQERTIFFKDD
                     GNYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIK
                     VNFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSALSKDPNEKRDHMVLL
                     EFVTAAGITLGMDELYK"
     primer_bind     complement(2411..2432)
                     /label=EGFP-N
                     /note="EGFP, reverse primer"
     primer_bind     complement(2672..2691)
                     /label=EXFP-R
                     /note="For distinguishing EGFP variants, reverse primer"
     primer_bind     3019..3040
                     /label=EGFP-C
                     /note="EGFP, forward primer"
     polyA_signal    3208..3329
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(3245..3264)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     3299..3318
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     rep_origin      complement(3336..3791)
                     /direction=LEFT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(3473..3494)
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     3685..3704
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     promoter        3818..3922
                     /gene="bla"
                     /label=AmpR promoter
     promoter        3924..4281
                     /label=SV40 promoter
                     /note="SV40 enhancer and early promoter"
     enhancer        3924..4115
                     /label=SV40 enhancer
                     /note="enhancer for the SV40 early promoter (Herr, 1993)"
     primer_bind     complement(3947..3967)
                     /label=pBABE 3'
                     /note="SV40 enhancer, reverse primer for pBABE vectors"
     rep_origin      4132..4267
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     CDS             4316..5110
                     /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             4316..5110
                     /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(4370..4389)
                     /label=Neo-R
                     /note="Neomycin resistance gene, reverse primer"
     primer_bind     4980..4999
                     /label=Neo-F
                     /note="Neomycin resistance gene, forward primer"
     primer_bind     complement(5298..5317)
                     /label=TK-pA-R
                     /note="Thymidine kinase polyA, reverse primer"
     polyA_signal    5342..5389
                     /label=HSV TK poly(A) signal
                     /note="herpes simplex virus thymidine kinase 
                     polyadenylation signal (Cole and Stacy, 1985)"
     rep_origin      5718..6306
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     6207..6226
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
ORIGIN
        1 tttgctcaca tgttctttcc tgcgttatcc cctgattctg tggataaccg tattaccgcc
       61 atgcattagt tattaatagt aatcaattac ggggtcatta gttcatagcc catatatgga
      121 gttccgcgtt acataactta cggtaaatgg cccgcctggc tgaccgccca acgacccccg
      181 cccattgacg tcaataatga cgtatgttcc catagtaacg ccaataggga ctttccattg
      241 acgtcaatgg gtggagtatt tacggtaaac tgcccacttg gcagtacatc aagtgtatca
      301 tatgccaagt acgcccccta ttgacgtcaa tgacggtaaa tggcccgcct ggcattatgc
      361 ccagtacatg accttatggg actttcctac ttggcagtac atctacgtat tagtcatcgc
      421 tattaccatg gtgatgcggt tttggcagta catcaatggg cgtggatagc ggtttgactc
      481 acggggattt ccaagtctcc accccattga cgtcaatggg agtttgtttt ggcaccaaaa
      541 tcaacgggac tttccaaaat gtcgtaacaa ctccgcccca ttgacgcaaa tgggcggtag
      601 gcgtgtacgg tgggaggtct atataagcag agctggttta gtgaaccgtc agatccgcta
      661 gcgctaccgg actcagatct cgagctcaag cttcgaattc ctgaggggcg caccgaccgc
      721 caccatgagt tcgttcagct acgagccgta cttttcgacc tcctacaagc ggcgctacgt
      781 ggagacgccc cgggtgcaca tctccagcgt gcgcagcggc tacagcacgg cgcgctctgc
      841 gtactccagc tactccgcgc ccgtctcctc ctctctgtcg gtgcgccgca gctactcatc
      901 cagctccggc tctttgatgc ccagcctgga gaacctcgat ctgagccagg tagccgccat
      961 cagcaacgac ctcaagtcta tccgcacaca ggagaaggca cagctgcagg acctcaacga
     1021 tcgcttcgcc agcttcatcg agcgcgtgca cgagctggag cagcagaaca aggtcctgga
     1081 agccgagctg ttggtgctgc gccagaagca ctcagagcct tcccgcttcc gcgccctgta
     1141 tgagcaggag atccgtgatc tgcgactggc ggccgaagac gccactaacg agaagcaggc
     1201 gctgcagggc gagcgcgagg ggctggagga gactctgcgc aacctgcagg ctcgctacga
     1261 ggaggaggtg ctgagccgcg aggacgccga gggccggctg atggaagccc gcaaaggcgc
     1321 ggatgaggct gcgctcgccc gcgccgagct ggagaagcgc atcgacagcc tgatggacga
     1381 gatagccttc ctgaaaaagg tgcacgagga agagatcgcc gagctgcagg ctcagatcca
     1441 gtatgctcag atctccgtgg agatggacgt gtcctccaag cccgacctct ccgccgctct
     1501 caaggacatc cgcgctcagt acgagaagct ggccgccaag aatatgcaga atgccgaaga
     1561 gtggttcaag agccgcttca cggtgctaac cgagagcgcc gccaagaaca ccgacgcagt
     1621 gcgcgctgcc aaggacgagg tgtcggaaag ccgccgccta cttaaggcta agaccctgga
     1681 gatcgaagcc tgccggggta tgaacgaagc tctagagaag cccctgcagg agctggagga
     1741 caagcagaat gcagacatca gcgccatgca ggacacaatc aacaaactgg agaatgagct
     1801 gcgaagcacg aagagcgaga tggccaggta cctgaaggag taccaggacc tcctcaatgt
     1861 caagatggca ttggacattg agattgcagc ttacaggaaa ctcttggaag gcgaagaaac
     1921 caggctcagt ttcaccagcg tgggtagcat aaccagcggc tactctcaga gctcgcaggt
     1981 ctttggccgt tctgcttaca gtggcttgca gagcagctcc tacttgatgt ctgctcgagc
     2041 attcccagcc tactatacca gccacgtcca ggaagagcag tcagaggtgg aggagaccat
     2101 tgaggctacg aaagctgagg aggccaagga tgagcccccc tctgaaggag aagcagaaga
     2161 ggaggagaag gagaaagagg agggggagga agaggaaggt gctgaggagg aagaagctgc
     2221 caaggatgag tctgaagatg ccaaagaaga agaaggtggt gagggtgaag aggaagacac
     2281 caaagagtca gaagaggaag agaagaaaga ggaaagtgct ggggaggagc aagctgctaa
     2341 gaagaaagat ggatccatcg ccaccatggt gagcaagggc gaggagctgt tcaccggggt
     2401 ggtgcccatc ctggtcgagc tggacggcga cgtaaacggc cacaagttca gcgtgtccgg
     2461 cgagggcgag ggcgatgcca cctacggcaa gctgaccctg aagttcatct gcaccaccgg
     2521 caagctgccc gtgccctggc ccaccctcgt gaccaccctg acctacggcg tgcagtgctt
     2581 cagccgctac cccgaccaca tgaagcagca cgacttcttc aagtccgcca tgcccgaagg
     2641 ctacgtccag gagcgcacca tcttcttcaa ggacgacggc aactacaaga cccgcgccga
     2701 ggtgaagttc gagggcgaca ccctggtgaa ccgcatcgag ctgaagggca tcgacttcaa
     2761 ggaggacggc aacatcctgg ggcacaagct ggagtacaac tacaacagcc acaacgtcta
     2821 tatcatggcc gacaagcaga agaacggcat caaggtgaac ttcaagatcc gccacaacat
     2881 cgaggacggc agcgtgcagc tcgccgacca ctaccagcag aacaccccca tcggcgacgg
     2941 ccccgtgctg ctgcccgaca accactacct gagcacccag tccgccctga gcaaagaccc
     3001 caacgagaag cgcgatcaca tggtcctgct ggagttcgtg accgccgccg ggatcactct
     3061 cggcatggac gagctgtaca agtaaagcgg ccgcgactct agatcataat cagccatacc
     3121 acatttgtag aggttttact tgctttaaaa aacctcccac acctccccct gaacctgaaa
     3181 cataaaatga atgcaattgt tgttgttaac ttgtttattg cagcttataa tggttacaaa
     3241 taaagcaata gcatcacaaa tttcacaaat aaagcatttt tttcactgca ttctagttgt
     3301 ggtttgtcca aactcatcaa tgtatcttaa ggcgtaaatt gtaagcgtta atattttgtt
     3361 aaaattcgcg ttaaattttt gttaaatcag ctcatttttt aaccaatagg ccgaaatcgg
     3421 caaaatccct tataaatcaa aagaatagac cgagataggg ttgagtgttg ttccagtttg
     3481 gaacaagagt ccactattaa agaacgtgga ctccaacgtc aaagggcgaa aaaccgtcta
     3541 tcagggcgat ggcccactac gtgaaccatc accctaatca agttttttgg ggtcgaggtg
     3601 ccgtaaagca ctaaatcgga accctaaagg gagcccccga tttagagctt gacggggaaa
     3661 gccggcgaac gtggcgagaa aggaagggaa gaaagcgaaa ggagcgggcg ctagggcgct
     3721 ggcaagtgta gcggtcacgc tgcgcgtaac caccacaccc gccgcgctta atgcgccgct
     3781 acagggcgcg tcaggtggca cttttcgggg aaatgtgcgc ggaaccccta tttgtttatt
     3841 tttctaaata cattcaaata tgtatccgct catgagacaa taaccctgat aaatgcttca
     3901 ataatattga aaaaggaaga gtcctgaggc ggaaagaacc agctgtggaa tgtgtgtcag
     3961 ttagggtgtg gaaagtcccc aggctcccca gcaggcagaa gtatgcaaag catgcatctc
     4021 aattagtcag caaccaggtg tggaaagtcc ccaggctccc cagcaggcag aagtatgcaa
     4081 agcatgcatc tcaattagtc agcaaccata gtcccgcccc taactccgcc catcccgccc
     4141 ctaactccgc ccagttccgc ccattctccg ccccatggct gactaatttt ttttatttat
     4201 gcagaggccg aggccgcctc ggcctctgag ctattccaga agtagtgagg aggctttttt
     4261 ggaggcctag gcttttgcaa agatcgatca agagacagga tgaggatcgt ttcgcatgat
     4321 tgaacaagat ggattgcacg caggttctcc ggccgcttgg gtggagaggc tattcggcta
     4381 tgactgggca caacagacaa tcggctgctc tgatgccgcc gtgttccggc tgtcagcgca
     4441 ggggcgcccg gttctttttg tcaagaccga cctgtccggt gccctgaatg aactgcaaga
     4501 cgaggcagcg cggctatcgt ggctggccac gacgggcgtt ccttgcgcag ctgtgctcga
     4561 cgttgtcact gaagcgggaa gggactggct gctattgggc gaagtgccgg ggcaggatct
     4621 cctgtcatct caccttgctc ctgccgagaa agtatccatc atggctgatg caatgcggcg
     4681 gctgcatacg cttgatccgg ctacctgccc attcgaccac caagcgaaac atcgcatcga
     4741 gcgagcacgt actcggatgg aagccggtct tgtcgatcag gatgatctgg acgaagagca
     4801 tcaggggctc gcgccagccg aactgttcgc caggctcaag gcgagcatgc ccgacggcga
     4861 ggatctcgtc gtgacccatg gcgatgcctg cttgccgaat atcatggtgg aaaatggccg
     4921 cttttctgga ttcatcgact gtggccggct gggtgtggcg gaccgctatc aggacatagc
     4981 gttggctacc cgtgatattg ctgaagagct tggcggcgaa tgggctgacc gcttcctcgt
     5041 gctttacggt atcgccgctc ccgattcgca gcgcatcgcc ttctatcgcc ttcttgacga
     5101 gttcttctga gcgggactct ggggttcgaa atgaccgacc aagcgacgcc caacctgcca
     5161 tcacgagatt tcgattccac cgccgccttc tatgaaaggt tgggcttcgg aatcgttttc
     5221 cgggacgccg gctggatgat cctccagcgc ggggatctca tgctggagtt cttcgcccac
     5281 cctaggggga ggctaactga aacacggaag gagacaatac cggaaggaac ccgcgctatg
     5341 acggcaataa aaagacagaa taaaacgcac ggtgttgggt cgtttgttca taaacgcggg
     5401 gttcggtccc agggctggca ctctgtcgat accccaccga gaccccattg gggccaatac
     5461 gcccgcgttt cttccttttc cccaccccac cccccaagtt cgggtgaagg cccagggctc
     5521 gcagccaacg tcggggcggc aggccctgcc atagcctcag gttactcata tatactttag
     5581 attgatttaa aacttcattt ttaatttaaa aggatctagg tgaagatcct ttttgataat
     5641 ctcatgacca aaatccctta acgtgagttt tcgttccact gagcgtcaga ccccgtagaa
     5701 aagatcaaag gatcttcttg agatcctttt tttctgcgcg taatctgctg cttgcaaaca
     5761 aaaaaaccac cgctaccagc ggtggtttgt ttgccggatc aagagctacc aactcttttt
     5821 ccgaaggtaa ctggcttcag cagagcgcag ataccaaata ctgttcttct agtgtagccg
     5881 tagttaggcc accacttcaa gaactctgta gcaccgccta catacctcgc tctgctaatc
     5941 ctgttaccag tggctgctgc cagtggcgat aagtcgtgtc ttaccgggtt ggactcaaga
     6001 cgatagttac cggataaggc gcagcggtcg ggctgaacgg ggggttcgtg cacacagccc
     6061 agcttggagc gaacgaccta caccgaactg agatacctac agcgtgagct atgagaaagc
     6121 gccacgcttc ccgaagggag aaaggcggac aggtatccgg taagcggcag ggtcggaaca
     6181 ggagagcgca cgagggagct tccaggggga aacgcctggt atctttatag tcctgtcggg
     6241 tttcgccacc tctgacttga gcgtcgattt ttgtgatgct cgtcaggggg gcggagccta
     6301 tggaaaaacg ccagcaacgc ggccttttta cggttcctgg ccttttgctg gcct
//
