LOCUS       sequence-425781-        4851 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Lipid droplet and ER localization of 1xHp of Spastin fused to 
            mApple.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 4851)
  AUTHORS   Li X, Gamuyao R, Wu ML, Cho WJ, King SV, Petersen RA, Stabley DR, 
            Lindow C, Climer LK, Shirinifard A, Ferrara F, Throm RE, Robinson 
            CG, Zhou Y, Carisey AF, Tebo AG, Chang CL
  TITLE     A fluorogenic complementation tool kit for interrogating lipid 
            droplet-organelle interaction.
  JOURNAL   J Cell Biol. 2024 Sep 2;223(9):e202311126. doi: 
            10.1083/jcb.202311126. Epub 2024 Jul 1.
  PUBMED    38949658
REFERENCE   2  (bases 1 to 4851)
  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: "J Cell 
            Biol."; date: "2024-09-2"; volume: "223(9):e202311126. doi"; pages: 
            " 10.1083/jcb.202311126. Epub 2024 Jul 1"
COMMENT     Sequence Label: mApple-1xHp
FEATURES             Location/Qualifiers
     source          1..4851
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     rep_origin      252..840
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     741..760
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     enhancer        1015..1318
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
     promoter        1319..1522
                     /label=CMV promoter
                     /note="human cytomegalovirus (CMV) immediate early 
                     promoter"
     primer_bind     1473..1493
                     /label=CMV-F
                     /note="Human CMV immediate early promoter, forward primer"
     regulatory      1561..1570
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     CDS             1567..2274
                     /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     1980..1999
                     /label=mCherry-F
                     /note="mCherry, forward primer"
     polyA_signal    2593..2714
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(2630..2649)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     2684..2703
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     rep_origin      complement(2721..3176)
                     /direction=LEFT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(2858..2879)
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     3070..3089
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     promoter        3203..3307
                     /gene="bla"
                     /label=AmpR promoter
     promoter        3309..3666
                     /label=SV40 promoter
                     /note="SV40 enhancer and early promoter"
     enhancer        3309..3500
                     /label=SV40 enhancer
                     /note="enhancer for the SV40 early promoter (Herr, 1993)"
     primer_bind     complement(3332..3352)
                     /label=pBABE 3'
                     /note="SV40 enhancer, reverse primer for pBABE vectors"
     rep_origin      3517..3652
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     CDS             3701..4495
                     /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             3701..4495
                     /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(3755..3774)
                     /label=Neo-R
                     /note="Neomycin resistance gene, reverse primer"
     primer_bind     4365..4384
                     /label=Neo-F
                     /note="Neomycin resistance gene, forward primer"
     primer_bind     complement(4683..4702)
                     /label=TK-pA-R
                     /note="Thymidine kinase polyA, reverse primer"
     polyA_signal    4727..4774
                     /label=HSV TK poly(A) signal
                     /note="herpes simplex virus thymidine kinase 
                     polyadenylation signal (Cole and Stacy, 1985)"
ORIGIN
        1 gtttcttcct tttccccacc ccacccccca agttcgggtg aaggcccagg gctcgcagcc
       61 aacgtcgggg cggcaggccc tgccatagcc tcaggttact catatatact ttagattgat
      121 ttaaaacttc atttttaatt taaaaggatc taggtgaaga tcctttttga taatctcatg
      181 accaaaatcc cttaacgtga gttttcgttc cactgagcgt cagaccccgt agaaaagatc
      241 aaaggatctt cttgagatcc tttttttctg cgcgtaatct gctgcttgca aacaaaaaaa
      301 ccaccgctac cagcggtggt ttgtttgccg gatcaagagc taccaactct ttttccgaag
      361 gtaactggct tcagcagagc gcagatacca aatactgttc ttctagtgta gccgtagtta
      421 ggccaccact tcaagaactc tgtagcaccg cctacatacc tcgctctgct aatcctgtta
      481 ccagtggctg ctgccagtgg cgataagtcg tgtcttaccg ggttggactc aagacgatag
      541 ttaccggata aggcgcagcg gtcgggctga acggggggtt cgtgcacaca gcccagcttg
      601 gagcgaacga cctacaccga actgagatac ctacagcgtg agctatgaga aagcgccacg
      661 cttcccgaag ggagaaaggc ggacaggtat ccggtaagcg gcagggtcgg aacaggagag
      721 cgcacgaggg agcttccagg gggaaacgcc tggtatcttt atagtcctgt cgggtttcgc
      781 cacctctgac ttgagcgtcg atttttgtga tgctcgtcag gggggcggag cctatggaaa
      841 aacgccagca acgcggcctt tttacggttc ctggcctttt gctggccttt tgctcacatg
      901 ttctttcctg cgttatcccc tgattctgtg gataaccgta ttaccgccat gcattagtta
      961 ttaatagtaa tcaattacgg ggtcattagt tcatagccca tatatggagt tccgcgttac
     1021 ataacttacg gtaaatggcc cgcctggctg accgcccaac gacccccgcc cattgacgtc
     1081 aataatgacg tatgttccca tagtaacgcc aatagggact ttccattgac gtcaatgggt
     1141 ggagtattta cggtaaactg cccacttggc agtacatcaa gtgtatcata tgccaagtac
     1201 gccccctatt gacgtcaatg acggtaaatg gcccgcctgg cattatgccc agtacatgac
     1261 cttatgggac tttcctactt ggcagtacat ctacgtatta gtcatcgcta ttaccatggt
     1321 gatgcggttt tggcagtaca tcaatgggcg tggatagcgg tttgactcac ggggatttcc
     1381 aagtctccac cccattgacg tcaatgggag tttgttttgg caccaaaatc aacgggactt
     1441 tccaaaatgt cgtaacaact ccgccccatt gacgcaaatg ggcggtaggc gtgtacggtg
     1501 ggaggtctat ataagcagag ctggtttagt gaaccgtcag atccgctagc gctaccggtc
     1561 gccaccatgg tgagcaaggg cgaggagaat aacatggcca tcatcaagga gttcatgcgc
     1621 ttcaaggtgc acatggaggg ctccgtgaac ggccacgagt tcgagatcga gggcgagggc
     1681 gagggccgcc cctacgaggc ctttcagacc gctaagctga aggtgaccaa gggtggcccc
     1741 ctgcccttcg cctgggacat cctgtcccct cagttcatgt acggctccaa ggtctacatt
     1801 aagcacccag ccgacatccc cgactacttc aagctgtcct tccccgaggg cttcaggtgg
     1861 gagcgcgtga tgaacttcga ggacggcggc attattcacg ttaaccagga ctcctccctg
     1921 caggacggcg tgttcatcta caaggtgaag ctgcgcggca ccaacttccc ctccgacggc
     1981 cccgtaatgc agaagaagac catgggctgg gaggcctccg aggagcggat gtaccccgag
     2041 gacggcgccc tgaagagcga gatcaagaag aggctgaagc tgaaggacgg cggccactac
     2101 gccgccgagg tcaagaccac ctacaaggcc aagaagcccg tgcagctgcc cggcgcctac
     2161 atcgtcgaca tcaagttgga catcgtgtcc cacaacgagg actacaccat cgtggaacag
     2221 tacgaacgcg ccgagggccg ccactccacc ggcggcatgg acgagctgta caagtccgga
     2281 ctcagatctc gagctcaagc ttcgaattct gagtcgccgc ataagcggaa cctgtactat
     2341 ttctcctacc cgctgtttgt aggcttcgcg ctgctgcgtt tggtcgcctt ccacctgggg
     2401 ctcctcttcg tgtggctctg ccagcgcttc tcccgcgccc tcatggcagc caagaggagc
     2461 taaggatcca ccggatctag ataactgatc ataatcagcc ataccacatt tgtagaggtt
     2521 ttacttgctt taaaaaacct cccacacctc cccctgaacc tgaaacataa aatgaatgca
     2581 attgttgttg ttaacttgtt tattgcagct tataatggtt acaaataaag caatagcatc
     2641 acaaatttca caaataaagc atttttttca ctgcattcta gttgtggttt gtccaaactc
     2701 atcaatgtat cttaacgcgt aaattgtaag cgttaatatt ttgttaaaat tcgcgttaaa
     2761 tttttgttaa atcagctcat tttttaacca ataggccgaa atcggcaaaa tcccttataa
     2821 atcaaaagaa tagaccgaga tagggttgag tgttgttcca gtttggaaca agagtccact
     2881 attaaagaac gtggactcca acgtcaaagg gcgaaaaacc gtctatcagg gcgatggccc
     2941 actacgtgaa ccatcaccct aatcaagttt tttggggtcg aggtgccgta aagcactaaa
     3001 tcggaaccct aaagggagcc cccgatttag agcttgacgg ggaaagccgg cgaacgtggc
     3061 gagaaaggaa gggaagaaag cgaaaggagc gggcgctagg gcgctggcaa gtgtagcggt
     3121 cacgctgcgc gtaaccacca cacccgccgc gcttaatgcg ccgctacagg gcgcgtcagg
     3181 tggcactttt cggggaaatg tgcgcggaac ccctatttgt ttatttttct aaatacattc
     3241 aaatatgtat ccgctcatga gacaataacc ctgataaatg cttcaataat attgaaaaag
     3301 gaagagtcct gaggcggaaa gaaccagctg tggaatgtgt gtcagttagg gtgtggaaag
     3361 tccccaggct ccccagcagg cagaagtatg caaagcatgc atctcaatta gtcagcaacc
     3421 aggtgtggaa agtccccagg ctccccagca ggcagaagta tgcaaagcat gcatctcaat
     3481 tagtcagcaa ccatagtccc gcccctaact ccgcccatcc cgcccctaac tccgcccagt
     3541 tccgcccatt ctccgcccca tggctgacta atttttttta tttatgcaga ggccgaggcc
     3601 gcctcggcct ctgagctatt ccagaagtag tgaggaggct tttttggagg cctaggcttt
     3661 tgcaaagatc gatcaagaga caggatgagg atcgtttcgc atgattgaac aagatggatt
     3721 gcacgcaggt tctccggccg cttgggtgga gaggctattc ggctatgact gggcacaaca
     3781 gacaatcggc tgctctgatg ccgccgtgtt ccggctgtca gcgcaggggc gcccggttct
     3841 ttttgtcaag accgacctgt ccggtgccct gaatgaactg caagacgagg cagcgcggct
     3901 atcgtggctg gccacgacgg gcgttccttg cgcagctgtg ctcgacgttg tcactgaagc
     3961 gggaagggac tggctgctat tgggcgaagt gccggggcag gatctcctgt catctcacct
     4021 tgctcctgcc gagaaagtat ccatcatggc tgatgcaatg cggcggctgc atacgcttga
     4081 tccggctacc tgcccattcg accaccaagc gaaacatcgc atcgagcgag cacgtactcg
     4141 gatggaagcc ggtcttgtcg atcaggatga tctggacgaa gagcatcagg ggctcgcgcc
     4201 agccgaactg ttcgccaggc tcaaggcgag catgcccgac ggcgaggatc tcgtcgtgac
     4261 ccatggcgat gcctgcttgc cgaatatcat ggtggaaaat ggccgctttt ctggattcat
     4321 cgactgtggc cggctgggtg tggcggaccg ctatcaggac atagcgttgg ctacccgtga
     4381 tattgctgaa gagcttggcg gcgaatgggc tgaccgcttc ctcgtgcttt acggtatcgc
     4441 cgctcccgat tcgcagcgca tcgccttcta tcgccttctt gacgagttct tctgagcggg
     4501 actctggggt tcgaaatgac cgaccaagcg acgcccaacc tgccatcacg agatttcgat
     4561 tccaccgccg ccttctatga aaggttgggc ttcggaatcg ttttccggga cgccggctgg
     4621 atgatcctcc agcgcgggga tctcatgctg gagttcttcg cccaccctag ggggaggcta
     4681 actgaaacac ggaaggagac aataccggaa ggaacccgcg ctatgacggc aataaaaaga
     4741 cagaataaaa cgcacggtgt tgggtcgttt gttcataaac gcggggttcg gtcccagggc
     4801 tggcactctg tcgatacccc accgagaccc cattggggcc aatacgcccg c
//
