LOCUS       sequence-425786-        5646 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Lipid droplet localization of 6xHp of Spastin fused to mApple.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 5646)
  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 5646)
  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-6xHp
FEATURES             Location/Qualifiers
     source          1..5646
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     promoter        40..243
                     /label=CMV promoter
                     /note="human cytomegalovirus (CMV) immediate early 
                     promoter"
     primer_bind     194..214
                     /label=CMV-F
                     /note="Human CMV immediate early promoter, forward primer"
     regulatory      282..291
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     CDS             288..995
                     /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     701..720
                     /label=mCherry-F
                     /note="mCherry, forward primer"
     polyA_signal    2109..2230
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(2146..2165)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     2200..2219
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     rep_origin      complement(2237..2692)
                     /direction=LEFT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(2374..2395)
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     2586..2605
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     promoter        2719..2823
                     /gene="bla"
                     /label=AmpR promoter
     promoter        2825..3182
                     /label=SV40 promoter
                     /note="SV40 enhancer and early promoter"
     enhancer        2825..3016
                     /label=SV40 enhancer
                     /note="enhancer for the SV40 early promoter (Herr, 1993)"
     primer_bind     complement(2848..2868)
                     /label=pBABE 3'
                     /note="SV40 enhancer, reverse primer for pBABE vectors"
     rep_origin      3033..3168
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     CDS             3217..4011
                     /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             3217..4011
                     /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(3271..3290)
                     /label=Neo-R
                     /note="Neomycin resistance gene, reverse primer"
     primer_bind     3881..3900
                     /label=Neo-F
                     /note="Neomycin resistance gene, forward primer"
     primer_bind     complement(4199..4218)
                     /label=TK-pA-R
                     /note="Thymidine kinase polyA, reverse primer"
     polyA_signal    4243..4290
                     /label=HSV TK poly(A) signal
                     /note="herpes simplex virus thymidine kinase 
                     polyadenylation signal (Cole and Stacy, 1985)"
     rep_origin      4619..5207
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     5108..5127
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     enhancer        join(5382..5646,1..39)
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
ORIGIN
        1 tggcagtaca tctacgtatt agtcatcgct attaccatgg tgatgcggtt ttggcagtac
       61 atcaatgggc gtggatagcg gtttgactca cggggatttc caagtctcca ccccattgac
      121 gtcaatggga gtttgttttg gcaccaaaat caacgggact ttccaaaatg tcgtaacaac
      181 tccgccccat tgacgcaaat gggcggtagg cgtgtacggt gggaggtcta tataagcaga
      241 gctggtttag tgaaccgtca gatccgctag cgctaccggt cgccaccatg gtgagcaagg
      301 gcgaggagaa taacatggcc atcatcaagg agttcatgcg cttcaaggtg cacatggagg
      361 gctccgtgaa cggccacgag ttcgagatcg agggcgaggg cgagggccgc ccctacgagg
      421 cctttcagac cgctaagctg aaggtgacca agggtggccc cctgcccttc gcctgggaca
      481 tcctgtcccc tcagttcatg tacggctcca aggtctacat taagcaccca gccgacatcc
      541 ccgactactt caagctgtcc ttccccgagg gcttcaggtg ggagcgcgtg atgaacttcg
      601 aggacggcgg cattattcac gttaaccagg actcctccct gcaggacggc gtgttcatct
      661 acaaggtgaa gctgcgcggc accaacttcc cctccgacgg ccccgtaatg cagaagaaga
      721 ccatgggctg ggaggcctcc gaggagcgga tgtaccccga ggacggcgcc ctgaagagcg
      781 agatcaagaa gaggctgaag ctgaaggacg gcggccacta cgccgccgag gtcaagacca
      841 cctacaaggc caagaagccc gtgcagctgc ccggcgccta catcgtcgac atcaagttgg
      901 acatcgtgtc ccacaacgag gactacacca tcgtggaaca gtacgaacgc gccgagggcc
      961 gccactccac cggcggcatg gacgagctgt acaagtccgg actcagatct cgagctcaag
     1021 catcgaattc cgaatccccg cacaaacgca acctgtacta cttctcttac cccctttttg
     1081 tcgggtttgc tctacttcga ttggttgctt ttcatcttgg attactgttt gtttggttgt
     1141 gtcaacgctt ttctagagcg ctgatggctg cgaaacgttc ggctaatagt gaatcgcccc
     1201 ataagaggaa cttatattac ttcagctatc ccttgttcgt gggtttcgcg ctacttcggc
     1261 tggtcgcgtt tcatctaggt ttgttgtttg tctggttatg tcaacggttc tcgcgagcgt
     1321 taatggcagc aaagaggtcg gcaaatagcg aatcgcctca taaacgcaac ctctattact
     1381 tctcttaccc gttgttcgtt ggcttcgctc tccttcggct agttgcgttt catcttggtc
     1441 ttctgttcgt gtggctttgc caaagatttt cgcgagcttt aatggcggct aagaggagtg
     1501 ccaatagcga atccccgcat aagcggaact tatactactt ctcttaccct ctttttgtag
     1561 gctttgctct actccggcta gtggccttcc atctaggcct actattcgtc tggctctgcc
     1621 agcgcttctc gcgagccttg atggcagcta agcgttccgc aaatagcgaa tcacctcata
     1681 agcggaatct gtactatttc tcatatcctc tcttcgtagg gttcgcacta ttacgtcttg
     1741 tagcatttca tctgggatta ttattcgtct ggttgtgcca gcgattttca cgagccttaa
     1801 tggctgcgaa gagaagcgca aattctgaat ctcctcacaa gcggaacctc tattacttta
     1861 gttacccgct cttcgtcggt ttcgcgttac tcaggctggt tgcattccac ctcggcttgc
     1921 tgttcgtatg gctttgccag agattctccc gggcgctaat ggcagccaag cgctcctaag
     1981 gatccaccgg atctagataa ctgatcataa tcagccatac cacatttgta gaggttttac
     2041 ttgctttaaa aaacctccca cacctccccc tgaacctgaa acataaaatg aatgcaattg
     2101 ttgttgttaa cttgtttatt gcagcttata atggttacaa ataaagcaat agcatcacaa
     2161 atttcacaaa taaagcattt ttttcactgc attctagttg tggtttgtcc aaactcatca
     2221 atgtatctta acgcgtaaat tgtaagcgtt aatattttgt taaaattcgc gttaaatttt
     2281 tgttaaatca gctcattttt taaccaatag gccgaaatcg gcaaaatccc ttataaatca
     2341 aaagaataga ccgagatagg gttgagtgtt gttccagttt ggaacaagag tccactatta
     2401 aagaacgtgg actccaacgt caaagggcga aaaaccgtct atcagggcga tggcccacta
     2461 cgtgaaccat caccctaatc aagttttttg gggtcgaggt gccgtaaagc actaaatcgg
     2521 aaccctaaag ggagcccccg atttagagct tgacggggaa agccggcgaa cgtggcgaga
     2581 aaggaaggga agaaagcgaa aggagcgggc gctagggcgc tggcaagtgt agcggtcacg
     2641 ctgcgcgtaa ccaccacacc cgccgcgctt aatgcgccgc tacagggcgc gtcaggtggc
     2701 acttttcggg gaaatgtgcg cggaacccct atttgtttat ttttctaaat acattcaaat
     2761 atgtatccgc tcatgagaca ataaccctga taaatgcttc aataatattg aaaaaggaag
     2821 agtcctgagg cggaaagaac cagctgtgga atgtgtgtca gttagggtgt ggaaagtccc
     2881 caggctcccc agcaggcaga agtatgcaaa gcatgcatct caattagtca gcaaccaggt
     2941 gtggaaagtc cccaggctcc ccagcaggca gaagtatgca aagcatgcat ctcaattagt
     3001 cagcaaccat agtcccgccc ctaactccgc ccatcccgcc cctaactccg cccagttccg
     3061 cccattctcc gccccatggc tgactaattt tttttattta tgcagaggcc gaggccgcct
     3121 cggcctctga gctattccag aagtagtgag gaggcttttt tggaggccta ggcttttgca
     3181 aagatcgatc aagagacagg atgaggatcg tttcgcatga ttgaacaaga tggattgcac
     3241 gcaggttctc cggccgcttg ggtggagagg ctattcggct atgactgggc acaacagaca
     3301 atcggctgct ctgatgccgc cgtgttccgg ctgtcagcgc aggggcgccc ggttcttttt
     3361 gtcaagaccg acctgtccgg tgccctgaat gaactgcaag acgaggcagc gcggctatcg
     3421 tggctggcca cgacgggcgt tccttgcgca gctgtgctcg acgttgtcac tgaagcggga
     3481 agggactggc tgctattggg cgaagtgccg gggcaggatc tcctgtcatc tcaccttgct
     3541 cctgccgaga aagtatccat catggctgat gcaatgcggc ggctgcatac gcttgatccg
     3601 gctacctgcc cattcgacca ccaagcgaaa catcgcatcg agcgagcacg tactcggatg
     3661 gaagccggtc ttgtcgatca ggatgatctg gacgaagagc atcaggggct cgcgccagcc
     3721 gaactgttcg ccaggctcaa ggcgagcatg cccgacggcg aggatctcgt cgtgacccat
     3781 ggcgatgcct gcttgccgaa tatcatggtg gaaaatggcc gcttttctgg attcatcgac
     3841 tgtggccggc tgggtgtggc ggaccgctat caggacatag cgttggctac ccgtgatatt
     3901 gctgaagagc ttggcggcga atgggctgac cgcttcctcg tgctttacgg tatcgccgct
     3961 cccgattcgc agcgcatcgc cttctatcgc cttcttgacg agttcttctg agcgggactc
     4021 tggggttcga aatgaccgac caagcgacgc ccaacctgcc atcacgagat ttcgattcca
     4081 ccgccgcctt ctatgaaagg ttgggcttcg gaatcgtttt ccgggacgcc ggctggatga
     4141 tcctccagcg cggggatctc atgctggagt tcttcgccca ccctaggggg aggctaactg
     4201 aaacacggaa ggagacaata ccggaaggaa cccgcgctat gacggcaata aaaagacaga
     4261 ataaaacgca cggtgttggg tcgtttgttc ataaacgcgg ggttcggtcc cagggctggc
     4321 actctgtcga taccccaccg agaccccatt ggggccaata cgcccgcgtt tcttcctttt
     4381 ccccacccca ccccccaagt tcgggtgaag gcccagggct cgcagccaac gtcggggcgg
     4441 caggccctgc catagcctca ggttactcat atatacttta gattgattta aaacttcatt
     4501 tttaatttaa aaggatctag gtgaagatcc tttttgataa tctcatgacc aaaatccctt
     4561 aacgtgagtt ttcgttccac tgagcgtcag accccgtaga aaagatcaaa ggatcttctt
     4621 gagatccttt ttttctgcgc gtaatctgct gcttgcaaac aaaaaaacca ccgctaccag
     4681 cggtggtttg tttgccggat caagagctac caactctttt tccgaaggta actggcttca
     4741 gcagagcgca gataccaaat actgttcttc tagtgtagcc gtagttaggc caccacttca
     4801 agaactctgt agcaccgcct acatacctcg ctctgctaat cctgttacca gtggctgctg
     4861 ccagtggcga taagtcgtgt cttaccgggt tggactcaag acgatagtta ccggataagg
     4921 cgcagcggtc gggctgaacg gggggttcgt gcacacagcc cagcttggag cgaacgacct
     4981 acaccgaact gagataccta cagcgtgagc tatgagaaag cgccacgctt cccgaaggga
     5041 gaaaggcgga caggtatccg gtaagcggca gggtcggaac aggagagcgc acgagggagc
     5101 ttccaggggg aaacgcctgg tatctttata gtcctgtcgg gtttcgccac ctctgacttg
     5161 agcgtcgatt tttgtgatgc tcgtcagggg ggcggagcct atggaaaaac gccagcaacg
     5221 cggccttttt acggttcctg gccttttgct ggccttttgc tcacatgttc tttcctgcgt
     5281 tatcccctga ttctgtggat aaccgtatta ccgccatgca ttagttatta atagtaatca
     5341 attacggggt cattagttca tagcccatat atggagttcc gcgttacata acttacggta
     5401 aatggcccgc ctggctgacc gcccaacgac ccccgcccat tgacgtcaat aatgacgtat
     5461 gttcccatag taacgccaat agggactttc cattgacgtc aatgggtgga gtatttacgg
     5521 taaactgccc acttggcagt acatcaagtg tatcatatgc caagtacgcc ccctattgac
     5581 gtcaatgacg gtaaatggcc cgcctggcat tatgcccagt acatgacctt atgggacttt
     5641 cctact
//
