LOCUS       sequence-419200-        4909 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  lipophagy reporter system based on the Keima-fluorophore.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 4909)
  AUTHORS   Chung J, Park J, Lai ZW, Lambert TJ, Richards RC, Zhang J, Walther 
            TC, Farese RV Jr
  TITLE     The Troyer syndrome protein spartin mediates selective autophagy of 
            lipid droplets.
  JOURNAL   Nat Cell Biol. 2023 Aug;25(8):1101-1110. doi: 
            10.1038/s41556-023-01178-w. Epub 2023 Jul 13.
  PUBMED    37443287
REFERENCE   2  (bases 1 to 4909)
  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: 
            "10.1038/s41556-023-01178-w"; journalName: "Nat Cell Biol"; date: 
            "2023-08"; volume: "25"; issue: "8"; pages: "1101-1110"
COMMENT     Sequence Label: pKeima-LiveDrop
FEATURES             Location/Qualifiers
     source          1..4909
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     enhancer        80..271
                     /label=SV40 enhancer
                     /note="enhancer for the SV40 early promoter (Herr, 1993)"
     primer_bind     228..248
                     /label=pBABE 3'
                     /note="SV40 enhancer, reverse primer for pBABE vectors"
     promoter        complement(273..377)
                     /gene="bla"
                     /label=AmpR promoter
     rep_origin      404..859
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(491..510)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     701..722
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     polyA_signal    866..987
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(877..896)
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     primer_bind     931..950
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     RBS             1335..1355
                     /note="efficient ribosome binding site from bacteriophage 
                     T7 gene 10 (Olins and Rangwala, 1989)"
     protein_bind    complement(1367..1391)
                     /gene="mutant version of attB"
                     /label=attB1
                     /bound_moiety="BP Clonase(TM)"
                     /note="recombination site for the Gateway(R) BP reaction"
     CDS             complement(join(1416..2075,2076..2078,2079..2081))
                     /codon_start=1
                     /product="humanized monomeric Keima-Red fluorescent 
                     protein"
                     /label=hmKeima-Red
                     /note="mammalian codon-optimized"
                     /translation="MVSVIAKQMTYKVYMSGTVNGHYFEVEGDGKGKPYEGEQTVKLTV
                     TKGGPLPFAWDILSPQLQYGSIPFTKYPEDIPDYFKQSFPEGYTWERSMNFEDGAVCTV
                     SNDSSIQGNCFIYNVKISGENFPPNGPVMQKKTQGWEPSTERLFARDGMLIGNDYMALK
                     LEGGGHYLCEFKSTYKAKKPVRMPGRHEIDRKLDVTSHNRDYTSVEQCEIAIARHSLLG
                     "
     regulatory      2078..2087
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     promoter        complement(2116..2319)
                     /label=CMV promoter
                     /note="human cytomegalovirus (CMV) immediate early 
                     promoter"
     primer_bind     complement(2145..2165)
                     /label=CMV-F
                     /note="Human CMV immediate early promoter, forward primer"
     enhancer        2320..2623
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
     rep_origin      complement(2798..3386)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(2878..2897)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     polyA_signal    3715..3762
                     /label=HSV TK poly(A) signal
                     /note="herpes simplex virus thymidine kinase 
                     polyadenylation signal (Cole and Stacy, 1985)"
     primer_bind     3787..3806
                     /label=TK-pA-R
                     /note="Thymidine kinase polyA, reverse primer"
     CDS             complement(3994..4788)
                     /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             complement(3994..4788)
                     /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(4105..4124)
                     /label=Neo-F
                     /note="Neomycin resistance gene, forward primer"
     primer_bind     4715..4734
                     /label=Neo-R
                     /note="Neomycin resistance gene, reverse primer"
     promoter        complement(join(4823..4909,1..271))
                     /label=SV40 promoter
                     /note="SV40 enhancer and early promoter"
     rep_origin      join(4837..4909,1..63)
                     /label=SV40 ori
                     /note="SV40 origin of replication"
ORIGIN
        1 aaaaaaaatt agtcagccat ggggcggaga atgggcggaa ctgggcggag ttaggggcgg
       61 gatgggcgga gttaggggcg ggactatggt tgctgactaa ttgagatgca tgctttgcat
      121 acttctgcct gctggggagc ctggggactt tccacacctg gttgctgact aattgagatg
      181 catgctttgc atacttctgc ctgctgggga gcctggggac tttccacacc ctaactgaca
      241 cacattccac agctggttct ttccgcctca ggactcttcc tttttcaata ttattgaagc
      301 atttatcagg gttattgtct catgagcgga tacatatttg aatgtattta gaaaaataaa
      361 caaatagggg ttccgcgcac atttccccga aaagtgccac ctgacgcgcc ctgtagcggc
      421 gcattaagcg cggcgggtgt ggtggttacg cgcagcgtga ccgctacact tgccagcgcc
      481 ctagcgcccg ctcctttcgc tttcttccct tcctttctcg ccacgttcgc cggctttccc
      541 cgtcaagctc taaatcgggg gctcccttta gggttccgat ttagtgcttt acggcacctc
      601 gaccccaaaa aacttgatta gggtgatggt tcacgtagtg ggccatcgcc ctgatagacg
      661 gtttttcgcc ctttgacgtt ggagtccacg ttctttaata gtggactctt gttccaaact
      721 ggaacaacac tcaaccctat ctcggtctat tcttttgatt tataagggat tttgccgatt
      781 tcggcctatt ggttaaaaaa tgagctgatt taacaaaaat ttaacgcgaa ttttaacaaa
      841 atattaacgc ttacaattta cgccttaaga tacattgatg agtttggaca aaccacaact
      901 agaatgcagt gaaaaaaatg ctttatttgt gaaatttgtg atgctattgc tttatttgta
      961 accattataa gctgcaataa acaagttaac aacaacaatt gcattcattt tatgtttcag
     1021 gttcaggggg aggtgtggga ggttttttaa agcaagtaaa acctctacaa atgtggtatg
     1081 gctgattatg atcagttatc tagatccggt ggatcccggg cccgcggtac cgtcgactgc
     1141 agaattcgaa gcttttaccg cttgaagggc ccatccttca agtatcccac tgcagccgtg
     1201 cagactgtta accacactac accaacgaag cataccaata cccggagggg catcaggatg
     1261 acgtagcgga tgaagaagcc gaacacccag atggaggtga ttttccagga aatgaactcg
     1321 tagtgggtga agggctcctt cttaaagtta aacaaggcgg ccgcggagcc tgcttttttg
     1381 tacaaacttg tataccggtg cgatcgagct cgagcgccca gcagggagtg gcgggcgatg
     1441 gcgatctcgc actgctccac gctggtgtag tcgcggttgt ggctggtcac gtccagcttg
     1501 cggtcgatct cgtggcggcc gggcatcctc acgggcttct tggccttgta ggtgctcttg
     1561 aactcgcaca ggtagtggcc gccgccctcc agcttcaggg ccatgtagtc gttgccgatc
     1621 agcattccgt cgcgggcgaa caggcgctcg gtgctgggct cccagccctg ggtcttcttc
     1681 tgcatcacgg ggccgttggg ggggaagttc tcgccgctga tcttcacgtt gtagatgaag
     1741 cagttgccct ggatgctgct gtcgttgctc acggtgcaca cggcgccgtc ctcgaagttc
     1801 atgctgcgct cccaggtgta gccctcgggg aagctctgct tgaagtagtc ggggatgtcc
     1861 tcggggtact tggtgaaggg gatgctgccg tactggagct gggggctcag gatgtcccag
     1921 gcgaagggca gggggccacc cttggtcacg gtcagcttca cggtctgctc gccctcgtag
     1981 ggcttgccct tgccgtcgcc ctccacctcg aagtagtggc cgttcacggt gccgctcatg
     2041 tacaccttgt aggtcatctg cttggcgatc acgctcacca tggtggcgct agcggatctg
     2101 acggttcact aaaccagctc tgcttatata gacctcccac cgtacacgcc taccgcccat
     2161 ttgcgtcaat ggggcggagt tgttacgaca ttttggaaag tcccgttgat tttggtgcca
     2221 aaacaaactc ccattgacgt caatggggtg gagacttgga aatccccgtg agtcaaaccg
     2281 ctatccacgc ccattgatgt actgccaaaa ccgcatcacc atggtaatag cgatgactaa
     2341 tacgtagatg tactgccaag taggaaagtc ccataaggtc atgtactggg cataatgcca
     2401 ggcgggccat ttaccgtcat tgacgtcaat agggggcgta cttggcatat gatacacttg
     2461 atgtactgcc aagtgggcag tttaccgtaa atactccacc cattgacgtc aatggaaagt
     2521 ccctattggc gttactatgg gaacatacgt cattattgac gtcaatgggc gggggtcgtt
     2581 gggcggtcag ccaggcgggc catttaccgt aagttatgta acgcggaact ccatatatgg
     2641 gctatgaact aatgaccccg taattgatta ctattaataa ctaatgcatg gcggtaatac
     2701 ggttatccac agaatcaggg gataacgcag gaaagaacat gtgagcaaaa ggccagcaaa
     2761 aggccaggaa ccgtaaaaag gccgcgttgc tggcgttttt ccataggctc cgcccccctg
     2821 acgagcatca caaaaatcga cgctcaagtc agaggtggcg aaacccgaca ggactataaa
     2881 gataccaggc gtttccccct ggaagctccc tcgtgcgctc tcctgttccg accctgccgc
     2941 ttaccggata cctgtccgcc tttctccctt cgggaagcgt ggcgctttct catagctcac
     3001 gctgtaggta tctcagttcg gtgtaggtcg ttcgctccaa gctgggctgt gtgcacgaac
     3061 cccccgttca gcccgaccgc tgcgccttat ccggtaacta tcgtcttgag tccaacccgg
     3121 taagacacga cttatcgcca ctggcagcag ccactggtaa caggattagc agagcgaggt
     3181 atgtaggcgg tgctacagag ttcttgaagt ggtggcctaa ctacggctac actagaagaa
     3241 cagtatttgg tatctgcgct ctgctgaagc cagttacctt cggaaaaaga gttggtagct
     3301 cttgatccgg caaacaaacc accgctggta gcggtggttt ttttgtttgc aagcagcaga
     3361 ttacgcgcag aaaaaaagga tctcaagaag atcctttgat cttttctacg gggtctgacg
     3421 ctcagtggaa cgaaaactca cgttaaggga ttttggtcat gagattatca aaaaggatct
     3481 tcacctagat ccttttaaat taaaaatgaa gttttaaatc aatctaaagt atatatgagt
     3541 aacctgaggc tatggcaggg cctgccgccc cgacgttggc tgcgagccct gggccttcac
     3601 ccgaacttgg ggggtggggt ggggaaaagg aagaaacgcg ggcgtattgg ccccaatggg
     3661 gtctcggtgg ggtatcgaca gagtgccagc cctgggaccg aaccccgcgt ttatgaacaa
     3721 acgacccaac accgtgcgtt ttattctgtc tttttattgc cgtcatagcg cgggttcctt
     3781 ccggtattgt ctccttccgt gtttcagtta gcctccccct agggtgggcg aagaactcca
     3841 gcatgagatc cccgcgctgg aggatcatcc agccggcgtc ccggaaaacg attccgaagc
     3901 ccaacctttc atagaaggcg gcggtggaat cgaaatctcg tgatggcagg ttgggcgtcg
     3961 cttggtcggt catttcgaac cccagagtcc cgctcagaag aactcgtcaa gaaggcgata
     4021 gaaggcgatg cgctgcgaat cgggagcggc gataccgtaa agcacgagga agcggtcagc
     4081 ccattcgccg ccaagctctt cagcaatatc acgggtagcc aacgctatgt cctgatagcg
     4141 gtccgccaca cccagccggc cacagtcgat gaatccagaa aagcggccat tttccaccat
     4201 gatattcggc aagcaggcat cgccatgggt cacgacgaga tcctcgccgt cgggcatgct
     4261 cgccttgagc ctggcgaaca gttcggctgg cgcgagcccc tgatgctctt cgtccagatc
     4321 atcctgatcg acaagaccgg cttccatccg agtacgtgct cgctcgatgc gatgtttcgc
     4381 ttggtggtcg aatgggcagg tagccggatc aagcgtatgc agccgccgca ttgcatcagc
     4441 catgatggat actttctcgg caggagcaag gtgagatgac aggagatcct gccccggcac
     4501 ttcgcccaat agcagccagt cccttcccgc ttcagtgaca acgtcgagca cagctgcgca
     4561 aggaacgccc gtcgtggcca gccacgatag ccgcgctgcc tcgtcttgca gttcattcag
     4621 ggcaccggac aggtcggtct tgacaaaaag aaccgggcgc ccctgcgctg acagccggaa
     4681 cacggcggca tcagagcagc cgattgtctg ttgtgcccag tcatagccga atagcctctc
     4741 cacccaagcg gccggagaac ctgcgtgcaa tccatcttgt tcaatcatgc gaaacgatcc
     4801 tcatcctgtc tcttgatcga tctttgcaaa agcctaggcc tccaaaaaag cctcctcact
     4861 acttctggaa tagctcagag gccgaggcgg cctcggcctc tgcataaat
//
