LOCUS       sequence-173592-        6082 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Expresses E112K spastin M1 and M87 in mammalian cells.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 6082)
  AUTHORS   Solowska JM, Garbern JY, Baas PW
  TITLE     Evaluation of loss of function as an explanation for SPG4-based 
            hereditary spastic paraplegia.
  JOURNAL   Hum Mol Genet. 2010 Jul 15;19(14):2767-79. doi: 10.1093/hmg/ddq177. 
            Epub 2010 Apr 29.
  PUBMED    20430936
REFERENCE   2  (bases 1 to 6082)
  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: 
            "10.1093/hmg/ddq177"; journalName: "Hum Mol Genet"; date: 
            "2010-07-15- 15"; volume: "19"; issue: "14"; pages: "2767-79"
COMMENT     Sequence Label: pCMV-Tag/E112K without 5'UTR
FEATURES             Location/Qualifiers
     source          1..6082
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     enhancer        1..304
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
     promoter        305..508
                     /label=CMV promoter
                     /note="human cytomegalovirus (CMV) immediate early 
                     promoter"
     primer_bind     459..479
                     /label=CMV-F
                     /note="Human CMV immediate early promoter, forward primer"
     promoter        554..572
                     /label=T3 promoter
                     /note="promoter for bacteriophage T3 RNA polymerase"
     primer_bind     complement(2517..2536)
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        complement(2518..2536)
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     polyA_signal    2810..2931
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(2847..2866)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     2901..2920
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     rep_origin      complement(2938..3393)
                     /direction=LEFT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(3075..3096)
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     3287..3306
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     promoter        3420..3524
                     /gene="bla"
                     /label=AmpR promoter
     promoter        3526..3883
                     /label=SV40 promoter
                     /note="SV40 enhancer and early promoter"
     enhancer        3526..3717
                     /label=SV40 enhancer
                     /note="enhancer for the SV40 early promoter (Herr, 1993)"
     primer_bind     complement(3549..3569)
                     /label=pBABE 3'
                     /note="SV40 enhancer, reverse primer for pBABE vectors"
     rep_origin      3734..3869
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     CDS             3918..4712
                     /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             3918..4712
                     /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(3972..3991)
                     /label=Neo-R
                     /note="Neomycin resistance gene, reverse primer"
     primer_bind     4582..4601
                     /label=Neo-F
                     /note="Neomycin resistance gene, forward primer"
     primer_bind     complement(4900..4919)
                     /label=TK-pA-R
                     /note="Thymidine kinase polyA, reverse primer"
     polyA_signal    4944..4991
                     /label=HSV TK poly(A) signal
                     /note="herpes simplex virus thymidine kinase 
                     polyadenylation signal (Cole and Stacy, 1985)"
     rep_origin      5320..5908
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     5809..5828
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
ORIGIN
        1 cgttacataa cttacggtaa atggcccgcc tggctgaccg cccaacgacc cccgcccatt
       61 gacgtcaata atgacgtatg ttcccatagt aacgccaata gggactttcc attgacgtca
      121 atgggtggag tatttacggt aaactgccca cttggcagta catcaagtgt atcatatgcc
      181 aagtacgccc cctattgacg tcaatgacgg taaatggccc gcctggcatt atgcccagta
      241 catgacctta tgggactttc ctacttggca gtacatctac gtattagtca tcgctattac
      301 catggtgatg cggttttggc agtacatcaa tgggcgtgga tagcggtttg actcacgggg
      361 atttccaagt ctccacccca ttgacgtcaa tgggagtttg ttttggcacc aaaatcaacg
      421 ggactttcca aaatgtcgta acaactccgc cccattgacg caaatgggcg gtaggcgtgt
      481 acggtgggag gtctatataa gcagagctgg tttagtgaac cgtcagatcc gctagcgatt
      541 acgccaagct cgaaattaac cctcactaaa gggaacaaaa gctggagatc gcggcggcgg
      601 cagtgagagc tgtgaatgaa ttctccgggt ggacgaggga agaagaaagg ctccggcggc
      661 gccagcaacc cggtgcctcc caggcctccg cccccttgcc tggcccccgc ccctcccgcc
      721 gccgggccgg cccctccgcc cgagtcgccg cataagcgga acctgtacta tttctcctac
      781 ccgctgtttg taggcttcgc gctgctgcgt ttggtcgcct tccacctggg gctcctcttc
      841 gtgtggctct gccagcgctt ctcccgcgcc ctcatggcag ccaagaggag ctccggggcc
      901 gcgccagcac ctgcctcggc ctcggccccg gcgccggtgc cgggcggcaa ggccgagcgc
      961 gtccgagtct tccacaaaca ggccttcgag tacatctcca ttgccctgcg catcgatgag
     1021 gatgagaaag caggacagaa ggagcaagct gtggaatggt ataagaaagg tattgaagaa
     1081 ctggaaaaag gaatagctgt tatagttaca ggacaaggtg aacagtgtga aagagctaga
     1141 cgccttcaag ctaaaatgat gactaatttg gttatggcca aggaccgctt acaacttcta
     1201 gagaagatgc aaccagtttt gccattttcc aagtcacaaa cggacgtcta taatgacagt
     1261 actaacttgg catgccgcaa tggacatctc cagtcagaaa gtggagctgt tccaaaaaga
     1321 aaagacccct taacacacac tagtaattca ctgcctcgtt caaaaacagt tatgaaaact
     1381 ggatctgcag gcctttcagg ccaccataga gcacctagtt acagtggttt atccatggtt
     1441 tctggagtga aacagggatc tggtcctgct cctaccactc ataagggtac tccgaaaaca
     1501 aataggacaa ataaaccttc tacccctaca actgctactc gtaagaaaaa agacttgaag
     1561 aattttagga atgtggacag caaccttgct aaccttataa tgaatgaaat tgtggacaat
     1621 ggaacagctg ttaaatttga tgatatagct ggtcaagact tggcaaaaca agcattgcaa
     1681 gaaattgtta ttcttccttc tctgaggcct gagttgttca cagggcttag agctcctgcc
     1741 agagggctgt tactctttgg tccacctggg aatgggaaga caatgctggc taaagcagta
     1801 gctgcagaat cgaatgcaac cttctttaat ataagtgctg caagtttaac ttcaaaatac
     1861 gtgggagaag gagagaaatt ggtgagggct ctttttgctg tggctcgaga acttcaacct
     1921 tctataattt ttatagatga agttgatagc cttttgtgtg aaagaagaga aggggagcac
     1981 gatgctagta gacgcctaaa aactgaattt ctaatagaat ttgatggtgt acagtctgct
     2041 ggagatgaca gagtacttgt aatgggtgca actaataggc cacaagagct tgatgaggct
     2101 gttctcaggc gtttcatcaa acgggtatat gtgtctttac caaatgagga gacaagacta
     2161 cttttgctta aaaatctgtt atgtaaacaa ggaagtccat tgacccaaaa agaactagca
     2221 caacttgcta gaatgactga tggatactca ggaagtgacc taacagcttt ggcaaaagat
     2281 gcagcactgg gtcctatccg agaactaaaa ccagaacagg tgaagaatat gtctgccagt
     2341 gagatgagaa atattcgatt atctgacttc actgaatcct tgaaaaaaat aaaacgcagc
     2401 gtcagccctc aaactttaga agcgtacata cgttggaaca aggactttgg agataccact
     2461 gtttaagggc ccggtacctt aattaattaa ggtaccaggt aagtgtaccc aattcgccct
     2521 atagtgagtc gtattacaat tcactcgatc gcccttccca acagttgcgc agcctgaatg
     2581 gcgaatggag atccaatttt taagtgtata atgtgttaaa ctactgattc taattgtttg
     2641 tgtattttag attcacagtc ccaaggctca tttcaggccc ctcagtcctc acagtctgtt
     2701 catgatcata atcagccata ccacatttgt agaggtttta cttgctttaa aaaacctccc
     2761 acacctcccc ctgaacctga aacataaaat gaatgcaatt gttgttgtta acttgtttat
     2821 tgcagcttat aatggttaca aataaagcaa tagcatcaca aatttcacaa ataaagcatt
     2881 tttttcactg cattctagtt gtggtttgtc caaactcatc aatgtatctt aacgcgtaaa
     2941 ttgtaagcgt taatattttg ttaaaattcg cgttaaattt ttgttaaatc agctcatttt
     3001 ttaaccaata ggccgaaatc ggcaaaatcc cttataaatc aaaagaatag accgagatag
     3061 ggttgagtgt tgttccagtt tggaacaaga gtccactatt aaagaacgtg gactccaacg
     3121 tcaaagggcg aaaaaccgtc tatcagggcg atggcccact acgtgaacca tcaccctaat
     3181 caagtttttt ggggtcgagg tgccgtaaag cactaaatcg gaaccctaaa gggagccccc
     3241 gatttagagc ttgacgggga aagccggcga acgtggcgag aaaggaaggg aagaaagcga
     3301 aaggagcggg cgctagggcg ctggcaagtg tagcggtcac gctgcgcgta accaccacac
     3361 ccgccgcgct taatgcgccg ctacagggcg cgtcaggtgg cacttttcgg ggaaatgtgc
     3421 gcggaacccc tatttgttta tttttctaaa tacattcaaa tatgtatccg ctcatgagac
     3481 aataaccctg ataaatgctt caataatatt gaaaaaggaa gaatcctgag gcggaaagaa
     3541 ccagctgtgg aatgtgtgtc agttagggtg tggaaagtcc ccaggctccc cagcaggcag
     3601 aagtatgcaa agcatgcatc tcaattagtc agcaaccagg tgtggaaagt ccccaggctc
     3661 cccagcaggc agaagtatgc aaagcatgca tctcaattag tcagcaacca tagtcccgcc
     3721 cctaactccg cccatcccgc ccctaactcc gcccagttcc gcccattctc cgccccatgg
     3781 ctgactaatt ttttttattt atgcagaggc cgaggccgcc tcggcctctg agctattcca
     3841 gaagtagtga ggaggctttt ttggaggcct aggcttttgc aaagatcgat caagagacag
     3901 gatgaggatc gtttcgcatg attgaacaag atggattgca cgcaggttct ccggccgctt
     3961 gggtggagag gctattcggc tatgactggg cacaacagac aatcggctgc tctgatgccg
     4021 ccgtgttccg gctgtcagcg caggggcgcc cggttctttt tgtcaagacc gacctgtccg
     4081 gtgccctgaa tgaactgcaa gacgaggcag cgcggctatc gtggctggcc acgacgggcg
     4141 ttccttgcgc agctgtgctc gacgttgtca ctgaagcggg aagggactgg ctgctattgg
     4201 gcgaagtgcc ggggcaggat ctcctgtcat ctcaccttgc tcctgccgag aaagtatcca
     4261 tcatggctga tgcaatgcgg cggctgcata cgcttgatcc ggctacctgc ccattcgacc
     4321 accaagcgaa acatcgcatc gagcgagcac gtactcggat ggaagccggt cttgtcgatc
     4381 aggatgatct ggacgaagaa catcaggggc tcgcgccagc cgaactgttc gccaggctca
     4441 aggcgagcat gcccgacggc gaggatctcg tcgtgaccca tggcgatgcc tgcttgccga
     4501 atatcatggt ggaaaatggc cgcttttctg gattcatcga ctgtggccgg ctgggtgtgg
     4561 cggaccgcta tcaggacata gcgttggcta cccgtgatat tgctgaagaa cttggcggcg
     4621 aatgggctga ccgcttcctc gtgctttacg gtatcgccgc tcccgattcg cagcgcatcg
     4681 ccttctatcg ccttcttgac gagttcttct gagcgggact ctggggttcg aaatgaccga
     4741 ccaagcgacg cccaacctgc catcacgaga tttcgattcc accgccgcct tctatgaaag
     4801 gttgggcttc ggaatcgttt tccgggacgc cggctggatg atcctccagc gcggggatct
     4861 catgctggag ttcttcgccc accctagggg gaggctaact gaaacacgga aggagacaat
     4921 accggaagga acccgcgcta tgacggcaat aaaaagacag aataaaacgc acggtgttgg
     4981 gtcgtttgtt cataaacgcg gggttcggtc ccagggctgg cactctgtcg ataccccacc
     5041 gagaccccat tggggccaat acgcccgcgt ttcttccttt tccccacccc accccccaag
     5101 ttcgggtgaa ggcccagggc tcgcagccaa cgtcggggcg gcaggccctg ccatagcctc
     5161 aggttactca tatatacttt agattgattt aaaacttcat ttttaattta aaaggatcta
     5221 ggtgaagatc ctttttgata atctcatgac caaaatccct taacgtgagt tttcgttcca
     5281 ctgagcgtca gaccccgtag aaaagatcaa aggatcttct tgagatcctt tttttctgcg
     5341 cgtaatctgc tgcttgcaaa caaaaaaacc accgctacca gcggtggttt gtttgccgga
     5401 tcaagagcta ccaactcttt ttccgaaggt aactggcttc agcagagcgc agataccaaa
     5461 tactgttctt ctagtgtagc cgtagttagg ccaccacttc aagaactctg tagcaccgcc
     5521 tacatacctc gctctgctaa tcctgttacc agtggctgct gccagtggcg ataagtcgtg
     5581 tcttaccggg ttggactcaa gacgatagtt accggataag gcgcagcggt cgggctgaac
     5641 ggggggttcg tgcacacagc ccagcttgga gcgaacgacc tacaccgaac tgagatacct
     5701 acagcgtgag ctatgagaaa gcgccacgct tcccgaaggg agaaaggcgg acaggtatcc
     5761 ggtaagcggc agggtcggaa caggagagcg cacgagggag cttccagggg gaaacgcctg
     5821 gtatctttat agtcctgtcg ggtttcgcca cctctgactt gagcgtcgat ttttgtgatg
     5881 ctcgtcaggg gggcggagcc tatggaaaaa cgccagcaac gcggcctttt tacggttcct
     5941 ggccttttgc tggccttttg ctcacatgtt ctttcctgcg ttatcccctg attctgtgga
     6001 taaccgtatt accgccatgc attagttatt aatagtaatc aattacgggg tcattagttc
     6061 atagcccata tatggagttc cg
//
