LOCUS       sequence-426327-        5220 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Mitochondria localization of low-affinity NFAST fused to 
            mApple-mito.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 5220)
  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 5220)
  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: NFAST(low)-mApple-mito
FEATURES             Location/Qualifiers
     source          1..5220
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     enhancer        101..404
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
     promoter        405..608
                     /label=CMV promoter
                     /note="human cytomegalovirus (CMV) immediate early 
                     promoter"
     primer_bind     559..579
                     /label=CMV-F
                     /note="Human CMV immediate early promoter, forward primer"
     regulatory      635..644
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     regulatory      1025..1034
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     CDS             1031..1738
                     /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     1444..1463
                     /label=mCherry-F
                     /note="mCherry, forward primer"
     polyA_signal    2048..2169
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(2085..2104)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     2139..2158
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     rep_origin      complement(2176..2631)
                     /direction=LEFT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(2313..2334)
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     2525..2544
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     promoter        2658..2762
                     /gene="bla"
                     /label=AmpR promoter
     promoter        2764..3121
                     /label=SV40 promoter
                     /note="SV40 enhancer and early promoter"
     enhancer        2764..2955
                     /label=SV40 enhancer
                     /note="enhancer for the SV40 early promoter (Herr, 1993)"
     primer_bind     complement(2787..2807)
                     /label=pBABE 3'
                     /note="SV40 enhancer, reverse primer for pBABE vectors"
     rep_origin      2972..3107
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     CDS             3156..3950
                     /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             3156..3950
                     /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(3210..3229)
                     /label=Neo-R
                     /note="Neomycin resistance gene, reverse primer"
     primer_bind     3820..3839
                     /label=Neo-F
                     /note="Neomycin resistance gene, forward primer"
     primer_bind     complement(4138..4157)
                     /label=TK-pA-R
                     /note="Thymidine kinase polyA, reverse primer"
     polyA_signal    4182..4229
                     /label=HSV TK poly(A) signal
                     /note="herpes simplex virus thymidine kinase 
                     polyadenylation signal (Cole and Stacy, 1985)"
     rep_origin      4558..5146
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     5047..5066
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
ORIGIN
        1 atcccctgat tctgtggata accgtattac cgccatgcat tagttattaa tagtaatcaa
       61 ttacggggtc attagttcat agcccatata tggagttccg cgttacataa cttacggtaa
      121 atggcccgcc tggctgaccg cccaacgacc cccgcccatt gacgtcaata atgacgtatg
      181 ttcccatagt aacgccaata gggactttcc attgacgtca atgggtggag tatttacggt
      241 aaactgccca cttggcagta catcaagtgt atcatatgcc aagtacgccc cctattgacg
      301 tcaatgacgg taaatggccc gcctggcatt atgcccagta catgacctta tgggactttc
      361 ctacttggca gtacatctac gtattagtca tcgctattac catggtgatg cggttttggc
      421 agtacatcaa tgggcgtgga tagcggtttg actcacgggg atttccaagt ctccacccca
      481 ttgacgtcaa tgggagtttg ttttggcacc aaaatcaacg ggactttcca aaatgtcgta
      541 acaactccgc cccattgacg caaatgggcg gtaggcgtgt acggtgggag gtctatataa
      601 gcagagctgg tttagtgaac cgtcagatcc gctagccacc atggagaccg tgagattcgg
      661 cggcgacgac atcgagaaca gcctggccaa gatggacgac aaggccctgg acaagctggc
      721 cttcggcgcc atccagctgg acggcaacgg caagatcatc cactacaacg ccgccgaggg
      781 caccatcacc ggcagagacc ccaagaccgt gatcggcaag aacttcttca ccgacgtggc
      841 ccccggcacc cagagcaagg agttccaggg cagattcaag gagggcgtgc agaagggcga
      901 cctgaacacc atgttcgagt ggatgatccc caccagcaga ggccccacca aggtgaaggt
      961 gcacatgaag aaggccatga cctccggagg aggcggcagc ggcggagggg gatctctacc
     1021 ggtcgccacc atggtgagca agggcgagga gaataacatg gccatcatca aggagttcat
     1081 gcgcttcaag gtgcacatgg agggctccgt gaacggccac gagttcgaga tcgagggcga
     1141 gggcgagggc cgcccctacg aggcctttca gaccgctaag ctgaaggtga ccaagggtgg
     1201 ccccctgccc ttcgcctggg acatcctgtc ccctcagttc atgtacggct ccaaggtcta
     1261 cattaagcac ccagccgaca tccccgacta cttcaagctg tccttccccg agggcttcag
     1321 gtgggagcgc gtgatgaact tcgaggacgg cggcattatt cacgttaacc aggactcctc
     1381 cctgcaggac ggcgtgttca tctacaaggt gaagctgcgc ggcaccaact tcccctccga
     1441 cggccccgta atgcagaaga agaccatggg ctgggaggcc tccgaggagc ggatgtaccc
     1501 cgaggacggc gccctgaaga gcgagatcaa gaagaggctg aagctgaagg acggcggcca
     1561 ctacgccgcc gaggtcaaga ccacctacaa ggccaagaag cccgtgcagc tgcccggcgc
     1621 ctacatcgtc gacatcaagt tggacatcgt gtcccacaac gaggactaca ccatcgtgga
     1681 acagtacgaa cgcgccgagg gccgccactc caccggcggc atggacgagc tgtacaagtc
     1741 cggactcaga tctcgagctc aagcttcgaa ttctttgcag gtgcagaatg gacctatagg
     1801 acatcgaggt gaaggggacc caagtggtat tcccatattt atggtgctgg tgccagtgtt
     1861 tgccctcacc atggtagcag cctgggcttt catgagatac cggcaacaac tttgataagg
     1921 atccaccgga tctagataac tgatcataat cagccatacc acatttgtag aggttttact
     1981 tgctttaaaa aacctcccac acctccccct gaacctgaaa cataaaatga atgcaattgt
     2041 tgttgttaac ttgtttattg cagcttataa tggttacaaa taaagcaata gcatcacaaa
     2101 tttcacaaat aaagcatttt tttcactgca ttctagttgt ggtttgtcca aactcatcaa
     2161 tgtatcttaa cgcgtaaatt gtaagcgtta atattttgtt aaaattcgcg ttaaattttt
     2221 gttaaatcag ctcatttttt aaccaatagg ccgaaatcgg caaaatccct tataaatcaa
     2281 aagaatagac cgagataggg ttgagtgttg ttccagtttg gaacaagagt ccactattaa
     2341 agaacgtgga ctccaacgtc aaagggcgaa aaaccgtcta tcagggcgat ggcccactac
     2401 gtgaaccatc accctaatca agttttttgg ggtcgaggtg ccgtaaagca ctaaatcgga
     2461 accctaaagg gagcccccga tttagagctt gacggggaaa gccggcgaac gtggcgagaa
     2521 aggaagggaa gaaagcgaaa ggagcgggcg ctagggcgct ggcaagtgta gcggtcacgc
     2581 tgcgcgtaac caccacaccc gccgcgctta atgcgccgct acagggcgcg tcaggtggca
     2641 cttttcgggg aaatgtgcgc ggaaccccta tttgtttatt tttctaaata cattcaaata
     2701 tgtatccgct catgagacaa taaccctgat aaatgcttca ataatattga aaaaggaaga
     2761 gtcctgaggc ggaaagaacc agctgtggaa tgtgtgtcag ttagggtgtg gaaagtcccc
     2821 aggctcccca gcaggcagaa gtatgcaaag catgcatctc aattagtcag caaccaggtg
     2881 tggaaagtcc ccaggctccc cagcaggcag aagtatgcaa agcatgcatc tcaattagtc
     2941 agcaaccata gtcccgcccc taactccgcc catcccgccc ctaactccgc ccagttccgc
     3001 ccattctccg ccccatggct gactaatttt ttttatttat gcagaggccg aggccgcctc
     3061 ggcctctgag ctattccaga agtagtgagg aggctttttt ggaggcctag gcttttgcaa
     3121 agatcgatca agagacagga tgaggatcgt ttcgcatgat tgaacaagat ggattgcacg
     3181 caggttctcc ggccgcttgg gtggagaggc tattcggcta tgactgggca caacagacaa
     3241 tcggctgctc tgatgccgcc gtgttccggc tgtcagcgca ggggcgcccg gttctttttg
     3301 tcaagaccga cctgtccggt gccctgaatg aactgcaaga cgaggcagcg cggctatcgt
     3361 ggctggccac gacgggcgtt ccttgcgcag ctgtgctcga cgttgtcact gaagcgggaa
     3421 gggactggct gctattgggc gaagtgccgg ggcaggatct cctgtcatct caccttgctc
     3481 ctgccgagaa agtatccatc atggctgatg caatgcggcg gctgcatacg cttgatccgg
     3541 ctacctgccc attcgaccac caagcgaaac atcgcatcga gcgagcacgt actcggatgg
     3601 aagccggtct tgtcgatcag gatgatctgg acgaagagca tcaggggctc gcgccagccg
     3661 aactgttcgc caggctcaag gcgagcatgc ccgacggcga ggatctcgtc gtgacccatg
     3721 gcgatgcctg cttgccgaat atcatggtgg aaaatggccg cttttctgga ttcatcgact
     3781 gtggccggct gggtgtggcg gaccgctatc aggacatagc gttggctacc cgtgatattg
     3841 ctgaagagct tggcggcgaa tgggctgacc gcttcctcgt gctttacggt atcgccgctc
     3901 ccgattcgca gcgcatcgcc ttctatcgcc ttcttgacga gttcttctga gcgggactct
     3961 ggggttcgaa atgaccgacc aagcgacgcc caacctgcca tcacgagatt tcgattccac
     4021 cgccgccttc tatgaaaggt tgggcttcgg aatcgttttc cgggacgccg gctggatgat
     4081 cctccagcgc ggggatctca tgctggagtt cttcgcccac cctaggggga ggctaactga
     4141 aacacggaag gagacaatac cggaaggaac ccgcgctatg acggcaataa aaagacagaa
     4201 taaaacgcac ggtgttgggt cgtttgttca taaacgcggg gttcggtccc agggctggca
     4261 ctctgtcgat accccaccga gaccccattg gggccaatac gcccgcgttt cttccttttc
     4321 cccaccccac cccccaagtt cgggtgaagg cccagggctc gcagccaacg tcggggcggc
     4381 aggccctgcc atagcctcag gttactcata tatactttag attgatttaa aacttcattt
     4441 ttaatttaaa aggatctagg tgaagatcct ttttgataat ctcatgacca aaatccctta
     4501 acgtgagttt tcgttccact gagcgtcaga ccccgtagaa aagatcaaag gatcttcttg
     4561 agatcctttt tttctgcgcg taatctgctg cttgcaaaca aaaaaaccac cgctaccagc
     4621 ggtggtttgt ttgccggatc aagagctacc aactcttttt ccgaaggtaa ctggcttcag
     4681 cagagcgcag ataccaaata ctgttcttct agtgtagccg tagttaggcc accacttcaa
     4741 gaactctgta gcaccgccta catacctcgc tctgctaatc ctgttaccag tggctgctgc
     4801 cagtggcgat aagtcgtgtc ttaccgggtt ggactcaaga cgatagttac cggataaggc
     4861 gcagcggtcg ggctgaacgg ggggttcgtg cacacagccc agcttggagc gaacgaccta
     4921 caccgaactg agatacctac agcgtgagct atgagaaagc gccacgcttc ccgaagggag
     4981 aaaggcggac aggtatccgg taagcggcag ggtcggaaca ggagagcgca cgagggagct
     5041 tccaggggga aacgcctggt atctttatag tcctgtcggg tttcgccacc tctgacttga
     5101 gcgtcgattt ttgtgatgct cgtcaggggg gcggagccta tggaaaaacg ccagcaacgc
     5161 ggccttttta cggttcctgg ccttttgctg gccttttgct cacatgttct ttcctgcgtt
//
