LOCUS       sequence-260031-        6354 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Expresses EGFP-tagged rat NFL containing the Charcot-Marie-Tooth 
            disease type 2E N98S 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-N98S-EGFP
FEATURES             Location/Qualifiers
     source          1..6354
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     enhancer        56..359
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
     promoter        360..563
                     /label=CMV promoter
                     /note="human cytomegalovirus (CMV) immediate early 
                     promoter"
     primer_bind     514..534
                     /label=CMV-F
                     /note="Human CMV immediate early promoter, forward primer"
     regulatory      2289..2298
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     CDS             join(2295..2297,2298..2300,2301..3014)
                     /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(2295..2297,2298..2300,2301..3011)
                     /codon_start=1
                     /product="enhanced GFP"
                     /label=EGFP
                     /translation="MVSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTL
                     KFICTTGKLPVPWPTLVTTLTYGVQCFSRYPDHMKQHDFFKSAMPEGYVQERTIFFKDD
                     GNYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIK
                     VNFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSALSKDPNEKRDHMVLL
                     EFVTAAGITLGMDELYK"
     primer_bind     complement(2340..2361)
                     /label=EGFP-N
                     /note="EGFP, reverse primer"
     primer_bind     complement(2601..2620)
                     /label=EXFP-R
                     /note="For distinguishing EGFP variants, reverse primer"
     primer_bind     2948..2969
                     /label=EGFP-C
                     /note="EGFP, forward primer"
     polyA_signal    3137..3258
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(3174..3193)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     3228..3247
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     rep_origin      complement(3265..3720)
                     /direction=LEFT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(3402..3423)
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     3614..3633
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     promoter        3747..3851
                     /gene="bla"
                     /label=AmpR promoter
     promoter        3853..4210
                     /label=SV40 promoter
                     /note="SV40 enhancer and early promoter"
     enhancer        3853..4044
                     /label=SV40 enhancer
                     /note="enhancer for the SV40 early promoter (Herr, 1993)"
     primer_bind     complement(3876..3896)
                     /label=pBABE 3'
                     /note="SV40 enhancer, reverse primer for pBABE vectors"
     rep_origin      4061..4196
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     CDS             4245..5039
                     /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             4245..5039
                     /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(4299..4318)
                     /label=Neo-R
                     /note="Neomycin resistance gene, reverse primer"
     primer_bind     4909..4928
                     /label=Neo-F
                     /note="Neomycin resistance gene, forward primer"
     primer_bind     complement(5227..5246)
                     /label=TK-pA-R
                     /note="Thymidine kinase polyA, reverse primer"
     polyA_signal    5271..5318
                     /label=HSV TK poly(A) signal
                     /note="herpes simplex virus thymidine kinase 
                     polyadenylation signal (Cole and Stacy, 1985)"
     rep_origin      5647..6235
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     6136..6155
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
ORIGIN
        1 attaatagta atcaattacg gggtcattag ttcatagccc atatatggag ttccgcgtta
       61 cataacttac ggtaaatggc ccgcctggct gaccgcccaa cgacccccgc ccattgacgt
      121 caataatgac gtatgttccc atagtaacgc caatagggac tttccattga cgtcaatggg
      181 tggagtattt acggtaaact gcccacttgg cagtacatca agtgtatcat atgccaagta
      241 cgccccctat tgacgtcaat gacggtaaat ggcccgcctg gcattatgcc cagtacatga
      301 ccttatggga ctttcctact tggcagtaca tctacgtatt agtcatcgct attaccatgg
      361 tgatgcggtt ttggcagtac atcaatgggc gtggatagcg gtttgactca cggggatttc
      421 caagtctcca ccccattgac gtcaatggga gtttgttttg gcaccaaaat caacgggact
      481 ttccaaaatg tcgtaacaac tccgccccat tgacgcaaat gggcggtagg cgtgtacggt
      541 gggaggtcta tataagcaga gctggtttag tgaaccgtca gatccgctag cgctaccgga
      601 ctcagatctc gagctcaagc ttcgaattcc tgaggggcgc accgaccgcc accatgagtt
      661 cgttcagcta cgagccgtac ttttcgacct cctacaagcg gcgctacgtg gagacgcccc
      721 gggtgcacat ctccagcgtg cgcagcggct acagcacggc gcgctctgcg tactccagct
      781 actccgcgcc cgtctcctcc tctctgtcgg tgcgccgcag ctactcatcc agctccggct
      841 ctttgatgcc cagcctggag aacctcgatc tgagccaggt agccgccatc agcaacgacc
      901 tcaagtctat ccgcacacag gagaaggcac agctgcagga cctcagcgat cgcttcgcca
      961 gcttcatcga gcgcgtgcac gagctggagc agcagaacaa ggtcctggaa gccgagctgt
     1021 tggtgctgcg ccagaagcac tcagagcctt cccgcttccg cgccctgtat gagcaggaga
     1081 tccgtgatct gcgactggcg gccgaagacg ccactaacga gaagcaggcg ctgcagggcg
     1141 agcgcgaggg gctggaggag actctgcgca acctgcaggc tcgctacgag gaggaggtgc
     1201 tgagccgcga ggacgccgag ggccggctga tggaagcccg caaaggcgcg gatgaggctg
     1261 cgctcgcccg cgccgagctg gagaagcgca tcgacagcct gatggacgag atagccttcc
     1321 tgaaaaaggt gcacgaggaa gagatcgccg agctgcaggc tcagatccag tatgctcaga
     1381 tctccgtgga gatggacgtg tcctccaagc ccgacctctc cgccgctctc aaggacatcc
     1441 gcgctcagta cgagaagctg gccgccaaga atatgcagaa tgccgaagag tggttcaaga
     1501 gccgcttcac ggtgctaacc gagagcgccg ccaagaacac cgacgcagtg cgcgctgcca
     1561 aggacgaggt gtcggaaagc cgccgcctac ttaaggctaa gaccctggag atcgaagcct
     1621 gccggggtat gaacgaagct ctagagaagc agctgcagga gctggaggac aagcagaatg
     1681 cagacatcag cgccatgcag gacacaatca acaaactgga gaatgagctg cgaagcacga
     1741 agagcgagat ggccaggtac ctgaaggagt accaggacct cctcaatgtc aagatggcat
     1801 tggacattga gattgcagct tacaggaaac tcttggaagg cgaagaaacc aggctcagtt
     1861 tcaccagcgt gggtagcata accagcggct actctcagag ctcgcaggtc tttggccgtt
     1921 ctgcttacag tggcttgcag agcagctcct acttgatgtc tgctcgagca ttcccagcct
     1981 actataccag ccacgtccag gaagagcagt cagaggtgga ggagaccatt gaggctacga
     2041 aagctgagga ggccaaggat gagcccccct ctgaaggaga agcagaagag gaggagaagg
     2101 agaaagagga gggggaggaa gaggaaggtg ctgaggagga agaagctgcc aaggatgagt
     2161 ctgaagatgc caaagaagaa gaaggtggtg agggtgaaga ggaagacacc aaagagtcag
     2221 aagaggaaga gaagaaagag gaaagtgctg gggaggagca agctgctaag aagaaagatg
     2281 gatccatcgc caccatggtg agcaagggcg aggagctgtt caccggggtg gtgcccatcc
     2341 tggtcgagct ggacggcgac gtaaacggcc acaagttcag cgtgtccggc gagggcgagg
     2401 gcgatgccac ctacggcaag ctgaccctga agttcatctg caccaccggc aagctgcccg
     2461 tgccctggcc caccctcgtg accaccctga cctacggcgt gcagtgcttc agccgctacc
     2521 ccgaccacat gaagcagcac gacttcttca agtccgccat gcccgaaggc tacgtccagg
     2581 agcgcaccat cttcttcaag gacgacggca actacaagac ccgcgccgag gtgaagttcg
     2641 agggcgacac cctggtgaac cgcatcgagc tgaagggcat cgacttcaag gaggacggca
     2701 acatcctggg gcacaagctg gagtacaact acaacagcca caacgtctat atcatggccg
     2761 acaagcagaa gaacggcatc aaggtgaact tcaagatccg ccacaacatc gaggacggca
     2821 gcgtgcagct cgccgaccac taccagcaga acacccccat cggcgacggc cccgtgctgc
     2881 tgcccgacaa ccactacctg agcacccagt ccgccctgag caaagacccc aacgagaagc
     2941 gcgatcacat ggtcctgctg gagttcgtga ccgccgccgg gatcactctc ggcatggacg
     3001 agctgtacaa gtaaagcggc cgcgactcta gatcataatc agccatacca catttgtaga
     3061 ggttttactt gctttaaaaa acctcccaca cctccccctg aacctgaaac ataaaatgaa
     3121 tgcaattgtt gttgttaact tgtttattgc agcttataat ggttacaaat aaagcaatag
     3181 catcacaaat ttcacaaata aagcattttt ttcactgcat tctagttgtg gtttgtccaa
     3241 actcatcaat gtatcttaag gcgtaaattg taagcgttaa tattttgtta aaattcgcgt
     3301 taaatttttg ttaaatcagc tcatttttta accaataggc cgaaatcggc aaaatccctt
     3361 ataaatcaaa agaatagacc gagatagggt tgagtgttgt tccagtttgg aacaagagtc
     3421 cactattaaa gaacgtggac tccaacgtca aagggcgaaa aaccgtctat cagggcgatg
     3481 gcccactacg tgaaccatca ccctaatcaa gttttttggg gtcgaggtgc cgtaaagcac
     3541 taaatcggaa ccctaaaggg agcccccgat ttagagcttg acggggaaag ccggcgaacg
     3601 tggcgagaaa ggaagggaag aaagcgaaag gagcgggcgc tagggcgctg gcaagtgtag
     3661 cggtcacgct gcgcgtaacc accacacccg ccgcgcttaa tgcgccgcta cagggcgcgt
     3721 caggtggcac ttttcgggga aatgtgcgcg gaacccctat ttgtttattt ttctaaatac
     3781 attcaaatat gtatccgctc atgagacaat aaccctgata aatgcttcaa taatattgaa
     3841 aaaggaagag tcctgaggcg gaaagaacca gctgtggaat gtgtgtcagt tagggtgtgg
     3901 aaagtcccca ggctccccag caggcagaag tatgcaaagc atgcatctca attagtcagc
     3961 aaccaggtgt ggaaagtccc caggctcccc agcaggcaga agtatgcaaa gcatgcatct
     4021 caattagtca gcaaccatag tcccgcccct aactccgccc atcccgcccc taactccgcc
     4081 cagttccgcc cattctccgc cccatggctg actaattttt tttatttatg cagaggccga
     4141 ggccgcctcg gcctctgagc tattccagaa gtagtgagga ggcttttttg gaggcctagg
     4201 cttttgcaaa gatcgatcaa gagacaggat gaggatcgtt tcgcatgatt gaacaagatg
     4261 gattgcacgc aggttctccg gccgcttggg tggagaggct attcggctat gactgggcac
     4321 aacagacaat cggctgctct gatgccgccg tgttccggct gtcagcgcag gggcgcccgg
     4381 ttctttttgt caagaccgac ctgtccggtg ccctgaatga actgcaagac gaggcagcgc
     4441 ggctatcgtg gctggccacg acgggcgttc cttgcgcagc tgtgctcgac gttgtcactg
     4501 aagcgggaag ggactggctg ctattgggcg aagtgccggg gcaggatctc ctgtcatctc
     4561 accttgctcc tgccgagaaa gtatccatca tggctgatgc aatgcggcgg ctgcatacgc
     4621 ttgatccggc tacctgccca ttcgaccacc aagcgaaaca tcgcatcgag cgagcacgta
     4681 ctcggatgga agccggtctt gtcgatcagg atgatctgga cgaagagcat caggggctcg
     4741 cgccagccga actgttcgcc aggctcaagg cgagcatgcc cgacggcgag gatctcgtcg
     4801 tgacccatgg cgatgcctgc ttgccgaata tcatggtgga aaatggccgc ttttctggat
     4861 tcatcgactg tggccggctg ggtgtggcgg accgctatca ggacatagcg ttggctaccc
     4921 gtgatattgc tgaagagctt ggcggcgaat gggctgaccg cttcctcgtg ctttacggta
     4981 tcgccgctcc cgattcgcag cgcatcgcct tctatcgcct tcttgacgag ttcttctgag
     5041 cgggactctg gggttcgaaa tgaccgacca agcgacgccc aacctgccat cacgagattt
     5101 cgattccacc gccgccttct atgaaaggtt gggcttcgga atcgttttcc gggacgccgg
     5161 ctggatgatc ctccagcgcg gggatctcat gctggagttc ttcgcccacc ctagggggag
     5221 gctaactgaa acacggaagg agacaatacc ggaaggaacc cgcgctatga cggcaataaa
     5281 aagacagaat aaaacgcacg gtgttgggtc gtttgttcat aaacgcgggg ttcggtccca
     5341 gggctggcac tctgtcgata ccccaccgag accccattgg ggccaatacg cccgcgtttc
     5401 ttccttttcc ccaccccacc ccccaagttc gggtgaaggc ccagggctcg cagccaacgt
     5461 cggggcggca ggccctgcca tagcctcagg ttactcatat atactttaga ttgatttaaa
     5521 acttcatttt taatttaaaa ggatctaggt gaagatcctt tttgataatc tcatgaccaa
     5581 aatcccttaa cgtgagtttt cgttccactg agcgtcagac cccgtagaaa agatcaaagg
     5641 atcttcttga gatccttttt ttctgcgcgt aatctgctgc ttgcaaacaa aaaaaccacc
     5701 gctaccagcg gtggtttgtt tgccggatca agagctacca actctttttc cgaaggtaac
     5761 tggcttcagc agagcgcaga taccaaatac tgttcttcta gtgtagccgt agttaggcca
     5821 ccacttcaag aactctgtag caccgcctac atacctcgct ctgctaatcc tgttaccagt
     5881 ggctgctgcc agtggcgata agtcgtgtct taccgggttg gactcaagac gatagttacc
     5941 ggataaggcg cagcggtcgg gctgaacggg gggttcgtgc acacagccca gcttggagcg
     6001 aacgacctac accgaactga gatacctaca gcgtgagcta tgagaaagcg ccacgcttcc
     6061 cgaagggaga aaggcggaca ggtatccggt aagcggcagg gtcggaacag gagagcgcac
     6121 gagggagctt ccagggggaa acgcctggta tctttatagt cctgtcgggt ttcgccacct
     6181 ctgacttgag cgtcgatttt tgtgatgctc gtcagggggg cggagcctat ggaaaaacgc
     6241 cagcaacgcg gcctttttac ggttcctggc cttttgctgg ccttttgctc acatgttctt
     6301 tcctgcgtta tcccctgatt ctgtggataa ccgtattacc gccatgcatt agtt
//
