LOCUS       sequence-258498-        6540 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  M1 SpastinE442Q expression.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 6540)
  AUTHORS   Chang CL, Weigel AV, Ioannou MS, Pasolli HA, Xu CS, Peale DR, 
            Shtengel G, Freeman M, Hess HF, Blackstone C, Lippincott-Schwartz J
  TITLE     Spastin tethers lipid droplets to peroxisomes and directs fatty acid
            trafficking through ESCRT-III.
  JOURNAL   J Cell Biol. 2019 Aug 5;218(8):2583-2599. doi: 
            10.1083/jcb.201902061. Epub 2019 Jun 21.
  PUBMED    31227594
REFERENCE   2  (bases 1 to 6540)
  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: 
            "10.1083/jcb.201902061"; journalName: "J Cell Biol"; date: 
            "2019-08-5- 5"; volume: "218"; issue: "8"; pages: "2583-2599"
COMMENT     Sequence Label: mApple-M1 SpastinE442Q
FEATURES             Location/Qualifiers
     source          1..6540
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     enhancer        16..319
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
     promoter        320..523
                     /label=CMV promoter
                     /note="human cytomegalovirus (CMV) immediate early 
                     promoter"
     primer_bind     474..494
                     /label=CMV-F
                     /note="Human CMV immediate early promoter, forward primer"
     regulatory      562..571
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     CDS             568..1275
                     /codon_start=1
                     /product="photostable monomeric derivative of DsRed (Shaner
                     et al., 2008)"
                     /label=mApple
                     /note="mammalian codon-optimized"
                     /translation="MVSKGEENNMAIIKEFMRFKVHMEGSVNGHEFEIEGEGEGRPYEA
                     FQTAKLKVTKGGPLPFAWDILSPQFMYGSKVYIKHPADIPDYFKLSFPEGFRWERVMNF
                     EDGGIIHVNQDSSLQDGVFIYKVKLRGTNFPSDGPVMQKKTMGWEASEERMYPEDGALK
                     SEIKKRLKLKDGGHYAAEVKTTYKAKKPVQLPGAYIVDIKLDIVSHNEDYTIVEQYERA
                     EGRHSTGGMDELYK"
     primer_bind     981..1000
                     /label=mCherry-F
                     /note="mCherry, forward primer"
     polyA_signal    3283..3404
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(3320..3339)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     3374..3393
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     rep_origin      complement(3411..3866)
                     /direction=LEFT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(3548..3569)
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     3760..3779
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     promoter        3893..3997
                     /gene="bla"
                     /label=AmpR promoter
     promoter        3999..4356
                     /label=SV40 promoter
                     /note="SV40 enhancer and early promoter"
     enhancer        3999..4190
                     /label=SV40 enhancer
                     /note="enhancer for the SV40 early promoter (Herr, 1993)"
     primer_bind     complement(4022..4042)
                     /label=pBABE 3'
                     /note="SV40 enhancer, reverse primer for pBABE vectors"
     rep_origin      4207..4342
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     CDS             4391..5185
                     /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             4391..5185
                     /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(4445..4464)
                     /label=Neo-R
                     /note="Neomycin resistance gene, reverse primer"
     primer_bind     5055..5074
                     /label=Neo-F
                     /note="Neomycin resistance gene, forward primer"
     primer_bind     complement(5373..5392)
                     /label=TK-pA-R
                     /note="Thymidine kinase polyA, reverse primer"
     polyA_signal    5417..5464
                     /label=HSV TK poly(A) signal
                     /note="herpes simplex virus thymidine kinase 
                     polyadenylation signal (Cole and Stacy, 1985)"
     rep_origin      5793..6381
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     6282..6301
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
ORIGIN
        1 atatatggag ttccgcgtta cataacttac ggtaaatggc ccgcctggct gaccgcccaa
       61 cgacccccgc ccattgacgt caataatgac gtatgttccc atagtaacgc caatagggac
      121 tttccattga cgtcaatggg tggagtattt acggtaaact gcccacttgg cagtacatca
      181 agtgtatcat atgccaagta cgccccctat tgacgtcaat gacggtaaat ggcccgcctg
      241 gcattatgcc cagtacatga ccttatggga ctttcctact tggcagtaca tctacgtatt
      301 agtcatcgct attaccatgg tgatgcggtt ttggcagtac atcaatgggc gtggatagcg
      361 gtttgactca cggggatttc caagtctcca ccccattgac gtcaatggga gtttgttttg
      421 gcaccaaaat caacgggact ttccaaaatg tcgtaacaac tccgccccat tgacgcaaat
      481 gggcggtagg cgtgtacggt gggaggtcta tataagcaga gctggtttag tgaaccgtca
      541 gatccgctag cgctaccggt cgccaccatg gtgagcaagg gcgaggagaa taacatggcc
      601 atcatcaagg agttcatgcg cttcaaggtg cacatggagg gctccgtgaa cggccacgag
      661 ttcgagatcg agggcgaggg cgagggccgc ccctacgagg cctttcagac cgctaagctg
      721 aaggtgacca agggtggccc cctgcccttc gcctgggaca tcctgtcccc tcagttcatg
      781 tacggctcca aggtctacat taagcaccca gccgacatcc ccgactactt caagctgtcc
      841 ttccccgagg gcttcaggtg ggagcgcgtg atgaacttcg aggacggcgg cattattcac
      901 gttaaccagg actcctccct gcaggacggc gtgttcatct acaaggtgaa gctgcgcggc
      961 accaacttcc cctccgacgg ccccgtaatg cagaagaaga ccatgggctg ggaggcctcc
     1021 gaggagcgga tgtaccccga ggacggcgcc ctgaagagcg agatcaagaa gaggctgaag
     1081 ctgaaggacg gcggccacta cgccgccgag gtcaagacca cctacaaggc caagaagccc
     1141 gtgcagctgc ccggcgccta catcgtcgac atcaagttgg acatcgtgtc ccacaacgag
     1201 gactacacca tcgtggaaca gtacgaacgc gccgagggcc gccactccac cggcggcatg
     1261 gacgagctgt acaagtccgg actcagatct cgagctcaag cttcgaattc tccgggtgga
     1321 cgagggaaga agaaaggctc cggcggcgcc agcaacccgg tgcctcccag gcctccgccc
     1381 ccttgcctgg cccccgcccc tcccgccgcc gggccggccc ctccgcccga gtcgccgcat
     1441 aagcggaacc tgtactattt ctcctacccg ctgtttgtag gcttcgcgct gctgcgtttg
     1501 gtcgccttcc acctggggct cctcttcgtg tggctctgcc agcgcttctc ccgcgccctc
     1561 gcggcagcca agaggagctc cggggccgcg ccagcacctg cctcggcctc ggccccggcg
     1621 ccggtgccgg gcggcgaggc cgagcgcgtc cgagtcttcc acaaacaggc cttcgagtac
     1681 atctccattg ccctgcgcat cgatgaggat gagaaagcag gacagaagga gcaagctgtg
     1741 gaatggtata agaaaggtat tgaagaactg gaaaaaggaa tagctgttat agttacagga
     1801 caaggtgaac agtgtgaaag agctagacgc cttcaagcta aaatgatgac taatttggtt
     1861 atggccaagg accgcttaca acttctagag aagatgcaac cagttttgcc attttccaag
     1921 tcacaaacgg acgtctataa tgacagtact aacttggcat gccgcaatgg acatctccag
     1981 tcagaaagtg gagctgttcc aaaaagaaaa gaccccttaa cacacactag taattcactg
     2041 cctcgttcaa aaacagttat gaaaactgga tctgcaggcc tttcaggcca ccatagagca
     2101 cctagttaca gtggtttatc catggtttct ggagtgaaac agggatctgg tcctgctcct
     2161 accactcata agggtactcc gaaaacaaat aggacaaata aaccttctac ccctacaact
     2221 gctactcgta agaaaaaaga cttgaagaat tttaggaatg tggacagcaa ccttgctaac
     2281 cttataatga atgaaattgt ggacaatgga acagctgtta aatttgatga tatagctggt
     2341 caagacttgg caaaacaagc attgcaagaa attgttattc ttccttctct gaggcctgag
     2401 ttgttcacag ggcttagagc tcctgccaga gggctgttac tctttggtcc acctgggaat
     2461 gggaagacaa tgctggctaa agcagtagct gcagaatcga atgcaacctt ctttaatata
     2521 agtgctgcaa gtttaacttc aaaatacgtg ggagaaggag agaaattggt gagggctctt
     2581 tttgctgtgg ctcgagaact tcaaccttct ataattttta tagatcaagt tgatagcctt
     2641 ttgtgtgaaa gaagagaagg ggagcacgat gctagtagac gcctaaaaac tgaatttcta
     2701 atagaatttg atggtgtaca gtctgctgga gatgacagag tacttgtaat gggtgcaact
     2761 aataggccac aagagcttga tgaggctgtt ctcaggcgtt tcatcaaacg ggtatatgtg
     2821 tctttaccaa atgaggagac aagactactt ttgcttaaaa atctgttatg taaacaagga
     2881 agtccattga cccaaaaaga actagcacaa cttgctagaa tgactgatgg atactcagga
     2941 agtgacctaa cagctttggc aaaagatgca gcactgggtc ctatccgaga actaaaacca
     3001 gaacaggtga agaatatgtc tgccagtgag atgagaaata ttcgattatc tgacttcact
     3061 gaatccttga aaaaaataaa acgcagcgtc agccctcaaa ctttagaagc gtacatacgt
     3121 tggaacaagg actttggaga taccactgtt taaggatcca ccggatctag ataactgatc
     3181 ataatcagcc ataccacatt tgtagaggtt ttacttgctt taaaaaacct cccacacctc
     3241 cccctgaacc tgaaacataa aatgaatgca attgttgttg ttaacttgtt tattgcagct
     3301 tataatggtt acaaataaag caatagcatc acaaatttca caaataaagc atttttttca
     3361 ctgcattcta gttgtggttt gtccaaactc atcaatgtat cttaacgcgt aaattgtaag
     3421 cgttaatatt ttgttaaaat tcgcgttaaa tttttgttaa atcagctcat tttttaacca
     3481 ataggccgaa atcggcaaaa tcccttataa atcaaaagaa tagaccgaga tagggttgag
     3541 tgttgttcca gtttggaaca agagtccact attaaagaac gtggactcca acgtcaaagg
     3601 gcgaaaaacc gtctatcagg gcgatggccc actacgtgaa ccatcaccct aatcaagttt
     3661 tttggggtcg aggtgccgta aagcactaaa tcggaaccct aaagggagcc cccgatttag
     3721 agcttgacgg ggaaagccgg cgaacgtggc gagaaaggaa gggaagaaag cgaaaggagc
     3781 gggcgctagg gcgctggcaa gtgtagcggt cacgctgcgc gtaaccacca cacccgccgc
     3841 gcttaatgcg ccgctacagg gcgcgtcagg tggcactttt cggggaaatg tgcgcggaac
     3901 ccctatttgt ttatttttct aaatacattc aaatatgtat ccgctcatga gacaataacc
     3961 ctgataaatg cttcaataat attgaaaaag gaagagtcct gaggcggaaa gaaccagctg
     4021 tggaatgtgt gtcagttagg gtgtggaaag tccccaggct ccccagcagg cagaagtatg
     4081 caaagcatgc atctcaatta gtcagcaacc aggtgtggaa agtccccagg ctccccagca
     4141 ggcagaagta tgcaaagcat gcatctcaat tagtcagcaa ccatagtccc gcccctaact
     4201 ccgcccatcc cgcccctaac tccgcccagt tccgcccatt ctccgcccca tggctgacta
     4261 atttttttta tttatgcaga ggccgaggcc gcctcggcct ctgagctatt ccagaagtag
     4321 tgaggaggct tttttggagg cctaggcttt tgcaaagatc gatcaagaga caggatgagg
     4381 atcgtttcgc atgattgaac aagatggatt gcacgcaggt tctccggccg cttgggtgga
     4441 gaggctattc ggctatgact gggcacaaca gacaatcggc tgctctgatg ccgccgtgtt
     4501 ccggctgtca gcgcaggggc gcccggttct ttttgtcaag accgacctgt ccggtgccct
     4561 gaatgaactg caagacgagg cagcgcggct atcgtggctg gccacgacgg gcgttccttg
     4621 cgcagctgtg ctcgacgttg tcactgaagc gggaagggac tggctgctat tgggcgaagt
     4681 gccggggcag gatctcctgt catctcacct tgctcctgcc gagaaagtat ccatcatggc
     4741 tgatgcaatg cggcggctgc atacgcttga tccggctacc tgcccattcg accaccaagc
     4801 gaaacatcgc atcgagcgag cacgtactcg gatggaagcc ggtcttgtcg atcaggatga
     4861 tctggacgaa gagcatcagg ggctcgcgcc agccgaactg ttcgccaggc tcaaggcgag
     4921 catgcccgac ggcgaggatc tcgtcgtgac ccatggcgat gcctgcttgc cgaatatcat
     4981 ggtggaaaat ggccgctttt ctggattcat cgactgtggc cggctgggtg tggcggaccg
     5041 ctatcaggac atagcgttgg ctacccgtga tattgctgaa gagcttggcg gcgaatgggc
     5101 tgaccgcttc ctcgtgcttt acggtatcgc cgctcccgat tcgcagcgca tcgccttcta
     5161 tcgccttctt gacgagttct tctgagcggg actctggggt tcgaaatgac cgaccaagcg
     5221 acgcccaacc tgccatcacg agatttcgat tccaccgccg ccttctatga aaggttgggc
     5281 ttcggaatcg ttttccggga cgccggctgg atgatcctcc agcgcgggga tctcatgctg
     5341 gagttcttcg cccaccctag ggggaggcta actgaaacac ggaaggagac aataccggaa
     5401 ggaacccgcg ctatgacggc aataaaaaga cagaataaaa cgcacggtgt tgggtcgttt
     5461 gttcataaac gcggggttcg gtcccagggc tggcactctg tcgatacccc accgagaccc
     5521 cattggggcc aatacgcccg cgtttcttcc ttttccccac cccacccccc aagttcgggt
     5581 gaaggcccag ggctcgcagc caacgtcggg gcggcaggcc ctgccatagc ctcaggttac
     5641 tcatatatac tttagattga tttaaaactt catttttaat ttaaaaggat ctaggtgaag
     5701 atcctttttg ataatctcat gaccaaaatc ccttaacgtg agttttcgtt ccactgagcg
     5761 tcagaccccg tagaaaagat caaaggatct tcttgagatc ctttttttct gcgcgtaatc
     5821 tgctgcttgc aaacaaaaaa accaccgcta ccagcggtgg tttgtttgcc ggatcaagag
     5881 ctaccaactc tttttccgaa ggtaactggc ttcagcagag cgcagatacc aaatactgtt
     5941 cttctagtgt agccgtagtt aggccaccac ttcaagaact ctgtagcacc gcctacatac
     6001 ctcgctctgc taatcctgtt accagtggct gctgccagtg gcgataagtc gtgtcttacc
     6061 gggttggact caagacgata gttaccggat aaggcgcagc ggtcgggctg aacggggggt
     6121 tcgtgcacac agcccagctt ggagcgaacg acctacaccg aactgagata cctacagcgt
     6181 gagctatgag aaagcgccac gcttcccgaa gggagaaagg cggacaggta tccggtaagc
     6241 ggcagggtcg gaacaggaga gcgcacgagg gagcttccag ggggaaacgc ctggtatctt
     6301 tatagtcctg tcgggtttcg ccacctctga cttgagcgtc gatttttgtg atgctcgtca
     6361 ggggggcgga gcctatggaa aaacgccagc aacgcggcct ttttacggtt cctggccttt
     6421 tgctggcctt ttgctcacat gttctttcct gcgttatccc ctgattctgt ggataaccgt
     6481 attaccgcca tgcattagtt attaatagta atcaattacg gggtcattag ttcatagccc
//
