LOCUS       sequence-175291-        6277 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Expression of mouse  GFP/WT spastin M85 in mammalian cells.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 6277)
  AUTHORS   Solowska JM, Morfini G, Falnikar A, Himes BT, Brady ST, Huang D, 
            Baas PW
  TITLE     Quantitative and functional analyses of spastin in the nervous 
            system: implications for hereditary spastic paraplegia.
  JOURNAL   J Neurosci. 2008 Feb 27;28(9):2147-57. doi: 
            10.1523/JNEUROSCI.3159-07.2008.
  PUBMED    18305248
REFERENCE   2  (bases 1 to 6277)
  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"; doi: 
            "10.1523/JNEUROSCI.3159-07"; journalName: "J Neurosci"; date: 
            "2008-02-27- 27"; volume: "28"; issue: "9"; pages: "2147-57"
COMMENT     Sequence Label: GFP/WT SP M85
FEATURES             Location/Qualifiers
     source          1..6277
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     enhancer        61..364
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
     promoter        365..568
                     /label=CMV promoter
                     /note="human cytomegalovirus (CMV) immediate early 
                     promoter"
     primer_bind     519..539
                     /label=CMV-F
                     /note="Human CMV immediate early promoter, forward primer"
     regulatory      607..616
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     CDS             join(613..615,616..618,619..1329)
                     /codon_start=1
                     /product="enhanced GFP"
                     /label=EGFP
                     /translation="MVSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTL
                     KFICTTGKLPVPWPTLVTTLTYGVQCFSRYPDHMKQHDFFKSAMPEGYVQERTIFFKDD
                     GNYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIK
                     VNFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSALSKDPNEKRDHMVLL
                     EFVTAAGITLGMDELYK"
     CDS             join(613..615,616..618,619..1329)
                     /codon_start=1
                     /product="the original enhanced GFP (Yang et al., 1996)"
                     /label=EGFP
                     /note="mammalian codon-optimized"
                     /translation="MVSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTL
                     KFICTTGKLPVPWPTLVTTLTYGVQCFSRYPDHMKQHDFFKSAMPEGYVQERTIFFKDD
                     GNYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIK
                     VNFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSALSKDPNEKRDHMVLL
                     EFVTAAGITLGMDELYK"
     primer_bind     complement(658..679)
                     /label=EGFP-N
                     /note="EGFP, reverse primer"
     primer_bind     complement(919..938)
                     /label=EXFP-R
                     /note="For distinguishing EGFP variants, reverse primer"
     primer_bind     1266..1287
                     /label=EGFP-C
                     /note="EGFP, forward primer"
     polyA_signal    3065..3186
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(3102..3121)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     3156..3175
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     rep_origin      complement(3193..3648)
                     /direction=LEFT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(3330..3351)
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     3542..3561
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     promoter        3675..3779
                     /gene="bla"
                     /label=AmpR promoter
     promoter        3781..4138
                     /label=SV40 promoter
                     /note="SV40 enhancer and early promoter"
     enhancer        3781..3972
                     /label=SV40 enhancer
                     /note="enhancer for the SV40 early promoter (Herr, 1993)"
     primer_bind     complement(3804..3824)
                     /label=pBABE 3'
                     /note="SV40 enhancer, reverse primer for pBABE vectors"
     rep_origin      3989..4124
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     CDS             4173..4967
                     /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             4173..4967
                     /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(4227..4246)
                     /label=Neo-R
                     /note="Neomycin resistance gene, reverse primer"
     primer_bind     4837..4856
                     /label=Neo-F
                     /note="Neomycin resistance gene, forward primer"
     primer_bind     complement(5155..5174)
                     /label=TK-pA-R
                     /note="Thymidine kinase polyA, reverse primer"
     polyA_signal    5199..5246
                     /label=HSV TK poly(A) signal
                     /note="herpes simplex virus thymidine kinase 
                     polyadenylation signal (Cole and Stacy, 1985)"
     rep_origin      5575..6163
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     6064..6083
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
ORIGIN
        1 tagttattaa tagtaatcaa ttacggggtc attagttcat agcccatata tggagttccg
       61 cgttacataa cttacggtaa atggcccgcc tggctgaccg cccaacgacc cccgcccatt
      121 gacgtcaata atgacgtatg ttcccatagt aacgccaata gggactttcc attgacgtca
      181 atgggtggag tatttacggt aaactgccca cttggcagta catcaagtgt atcatatgcc
      241 aagtacgccc cctattgacg tcaatgacgg taaatggccc gcctggcatt atgcccagta
      301 catgacctta tgggactttc ctacttggca gtacatctac gtattagtca tcgctattac
      361 catggtgatg cggttttggc agtacatcaa tgggcgtgga tagcggtttg actcacgggg
      421 atttccaagt ctccacccca ttgacgtcaa tgggagtttg ttttggcacc aaaatcaacg
      481 ggactttcca aaatgtcgta acaactccgc cccattgacg caaatgggcg gtaggcgtgt
      541 acggtgggag gtctatataa gcagagctgg tttagtgaac cgtcagatcc gctagcgcta
      601 ccggtcgcca ccatggtgag caagggcgag gagctgttca ccggggtggt gcccatcctg
      661 gtcgagctgg acggcgacgt aaacggccac aagttcagcg tgtccggcga gggcgagggc
      721 gatgccacct acggcaagct gaccctgaag ttcatctgca ccaccggcaa gctgcccgtg
      781 ccctggccca ccctcgtgac caccctgacc tacggcgtgc agtgcttcag ccgctacccc
      841 gaccacatga agcagcacga cttcttcaag tccgccatgc ccgaaggcta cgtccaggag
      901 cgcaccatct tcttcaagga cgacggcaac tacaagaccc gcgccgaggt gaagttcgag
      961 ggcgacaccc tggtgaaccg catcgagctg aagggcatcg acttcaagga ggacggcaac
     1021 atcctggggc acaagctgga gtacaactac aacagccaca acgtctatat catggccgac
     1081 aagcagaaga acggcatcaa ggtgaacttc aagatccgcc acaacatcga ggacggcagc
     1141 gtgcagctcg ccgaccacta ccagcagaac acccccatcg gcgacggccc cgtgctgctg
     1201 cccgacaacc actacctgag cacccagtcc gccctgagca aagaccccaa cgagaagcgc
     1261 gatcacatgg tcctgctgga gttcgtgacc gccgccggga tcactctcgg catggacgag
     1321 ctgtacaagt ccggactcag atccgccgcc aagaggagct ccgggaccgc gccggcgccc
     1381 gcctcgccct cgcccccagc gcccggaccg ggtggcgagg ccgagagcgt ccgcgtcttc
     1441 cacaagcagg ccttcgagta catctccatt gccctgcgca tcgacgagga agagaaagga
     1501 cagaaggaac aagctgtgga atggtataag aaaggtatcg aagaactgga aaaaggaatc
     1561 gctgttatag ttacgggcca aggtgaacag tatgaaagag ctagacgtct tcaagccaaa
     1621 atgatgacta atttagttat ggccaaggac cgtttacaac ttctagagaa gctgcaacca
     1681 gttttgcaat tttccaagtc acagacggac gtctataacg agagtactaa cctgacatgc
     1741 cgcaatggac atctccagtc agaaagtgga gcagttccga agaggaaaga ccccttaaca
     1801 catgctagta attcattgcc tcgatcaaaa actgtcctga aaagtggctc cgcagggctc
     1861 tccggtcacc acagggcgcc tagttgcagt ggtttgtcca tggtttctgg agcaagaccg
     1921 ggacctggtc ctgcagctac cacacataag ggtactccaa aaccaaatag aaccaacaaa
     1981 ccttctactc ccacaactgc agttcggaaa aagaaagact tgaaaaattt taggaatgtg
     2041 gacagcaatc ttgctaacct tataatgaat gaaattgttg acaatgggac agctgttaag
     2101 tttgatgaca tagccgggca ggagctggca aagcaagcgc tgcaggagat tgtcatcctt
     2161 ccttctctgc ggcctgagtt gttcacaggg ctcagagctc ctgctagagg cttgttactc
     2221 ttcggtccgc caggaaacgg aaaaacaatg ctggctaaag cagtagctgc agagtctaat
     2281 gcaacttttt tcaacataag tgctgccagt ttaacttcaa aatatgtggg agaaggagag
     2341 aaattggtga gagctctctt tgctgtggct cgagaacttc aaccatctat aatttttata
     2401 gatgaagttg acagtctttt gtgtgagaga cgggaagggg agcacgacgc tagcagacgg
     2461 ctaaagacgg aatttttaat agaattcgac ggggtgcaat ctgctggaga tgacagagta
     2521 cttgtaatgg gtgcaactaa caggccccaa gagcttgatg aagctgttct caggcgtttc
     2581 attaaacggg tatatgtgtc cttaccaaat gaggagacaa gactccttct gcttaaaaac
     2641 ctgttgtgta aacaaggaag tccactgacc caaaaagaac tcgcacagct tgctagaatg
     2701 accgatggat actctggaag tgatctgacc gctttggcca aggatgcagc cctgggtcct
     2761 atccgagaac tgaagccaga gcaggtgaag aatatgtctg ccagtgagat gagaaatatt
     2821 cgattatctg acttcacaga atccttaaaa aagataaaac gcagtgtgag tcctcagacc
     2881 ttagaagcat acatacgctg gaacaaggac tttggagaca ccactgttta agatgggatc
     2941 caccggatct agataactga tcataatcag ccataccaca tttgtagagg ttttacttgc
     3001 tttaaaaaac ctcccacacc tccccctgaa cctgaaacat aaaatgaatg caattgttgt
     3061 tgttaacttg tttattgcag cttataatgg ttacaaataa agcaatagca tcacaaattt
     3121 cacaaataaa gcattttttt cactgcattc tagttgtggt ttgtccaaac tcatcaatgt
     3181 atcttaacgc gtaaattgta agcgttaata ttttgttaaa attcgcgtta aatttttgtt
     3241 aaatcagctc attttttaac caataggccg aaatcggcaa aatcccttat aaatcaaaag
     3301 aatagaccga gatagggttg agtgttgttc cagtttggaa caagagtcca ctattaaaga
     3361 acgtggactc caacgtcaaa gggcgaaaaa ccgtctatca gggcgatggc ccactacgtg
     3421 aaccatcacc ctaatcaagt tttttggggt cgaggtgccg taaagcacta aatcggaacc
     3481 ctaaagggag cccccgattt agagcttgac ggggaaagcc ggcgaacgtg gcgagaaagg
     3541 aagggaagaa agcgaaagga gcgggcgcta gggcgctggc aagtgtagcg gtcacgctgc
     3601 gcgtaaccac cacacccgcc gcgcttaatg cgccgctaca gggcgcgtca ggtggcactt
     3661 ttcggggaaa tgtgcgcgga acccctattt gtttattttt ctaaatacat tcaaatatgt
     3721 atccgctcat gagacaataa ccctgataaa tgcttcaata atattgaaaa aggaagagtc
     3781 ctgaggcgga aagaaccagc tgtggaatgt gtgtcagtta gggtgtggaa agtccccagg
     3841 ctccccagca ggcagaagta tgcaaagcat gcatctcaat tagtcagcaa ccaggtgtgg
     3901 aaagtcccca ggctccccag caggcagaag tatgcaaagc atgcatctca attagtcagc
     3961 aaccatagtc ccgcccctaa ctccgcccat cccgccccta actccgccca gttccgccca
     4021 ttctccgccc catggctgac taattttttt tatttatgca gaggccgagg ccgcctcggc
     4081 ctctgagcta ttccagaagt agtgaggagg cttttttgga ggcctaggct tttgcaaaga
     4141 tcgatcaaga gacaggatga ggatcgtttc gcatgattga acaagatgga ttgcacgcag
     4201 gttctccggc cgcttgggtg gagaggctat tcggctatga ctgggcacaa cagacaatcg
     4261 gctgctctga tgccgccgtg ttccggctgt cagcgcaggg gcgcccggtt ctttttgtca
     4321 agaccgacct gtccggtgcc ctgaatgaac tgcaagacga ggcagcgcgg ctatcgtggc
     4381 tggccacgac gggcgttcct tgcgcagctg tgctcgacgt tgtcactgaa gcgggaaggg
     4441 actggctgct attgggcgaa gtgccggggc aggatctcct gtcatctcac cttgctcctg
     4501 ccgagaaagt atccatcatg gctgatgcaa tgcggcggct gcatacgctt gatccggcta
     4561 cctgcccatt cgaccaccaa gcgaaacatc gcatcgagcg agcacgtact cggatggaag
     4621 ccggtcttgt cgatcaggat gatctggacg aagagcatca ggggctcgcg ccagccgaac
     4681 tgttcgccag gctcaaggcg agcatgcccg acggcgagga tctcgtcgtg acccatggcg
     4741 atgcctgctt gccgaatatc atggtggaaa atggccgctt ttctggattc atcgactgtg
     4801 gccggctggg tgtggcggac cgctatcagg acatagcgtt ggctacccgt gatattgctg
     4861 aagagcttgg cggcgaatgg gctgaccgct tcctcgtgct ttacggtatc gccgctcccg
     4921 attcgcagcg catcgccttc tatcgccttc ttgacgagtt cttctgagcg ggactctggg
     4981 gttcgaaatg accgaccaag cgacgcccaa cctgccatca cgagatttcg attccaccgc
     5041 cgccttctat gaaaggttgg gcttcggaat cgttttccgg gacgccggct ggatgatcct
     5101 ccagcgcggg gatctcatgc tggagttctt cgcccaccct agggggaggc taactgaaac
     5161 acggaaggag acaataccgg aaggaacccg cgctatgacg gcaataaaaa gacagaataa
     5221 aacgcacggt gttgggtcgt ttgttcataa acgcggggtt cggtcccagg gctggcactc
     5281 tgtcgatacc ccaccgagac cccattgggg ccaatacgcc cgcgtttctt ccttttcccc
     5341 accccacccc ccaagttcgg gtgaaggccc agggctcgca gccaacgtcg gggcggcagg
     5401 ccctgccata gcctcaggtt actcatatat actttagatt gatttaaaac ttcattttta
     5461 atttaaaagg atctaggtga agatcctttt tgataatctc atgaccaaaa tcccttaacg
     5521 tgagttttcg ttccactgag cgtcagaccc cgtagaaaag atcaaaggat cttcttgaga
     5581 tccttttttt ctgcgcgtaa tctgctgctt gcaaacaaaa aaaccaccgc taccagcggt
     5641 ggtttgtttg ccggatcaag agctaccaac tctttttccg aaggtaactg gcttcagcag
     5701 agcgcagata ccaaatactg ttcttctagt gtagccgtag ttaggccacc acttcaagaa
     5761 ctctgtagca ccgcctacat acctcgctct gctaatcctg ttaccagtgg ctgctgccag
     5821 tggcgataag tcgtgtctta ccgggttgga ctcaagacga tagttaccgg ataaggcgca
     5881 gcggtcgggc tgaacggggg gttcgtgcac acagcccagc ttggagcgaa cgacctacac
     5941 cgaactgaga tacctacagc gtgagctatg agaaagcgcc acgcttcccg aagggagaaa
     6001 ggcggacagg tatccggtaa gcggcagggt cggaacagga gagcgcacga gggagcttcc
     6061 agggggaaac gcctggtatc tttatagtcc tgtcgggttt cgccacctct gacttgagcg
     6121 tcgatttttg tgatgctcgt caggggggcg gagcctatgg aaaaacgcca gcaacgcggc
     6181 ctttttacgg ttcctggcct tttgctggcc ttttgctcac atgttctttc ctgcgttatc
     6241 ccctgattct gtggataacc gtattaccgc catgcat
//
