LOCUS       sequence-260029-        6354 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Expresses EGFP-tagged rat NFL containing the Charcot-Marie-Tooth 
            disease type 2E E397K 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-E397K-EGFP
FEATURES             Location/Qualifiers
     source          1..6354
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     enhancer        77..380
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
     promoter        381..584
                     /label=CMV promoter
                     /note="human cytomegalovirus (CMV) immediate early 
                     promoter"
     primer_bind     535..555
                     /label=CMV-F
                     /note="Human CMV immediate early promoter, forward primer"
     regulatory      2310..2319
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     CDS             join(2316..2318,2319..2321,2322..3035)
                     /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(2316..2318,2319..2321,2322..3032)
                     /codon_start=1
                     /product="enhanced GFP"
                     /label=EGFP
                     /translation="MVSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTL
                     KFICTTGKLPVPWPTLVTTLTYGVQCFSRYPDHMKQHDFFKSAMPEGYVQERTIFFKDD
                     GNYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIK
                     VNFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSALSKDPNEKRDHMVLL
                     EFVTAAGITLGMDELYK"
     primer_bind     complement(2361..2382)
                     /label=EGFP-N
                     /note="EGFP, reverse primer"
     primer_bind     complement(2622..2641)
                     /label=EXFP-R
                     /note="For distinguishing EGFP variants, reverse primer"
     primer_bind     2969..2990
                     /label=EGFP-C
                     /note="EGFP, forward primer"
     polyA_signal    3158..3279
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(3195..3214)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     3249..3268
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     rep_origin      complement(3286..3741)
                     /direction=LEFT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(3423..3444)
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     3635..3654
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     promoter        3768..3872
                     /gene="bla"
                     /label=AmpR promoter
     promoter        3874..4231
                     /label=SV40 promoter
                     /note="SV40 enhancer and early promoter"
     enhancer        3874..4065
                     /label=SV40 enhancer
                     /note="enhancer for the SV40 early promoter (Herr, 1993)"
     primer_bind     complement(3897..3917)
                     /label=pBABE 3'
                     /note="SV40 enhancer, reverse primer for pBABE vectors"
     rep_origin      4082..4217
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     CDS             4266..5060
                     /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             4266..5060
                     /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(4320..4339)
                     /label=Neo-R
                     /note="Neomycin resistance gene, reverse primer"
     primer_bind     4930..4949
                     /label=Neo-F
                     /note="Neomycin resistance gene, forward primer"
     primer_bind     complement(5248..5267)
                     /label=TK-pA-R
                     /note="Thymidine kinase polyA, reverse primer"
     polyA_signal    5292..5339
                     /label=HSV TK poly(A) signal
                     /note="herpes simplex virus thymidine kinase 
                     polyadenylation signal (Cole and Stacy, 1985)"
     rep_origin      5668..6256
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     6157..6176
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
ORIGIN
        1 tattaccgcc atgcattagt tattaatagt aatcaattac ggggtcatta gttcatagcc
       61 catatatgga gttccgcgtt acataactta cggtaaatgg cccgcctggc tgaccgccca
      121 acgacccccg cccattgacg tcaataatga cgtatgttcc catagtaacg ccaataggga
      181 ctttccattg acgtcaatgg gtggagtatt tacggtaaac tgcccacttg gcagtacatc
      241 aagtgtatca tatgccaagt acgcccccta ttgacgtcaa tgacggtaaa tggcccgcct
      301 ggcattatgc ccagtacatg accttatggg actttcctac ttggcagtac atctacgtat
      361 tagtcatcgc tattaccatg gtgatgcggt tttggcagta catcaatggg cgtggatagc
      421 ggtttgactc acggggattt ccaagtctcc accccattga cgtcaatggg agtttgtttt
      481 ggcaccaaaa tcaacgggac tttccaaaat gtcgtaacaa ctccgcccca ttgacgcaaa
      541 tgggcggtag gcgtgtacgg tgggaggtct atataagcag agctggttta gtgaaccgtc
      601 agatccgcta gcgctaccgg actcagatct cgagctcaag cttcgaattc ctgaggggcg
      661 caccgaccgc caccatgagt tcgttcagct acgagccgta cttttcgacc tcctacaagc
      721 ggcgctacgt ggagacgccc cgggtgcaca tctccagcgt gcgcagcggc tacagcacgg
      781 cgcgctctgc gtactccagc tactccgcgc ccgtctcctc ctctctgtcg gtgcgccgca
      841 gctactcatc cagctccggc tctttgatgc ccagcctgga gaacctcgat ctgagccagg
      901 tagccgccat cagcaacgac ctcaagtcta tccgcacaca ggagaaggca cagctgcagg
      961 acctcaacga tcgcttcgcc agcttcatcg agcgcgtgca cgagctggag cagcagaaca
     1021 aggtcctgga agccgagctg ttggtgctgc gccagaagca ctcagagcct tcccgcttcc
     1081 gcgccctgta tgagcaggag atccgtgatc tgcgactggc ggccgaagac gccactaacg
     1141 agaagcaggc gctgcagggc gagcgcgagg ggctggagga gactctgcgc aacctgcagg
     1201 ctcgctacga ggaggaggtg ctgagccgcg aggacgccga gggccggctg atggaagccc
     1261 gcaaaggcgc ggatgaggct gcgctcgccc gcgccgagct ggagaagcgc atcgacagcc
     1321 tgatggacga gatagccttc ctgaaaaagg tgcacgagga agagatcgcc gagctgcagg
     1381 ctcagatcca gtatgctcag atctccgtgg agatggacgt gtcctccaag cccgacctct
     1441 ccgccgctct caaggacatc cgcgctcagt acgagaagct ggccgccaag aatatgcaga
     1501 atgccgaaga gtggttcaag agccgcttca cggtgctaac cgagagcgcc gccaagaaca
     1561 ccgacgcagt gcgcgctgcc aaggacgagg tgtcggaaag ccgccgccta cttaaggcta
     1621 agaccctgga gatcgaagcc tgccggggta tgaacgaagc tctagagaag cagctgcagg
     1681 agctggagga caagcagaat gcagacatca gcgccatgca ggacacaatc aacaaactgg
     1741 agaatgagct gcgaagcacg aagagcgaga tggccaggta cctgaaggag taccaggacc
     1801 tcctcaatgt caagatggca ttggacattg agattgcagc ttacaggaaa ctcttggaag
     1861 gcaaagaaac caggctcagt ttcaccagcg tgggtagcat aaccagcggc tactctcaga
     1921 gctcgcaggt ctttggccgt tctgcttaca gtggcttgca gagcagctcc tacttgatgt
     1981 ctgctcgagc attcccagcc tactatacca gccacgtcca ggaagagcag tcagaggtgg
     2041 aggagaccat tgaggctacg aaagctgagg aggccaagga tgagcccccc tctgaaggag
     2101 aagcagaaga ggaggagaag gagaaagagg agggggagga agaggaaggt gctgaggagg
     2161 aagaagctgc caaggatgag tctgaagatg ccaaagaaga agaaggtggt gagggtgaag
     2221 aggaagacac caaagagtca gaagaggaag agaagaaaga ggaaagtgct ggggaggagc
     2281 aagctgctaa gaagaaagat ggatccatcg ccaccatggt gagcaagggc gaggagctgt
     2341 tcaccggggt ggtgcccatc ctggtcgagc tggacggcga cgtaaacggc cacaagttca
     2401 gcgtgtccgg cgagggcgag ggcgatgcca cctacggcaa gctgaccctg aagttcatct
     2461 gcaccaccgg caagctgccc gtgccctggc ccaccctcgt gaccaccctg acctacggcg
     2521 tgcagtgctt cagccgctac cccgaccaca tgaagcagca cgacttcttc aagtccgcca
     2581 tgcccgaagg ctacgtccag gagcgcacca tcttcttcaa ggacgacggc aactacaaga
     2641 cccgcgccga ggtgaagttc gagggcgaca ccctggtgaa ccgcatcgag ctgaagggca
     2701 tcgacttcaa ggaggacggc aacatcctgg ggcacaagct ggagtacaac tacaacagcc
     2761 acaacgtcta tatcatggcc gacaagcaga agaacggcat caaggtgaac ttcaagatcc
     2821 gccacaacat cgaggacggc agcgtgcagc tcgccgacca ctaccagcag aacaccccca
     2881 tcggcgacgg ccccgtgctg ctgcccgaca accactacct gagcacccag tccgccctga
     2941 gcaaagaccc caacgagaag cgcgatcaca tggtcctgct ggagttcgtg accgccgccg
     3001 ggatcactct cggcatggac gagctgtaca agtaaagcgg ccgcgactct agatcataat
     3061 cagccatacc acatttgtag aggttttact tgctttaaaa aacctcccac acctccccct
     3121 gaacctgaaa cataaaatga atgcaattgt tgttgttaac ttgtttattg cagcttataa
     3181 tggttacaaa taaagcaata gcatcacaaa tttcacaaat aaagcatttt tttcactgca
     3241 ttctagttgt ggtttgtcca aactcatcaa tgtatcttaa ggcgtaaatt gtaagcgtta
     3301 atattttgtt aaaattcgcg ttaaattttt gttaaatcag ctcatttttt aaccaatagg
     3361 ccgaaatcgg caaaatccct tataaatcaa aagaatagac cgagataggg ttgagtgttg
     3421 ttccagtttg gaacaagagt ccactattaa agaacgtgga ctccaacgtc aaagggcgaa
     3481 aaaccgtcta tcagggcgat ggcccactac gtgaaccatc accctaatca agttttttgg
     3541 ggtcgaggtg ccgtaaagca ctaaatcgga accctaaagg gagcccccga tttagagctt
     3601 gacggggaaa gccggcgaac gtggcgagaa aggaagggaa gaaagcgaaa ggagcgggcg
     3661 ctagggcgct ggcaagtgta gcggtcacgc tgcgcgtaac caccacaccc gccgcgctta
     3721 atgcgccgct acagggcgcg tcaggtggca cttttcgggg aaatgtgcgc ggaaccccta
     3781 tttgtttatt tttctaaata cattcaaata tgtatccgct catgagacaa taaccctgat
     3841 aaatgcttca ataatattga aaaaggaaga gtcctgaggc ggaaagaacc agctgtggaa
     3901 tgtgtgtcag ttagggtgtg gaaagtcccc aggctcccca gcaggcagaa gtatgcaaag
     3961 catgcatctc aattagtcag caaccaggtg tggaaagtcc ccaggctccc cagcaggcag
     4021 aagtatgcaa agcatgcatc tcaattagtc agcaaccata gtcccgcccc taactccgcc
     4081 catcccgccc ctaactccgc ccagttccgc ccattctccg ccccatggct gactaatttt
     4141 ttttatttat gcagaggccg aggccgcctc ggcctctgag ctattccaga agtagtgagg
     4201 aggctttttt ggaggcctag gcttttgcaa agatcgatca agagacagga tgaggatcgt
     4261 ttcgcatgat tgaacaagat ggattgcacg caggttctcc ggccgcttgg gtggagaggc
     4321 tattcggcta tgactgggca caacagacaa tcggctgctc tgatgccgcc gtgttccggc
     4381 tgtcagcgca ggggcgcccg gttctttttg tcaagaccga cctgtccggt gccctgaatg
     4441 aactgcaaga cgaggcagcg cggctatcgt ggctggccac gacgggcgtt ccttgcgcag
     4501 ctgtgctcga cgttgtcact gaagcgggaa gggactggct gctattgggc gaagtgccgg
     4561 ggcaggatct cctgtcatct caccttgctc ctgccgagaa agtatccatc atggctgatg
     4621 caatgcggcg gctgcatacg cttgatccgg ctacctgccc attcgaccac caagcgaaac
     4681 atcgcatcga gcgagcacgt actcggatgg aagccggtct tgtcgatcag gatgatctgg
     4741 acgaagagca tcaggggctc gcgccagccg aactgttcgc caggctcaag gcgagcatgc
     4801 ccgacggcga ggatctcgtc gtgacccatg gcgatgcctg cttgccgaat atcatggtgg
     4861 aaaatggccg cttttctgga ttcatcgact gtggccggct gggtgtggcg gaccgctatc
     4921 aggacatagc gttggctacc cgtgatattg ctgaagagct tggcggcgaa tgggctgacc
     4981 gcttcctcgt gctttacggt atcgccgctc ccgattcgca gcgcatcgcc ttctatcgcc
     5041 ttcttgacga gttcttctga gcgggactct ggggttcgaa atgaccgacc aagcgacgcc
     5101 caacctgcca tcacgagatt tcgattccac cgccgccttc tatgaaaggt tgggcttcgg
     5161 aatcgttttc cgggacgccg gctggatgat cctccagcgc ggggatctca tgctggagtt
     5221 cttcgcccac cctaggggga ggctaactga aacacggaag gagacaatac cggaaggaac
     5281 ccgcgctatg acggcaataa aaagacagaa taaaacgcac ggtgttgggt cgtttgttca
     5341 taaacgcggg gttcggtccc agggctggca ctctgtcgat accccaccga gaccccattg
     5401 gggccaatac gcccgcgttt cttccttttc cccaccccac cccccaagtt cgggtgaagg
     5461 cccagggctc gcagccaacg tcggggcggc aggccctgcc atagcctcag gttactcata
     5521 tatactttag attgatttaa aacttcattt ttaatttaaa aggatctagg tgaagatcct
     5581 ttttgataat ctcatgacca aaatccctta acgtgagttt tcgttccact gagcgtcaga
     5641 ccccgtagaa aagatcaaag gatcttcttg agatcctttt tttctgcgcg taatctgctg
     5701 cttgcaaaca aaaaaaccac cgctaccagc ggtggtttgt ttgccggatc aagagctacc
     5761 aactcttttt ccgaaggtaa ctggcttcag cagagcgcag ataccaaata ctgttcttct
     5821 agtgtagccg tagttaggcc accacttcaa gaactctgta gcaccgccta catacctcgc
     5881 tctgctaatc ctgttaccag tggctgctgc cagtggcgat aagtcgtgtc ttaccgggtt
     5941 ggactcaaga cgatagttac cggataaggc gcagcggtcg ggctgaacgg ggggttcgtg
     6001 cacacagccc agcttggagc gaacgaccta caccgaactg agatacctac agcgtgagct
     6061 atgagaaagc gccacgcttc ccgaagggag aaaggcggac aggtatccgg taagcggcag
     6121 ggtcggaaca ggagagcgca cgagggagct tccaggggga aacgcctggt atctttatag
     6181 tcctgtcggg tttcgccacc tctgacttga gcgtcgattt ttgtgatgct cgtcaggggg
     6241 gcggagccta tggaaaaacg ccagcaacgc ggccttttta cggttcctgg ccttttgctg
     6301 gccttttgct cacatgttct ttcctgcgtt atcccctgat tctgtggata accg
//
