LOCUS       sequence-425785-        5682 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Lipid droplet localization of APEX2-6xHp for electron microscopy.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 5682)
  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 5682)
  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: APEX2-6xHp
FEATURES             Location/Qualifiers
     source          1..5682
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     29..49
                     /label=CMV-F
                     /note="Human CMV immediate early promoter, forward primer"
     regulatory      105..114
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     CDS             111..860
                     /codon_start=1
                     /product="soybean ascorbate peroxidase, engineered to serve
                     as a monomeric tag for electron microscopy and live-cell 
                     proteomics (Lam et al., 2015)"
                     /label=APEX2
                     /note="more active than the original APEX due to the A134P 
                     mutation"
                     /translation="MGKSYPTVSADYQDAVEKAKKKLRGFIAEKRCAPLMLRLAFHSAG
                     TFDKGTKTGGPFGTIKHPAELAHSANNGLDIAVRLLEPLKAEFPILSYADFYQLAGVVA
                     VEVTGGPKVPFHPGREDKPEPPPEGRLPDPTKGSDHLRDVFGKAMGLTDQDIVALSGGH
                     TIGAAHKERSGFEGPWTSNPLIFDNSYFTELLSGEKEGLLQLPSDKALLSDPVFRPLVD
                     KYAADEDAFFADYAEAHQKLSELGFADA"
     polyA_signal    1980..2101
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(2017..2036)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     2071..2090
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     rep_origin      complement(2108..2563)
                     /direction=LEFT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(2245..2266)
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     2457..2476
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     promoter        2590..2694
                     /gene="bla"
                     /label=AmpR promoter
     promoter        2696..3053
                     /label=SV40 promoter
                     /note="SV40 enhancer and early promoter"
     enhancer        2696..2887
                     /label=SV40 enhancer
                     /note="enhancer for the SV40 early promoter (Herr, 1993)"
     primer_bind     complement(2719..2739)
                     /label=pBABE 3'
                     /note="SV40 enhancer, reverse primer for pBABE vectors"
     rep_origin      2904..3039
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     CDS             3088..3882
                     /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             3088..3882
                     /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(3142..3161)
                     /label=Neo-R
                     /note="Neomycin resistance gene, reverse primer"
     primer_bind     3752..3771
                     /label=Neo-F
                     /note="Neomycin resistance gene, forward primer"
     primer_bind     complement(4070..4089)
                     /label=TK-pA-R
                     /note="Thymidine kinase polyA, reverse primer"
     polyA_signal    4114..4161
                     /label=HSV TK poly(A) signal
                     /note="herpes simplex virus thymidine kinase 
                     polyadenylation signal (Cole and Stacy, 1985)"
     rep_origin      4490..5078
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     4979..4998
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     enhancer        5253..5556
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
     promoter        join(5557..5682,1..78)
                     /label=CMV promoter
                     /note="human cytomegalovirus (CMV) immediate early 
                     promoter"
ORIGIN
        1 aaatgtcgta acaactccgc cccattgacg caaatgggcg gtaggcgtgt acggtgggag
       61 gtctatataa gcagagctgg tttagtgaac cgtcagatcc gctagccacc atgggaaagt
      121 cttacccaac tgtgagtgct gattaccagg acgccgttga gaaggcgaag aagaagctca
      181 gaggcttcat cgctgagaag agatgcgctc ctctaatgct ccgtttggca ttccactctg
      241 ctggaacctt tgacaagggc acgaagaccg gtggaccctt cggaaccatc aagcaccctg
      301 ccgaactggc tcacagcgct aacaacggtc ttgacatcgc tgttaggctt ttggagccac
      361 tcaaggcgga gttccctatt ttgagctacg ccgatttcta ccagttggct ggcgttgttg
      421 ccgttgaggt cacgggtgga cctaaggttc cattccaccc tggaagagag gacaagcctg
      481 agccaccacc agagggtcgc ttgcccgatc ccactaaggg ttctgaccat ttgagagatg
      541 tgtttggcaa agctatgggg cttactgacc aagatatcgt tgctctatct gggggtcaca
      601 ctattggagc tgcacacaag gagcgttctg gatttgaggg tccctggacc tctaatcctc
      661 ttattttcga caactcatac ttcacggagt tgttgagtgg tgagaaggaa ggtctccttc
      721 agctaccttc tgacaaggct cttttgtctg accctgtatt ccgccctctc gttgacaaat
      781 atgcagcgga cgaagatgcc ttctttgctg attacgctga ggctcaccaa aagctttccg
      841 agcttgggtt tgctgatgcc tcagctggag gcagtgcagg tggctctgcc ggagggaatt
      901 ccgaatcccc gcacaaacgc aacctgtact acttctctta cccccttttt gtcgggtttg
      961 ctctacttcg attggttgct tttcatcttg gattactgtt tgtttggttg tgtcaacgct
     1021 tttctagagc gctgatggct gcgaaacgtt cggctaatag tgaatcgccc cataagagga
     1081 acttatatta cttcagctat cccttgttcg tgggtttcgc gctacttcgg ctggtcgcgt
     1141 ttcatctagg tttgttgttt gtctggttat gtcaacggtt ctcgcgagcg ttaatggcag
     1201 caaagaggtc ggcaaatagc gaatcgcctc ataaacgcaa cctctattac ttctcttacc
     1261 cgttgttcgt tggcttcgct ctccttcggc tagttgcgtt tcatcttggt cttctgttcg
     1321 tgtggctttg ccaaagattt tcgcgagctt taatggcggc taagaggagt gccaatagcg
     1381 aatccccgca taagcggaac ttatactact tctcttaccc tctttttgta ggctttgctc
     1441 tactccggct agtggccttc catctaggcc tactattcgt ctggctctgc cagcgcttct
     1501 cgcgagcctt gatggcagct aagcgttccg caaatagcga atcacctcat aagcggaatc
     1561 tgtactattt ctcatatcct ctcttcgtag ggttcgcact attacgtctt gtagcatttc
     1621 atctgggatt attattcgtc tggttgtgcc agcgattttc acgagcctta atggctgcga
     1681 agagaagcgc aaattctgaa tctcctcaca agcggaacct ctattacttt agttacccgc
     1741 tcttcgtcgg tttcgcgtta ctcaggctgg ttgcattcca cctcggcttg ctgttcgtat
     1801 ggctttgcca gagattctcc cgggcgctaa tggcagccaa gcgctcctaa ggatccaccg
     1861 gatctagata actgatcata atcagccata ccacatttgt agaggtttta cttgctttaa
     1921 aaaacctccc acacctcccc ctgaacctga aacataaaat gaatgcaatt gttgttgtta
     1981 acttgtttat tgcagcttat aatggttaca aataaagcaa tagcatcaca aatttcacaa
     2041 ataaagcatt tttttcactg cattctagtt gtggtttgtc caaactcatc aatgtatctt
     2101 aacgcgtaaa ttgtaagcgt taatattttg ttaaaattcg cgttaaattt ttgttaaatc
     2161 agctcatttt ttaaccaata ggccgaaatc ggcaaaatcc cttataaatc aaaagaatag
     2221 accgagatag ggttgagtgt tgttccagtt tggaacaaga gtccactatt aaagaacgtg
     2281 gactccaacg tcaaagggcg aaaaaccgtc tatcagggcg atggcccact acgtgaacca
     2341 tcaccctaat caagtttttt ggggtcgagg tgccgtaaag cactaaatcg gaaccctaaa
     2401 gggagccccc gatttagagc ttgacgggga aagccggcga acgtggcgag aaaggaaggg
     2461 aagaaagcga aaggagcggg cgctagggcg ctggcaagtg tagcggtcac gctgcgcgta
     2521 accaccacac ccgccgcgct taatgcgccg ctacagggcg cgtcaggtgg cacttttcgg
     2581 ggaaatgtgc gcggaacccc tatttgttta tttttctaaa tacattcaaa tatgtatccg
     2641 ctcatgagac aataaccctg ataaatgctt caataatatt gaaaaaggaa gagtcctgag
     2701 gcggaaagaa ccagctgtgg aatgtgtgtc agttagggtg tggaaagtcc ccaggctccc
     2761 cagcaggcag aagtatgcaa agcatgcatc tcaattagtc agcaaccagg tgtggaaagt
     2821 ccccaggctc cccagcaggc agaagtatgc aaagcatgca tctcaattag tcagcaacca
     2881 tagtcccgcc cctaactccg cccatcccgc ccctaactcc gcccagttcc gcccattctc
     2941 cgccccatgg ctgactaatt ttttttattt atgcagaggc cgaggccgcc tcggcctctg
     3001 agctattcca gaagtagtga ggaggctttt ttggaggcct aggcttttgc aaagatcgat
     3061 caagagacag gatgaggatc gtttcgcatg attgaacaag atggattgca cgcaggttct
     3121 ccggccgctt gggtggagag gctattcggc tatgactggg cacaacagac aatcggctgc
     3181 tctgatgccg ccgtgttccg gctgtcagcg caggggcgcc cggttctttt tgtcaagacc
     3241 gacctgtccg gtgccctgaa tgaactgcaa gacgaggcag cgcggctatc gtggctggcc
     3301 acgacgggcg ttccttgcgc agctgtgctc gacgttgtca ctgaagcggg aagggactgg
     3361 ctgctattgg gcgaagtgcc ggggcaggat ctcctgtcat ctcaccttgc tcctgccgag
     3421 aaagtatcca tcatggctga tgcaatgcgg cggctgcata cgcttgatcc ggctacctgc
     3481 ccattcgacc accaagcgaa acatcgcatc gagcgagcac gtactcggat ggaagccggt
     3541 cttgtcgatc aggatgatct ggacgaagag catcaggggc tcgcgccagc cgaactgttc
     3601 gccaggctca aggcgagcat gcccgacggc gaggatctcg tcgtgaccca tggcgatgcc
     3661 tgcttgccga atatcatggt ggaaaatggc cgcttttctg gattcatcga ctgtggccgg
     3721 ctgggtgtgg cggaccgcta tcaggacata gcgttggcta cccgtgatat tgctgaagag
     3781 cttggcggcg aatgggctga ccgcttcctc gtgctttacg gtatcgccgc tcccgattcg
     3841 cagcgcatcg ccttctatcg ccttcttgac gagttcttct gagcgggact ctggggttcg
     3901 aaatgaccga ccaagcgacg cccaacctgc catcacgaga tttcgattcc accgccgcct
     3961 tctatgaaag gttgggcttc ggaatcgttt tccgggacgc cggctggatg atcctccagc
     4021 gcggggatct catgctggag ttcttcgccc accctagggg gaggctaact gaaacacgga
     4081 aggagacaat accggaagga acccgcgcta tgacggcaat aaaaagacag aataaaacgc
     4141 acggtgttgg gtcgtttgtt cataaacgcg gggttcggtc ccagggctgg cactctgtcg
     4201 ataccccacc gagaccccat tggggccaat acgcccgcgt ttcttccttt tccccacccc
     4261 accccccaag ttcgggtgaa ggcccagggc tcgcagccaa cgtcggggcg gcaggccctg
     4321 ccatagcctc aggttactca tatatacttt agattgattt aaaacttcat ttttaattta
     4381 aaaggatcta ggtgaagatc ctttttgata atctcatgac caaaatccct taacgtgagt
     4441 tttcgttcca ctgagcgtca gaccccgtag aaaagatcaa aggatcttct tgagatcctt
     4501 tttttctgcg cgtaatctgc tgcttgcaaa caaaaaaacc accgctacca gcggtggttt
     4561 gtttgccgga tcaagagcta ccaactcttt ttccgaaggt aactggcttc agcagagcgc
     4621 agataccaaa tactgttctt ctagtgtagc cgtagttagg ccaccacttc aagaactctg
     4681 tagcaccgcc tacatacctc gctctgctaa tcctgttacc agtggctgct gccagtggcg
     4741 ataagtcgtg tcttaccggg ttggactcaa gacgatagtt accggataag gcgcagcggt
     4801 cgggctgaac ggggggttcg tgcacacagc ccagcttgga gcgaacgacc tacaccgaac
     4861 tgagatacct acagcgtgag ctatgagaaa gcgccacgct tcccgaaggg agaaaggcgg
     4921 acaggtatcc ggtaagcggc agggtcggaa caggagagcg cacgagggag cttccagggg
     4981 gaaacgcctg gtatctttat agtcctgtcg ggtttcgcca cctctgactt gagcgtcgat
     5041 ttttgtgatg ctcgtcaggg gggcggagcc tatggaaaaa cgccagcaac gcggcctttt
     5101 tacggttcct ggccttttgc tggccttttg ctcacatgtt ctttcctgcg ttatcccctg
     5161 attctgtgga taaccgtatt accgccatgc attagttatt aatagtaatc aattacgggg
     5221 tcattagttc atagcccata tatggagttc cgcgttacat aacttacggt aaatggcccg
     5281 cctggctgac cgcccaacga cccccgccca ttgacgtcaa taatgacgta tgttcccata
     5341 gtaacgccaa tagggacttt ccattgacgt caatgggtgg agtatttacg gtaaactgcc
     5401 cacttggcag tacatcaagt gtatcatatg ccaagtacgc cccctattga cgtcaatgac
     5461 ggtaaatggc ccgcctggca ttatgcccag tacatgacct tatgggactt tcctacttgg
     5521 cagtacatct acgtattagt catcgctatt accatggtga tgcggttttg gcagtacatc
     5581 aatgggcgtg gatagcggtt tgactcacgg ggatttccaa gtctccaccc cattgacgtc
     5641 aatgggagtt tgttttggca ccaaaatcaa cgggactttc ca
//
