LOCUS       sequence-387854-        5717 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Expressing fluorescent SNAPf-mTurquoise2 fusion protein in mammalian
            cells with localization in the mitochondria..
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 5717)
  AUTHORS   Martin A, Rivera-Fuentes P
  TITLE     A general strategy to develop fluorogenic polymethine dyes for 
            bioimaging.
  JOURNAL   Nat Chem. 2024 Jan;16(1):28-35. doi: 10.1038/s41557-023-01367-y. 
            Epub 2023 Nov 27.
  PUBMED    38012391
REFERENCE   2  (bases 1 to 5717)
  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/s41557-023-01367-y"; journalName: "Nat Chem"; date: 
            "2024-01"; volume: "16"; issue: "1"; pages: "28-35"
COMMENT     Sequence Label: TOM20-SNAPf-mTurquoise2
FEATURES             Location/Qualifiers
     source          1..5717
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     enhancer        61..364
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
     promoter        365..568
                     /label=CMV promoter
                     /note="human cytomegalovirus (CMV) immediate early 
                     promoter"
     primer_bind     519..539
                     /label=CMV-F
                     /note="Human CMV immediate early promoter, forward primer"
     CDS             1084..1629
                     /codon_start=1
                     /product="human O6-alkylguanine-DNA-alkyltransferase 
                     variant that forms covalent bonds with benzylguanine 
                     derivatives"
                     /label=SNAP-tag(R)
                     /note="fast-reacting variant, optimized for mammalian 
                     expression"
                     /translation="MDKDCEMKRTTLDSPLGKLELSGCEQGLHRIIFLGKGTSAADAVE
                     VPAPAAVLGGPEPLMQATAWLNAYFHQPEAIEEFPVPALHHPVFQQESFTRQVLWKLLK
                     VVKFGEVISYSHLAALAGNPAATAAVKTALSGNPVPILIPCHRVVQGDLDVGGYEGGLA
                     VKEWLLAHEGHRLGKPGLG"
     CDS             join(1666..1668,1669..2382)
                     /codon_start=1
                     /product="enhanced monomeric variant of CFP (Goedhart et 
                     al., 2012)"
                     /label=mTurquoise2
                     /note="mammalian codon-optimized"
                     /translation="VSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTLK
                     FICTTGKLPVPWPTLVTTLSWGVQCFARYPDHMKQHDFFKSAMPEGYVQERTIFFKDDG
                     NYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYFSDNVYITADKQKNGIKA
                     NFKIRHNIEDGGVQLADHYQQNTPIGDGPVLLPDNHYLSTQSKLSKDPNEKRDHMVLLE
                     FVTAAGITLGMDELYK"
     primer_bind     complement(1708..1729)
                     /label=EGFP-N
                     /note="EGFP, reverse primer"
     primer_bind     complement(1969..1988)
                     /label=EXFP-R
                     /note="For distinguishing EGFP variants, reverse primer"
     primer_bind     2316..2337
                     /label=EGFP-C
                     /note="EGFP, forward primer"
     polyA_signal    2505..2626
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(2542..2561)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     2596..2615
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     rep_origin      complement(2633..3088)
                     /direction=LEFT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(2770..2791)
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     2982..3001
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     promoter        3115..3219
                     /gene="bla"
                     /label=AmpR promoter
     promoter        3221..3578
                     /label=SV40 promoter
                     /note="SV40 enhancer and early promoter"
     enhancer        3221..3412
                     /label=SV40 enhancer
                     /note="enhancer for the SV40 early promoter (Herr, 1993)"
     primer_bind     complement(3244..3264)
                     /label=pBABE 3'
                     /note="SV40 enhancer, reverse primer for pBABE vectors"
     rep_origin      3429..3564
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     CDS             3613..4407
                     /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             3613..4407
                     /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(3667..3686)
                     /label=Neo-R
                     /note="Neomycin resistance gene, reverse primer"
     primer_bind     4277..4296
                     /label=Neo-F
                     /note="Neomycin resistance gene, forward primer"
     primer_bind     complement(4595..4614)
                     /label=TK-pA-R
                     /note="Thymidine kinase polyA, reverse primer"
     polyA_signal    4639..4686
                     /label=HSV TK poly(A) signal
                     /note="herpes simplex virus thymidine kinase 
                     polyadenylation signal (Cole and Stacy, 1985)"
     rep_origin      5015..5603
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     5504..5523
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
ORIGIN
        1 tagttattaa tagtaatcaa ttacggggtc attagttcat agcccatata tggagttccg
       61 cgttacataa cttacggtaa atggcccgcc tggctgaccg cccaacgacc cccgcccatt
      121 gacgtcaata atgacgtatg ttcccatagt aacgccaata gggactttcc attgacgtca
      181 atgggtggag tatttacggt aaactgccca cttggcagta catcaagtgt atcatatgcc
      241 aagtacgccc cctattgacg tcaatgacgg taaatggccc gcctggcatt atgcccagta
      301 catgacctta tgggactttc ctacttggca gtacatctac gtattagtca tcgctattac
      361 catggtgatg cggttttggc agtacatcaa tgggcgtgga tagcggtttg actcacgggg
      421 atttccaagt ctccacccca ttgacgtcaa tgggagtttg ttttggcacc aaaatcaacg
      481 ggactttcca aaatgtcgta acaactccgc cccattgacg caaatgggcg gtaggcgtgt
      541 acggtgggag gtctatataa gcagagctgg tttagtgaac cgtcagatcc gctagcgcta
      601 ccggactcaa tggtgggtcg gaacagcgcc atcgccgccg gtgtatgcgg ggcccttttc
      661 attgggtact gcatctactt cgaccgcaaa agacgaagtg accccaactt caagaacagg
      721 cttcgagaac gaagaaagaa acagaagctt gccaaggaga gagctgggct ttccaagtta
      781 cctgacctta aagatgctga agctgttcag aagttcttcc ttgaagaaat acagcttggt
      841 gaagagttac tagctcaagg tgaatatgag aagggcgtag accatctgac aaatgcaatt
      901 gctgtgtgtg gacagccaca gcagttactg caggtcttac agcaaactct tccaccacca
      961 gtgttccaga tgcttctgac taagctccca acaattagtc agagaattgt aagtgctcag
     1021 agcttggctg aagatgatgt ggaagcgcca gcatttaaat ctgtacagac cggtgaattc
     1081 accatggaca aagactgcga aatgaagcgc accaccctgg atagccctct gggcaagctg
     1141 gaactgtctg ggtgcgaaca gggcctgcac cgtatcatct tcctgggcaa aggaacatct
     1201 gccgccgacg ccgtggaagt gcctgcccca gccgccgtgc tgggcggacc agagccactg
     1261 atgcaggcca ccgcctggct caacgcctac tttcaccagc ctgaggccat cgaggagttc
     1321 cctgtgccag ccctgcacca cccagtgttc cagcaggaga gctttacccg ccaggtgctg
     1381 tggaaactgc tgaaagtggt gaagttcgga gaggtcatca gctacagcca cctggccgcc
     1441 ctggccggca atcccgccgc caccgccgcc gtgaaaaccg ccctgagcgg aaatcccgtg
     1501 cccattctga tcccctgcca ccgggtggtg cagggcgacc tggacgtggg gggctacgag
     1561 ggcgggctcg ccgtgaaaga gtggctgctg gcccacgagg gccacagact gggcaagcct
     1621 gggctgggtc ctgcaggcgg atccgcgttt aaactcgagg ttaatgtgag caagggcgag
     1681 gagctgttca ccggggtggt gcccatcctg gtcgagctgg acggcgacgt aaacggccac
     1741 aagttcagcg tgtccggcga gggcgagggc gatgccacct acggcaagct gaccctgaag
     1801 ttcatctgca ccaccggcaa gctgcccgtg ccctggccca ccctcgtgac caccctgtcc
     1861 tggggcgtgc agtgcttcgc ccgctacccc gaccacatga agcagcacga cttcttcaag
     1921 tccgccatgc ccgaaggcta cgtccaggag cgcaccatct tcttcaagga cgacggcaac
     1981 tacaagaccc gcgccgaggt gaagttcgag ggcgacaccc tggtgaaccg catcgagctg
     2041 aagggcatcg acttcaagga ggacggcaac atcctggggc acaagctgga gtacaactac
     2101 tttagcgaca acgtctatat caccgccgac aagcagaaga acggcatcaa ggccaacttc
     2161 aagatccgcc acaacatcga ggacggcggc gtgcagctcg ccgaccacta ccagcagaac
     2221 acccccatcg gcgacggccc cgtgctgctg cccgacaacc actacctgag cacccagtcc
     2281 aagctgagca aagaccccaa cgagaagcgc gatcacatgg tcctgctgga gttcgtgacc
     2341 gccgccggga tcactctcgg catggacgag ctgtacaagt aaagcggccg cgactctaga
     2401 tcataatcag ccataccaca tttgtagagg ttttacttgc tttaaaaaac ctcccacacc
     2461 tccccctgaa cctgaaacat aaaatgaatg caattgttgt tgttaacttg tttattgcag
     2521 cttataatgg ttacaaataa agcaatagca tcacaaattt cacaaataaa gcattttttt
     2581 cactgcattc tagttgtggt ttgtccaaac tcatcaatgt atcttaaggc gtaaattgta
     2641 agcgttaata ttttgttaaa attcgcgtta aatttttgtt aaatcagctc attttttaac
     2701 caataggccg aaatcggcaa aatcccttat aaatcaaaag aatagaccga gatagggttg
     2761 agtgttgttc cagtttggaa caagagtcca ctattaaaga acgtggactc caacgtcaaa
     2821 gggcgaaaaa ccgtctatca gggcgatggc ccactacgtg aaccatcacc ctaatcaagt
     2881 tttttggggt cgaggtgccg taaagcacta aatcggaacc ctaaagggag cccccgattt
     2941 agagcttgac ggggaaagcc ggcgaacgtg gcgagaaagg aagggaagaa agcgaaagga
     3001 gcgggcgcta gggcgctggc aagtgtagcg gtcacgctgc gcgtaaccac cacacccgcc
     3061 gcgcttaatg cgccgctaca gggcgcgtca ggtggcactt ttcggggaaa tgtgcgcgga
     3121 acccctattt gtttattttt ctaaatacat tcaaatatgt atccgctcat gagacaataa
     3181 ccctgataaa tgcttcaata atattgaaaa aggaagagtc ctgaggcgga aagaaccagc
     3241 tgtggaatgt gtgtcagtta gggtgtggaa agtccccagg ctccccagca ggcagaagta
     3301 tgcaaagcat gcatctcaat tagtcagcaa ccaggtgtgg aaagtcccca ggctccccag
     3361 caggcagaag tatgcaaagc atgcatctca attagtcagc aaccatagtc ccgcccctaa
     3421 ctccgcccat cccgccccta actccgccca gttccgccca ttctccgccc catggctgac
     3481 taattttttt tatttatgca gaggccgagg ccgcctcggc ctctgagcta ttccagaagt
     3541 agtgaggagg cttttttgga ggcctaggct tttgcaaaga tcgatcaaga gacaggatga
     3601 ggatcgtttc gcatgattga acaagatgga ttgcacgcag gttctccggc cgcttgggtg
     3661 gagaggctat tcggctatga ctgggcacaa cagacaatcg gctgctctga tgccgccgtg
     3721 ttccggctgt cagcgcaggg gcgcccggtt ctttttgtca agaccgacct gtccggtgcc
     3781 ctgaatgaac tgcaagacga ggcagcgcgg ctatcgtggc tggccacgac gggcgttcct
     3841 tgcgcagctg tgctcgacgt tgtcactgaa gcgggaaggg actggctgct attgggcgaa
     3901 gtgccggggc aggatctcct gtcatctcac cttgctcctg ccgagaaagt atccatcatg
     3961 gctgatgcaa tgcggcggct gcatacgctt gatccggcta cctgcccatt cgaccaccaa
     4021 gcgaaacatc gcatcgagcg agcacgtact cggatggaag ccggtcttgt cgatcaggat
     4081 gatctggacg aagagcatca ggggctcgcg ccagccgaac tgttcgccag gctcaaggcg
     4141 agcatgcccg acggcgagga tctcgtcgtg acccatggcg atgcctgctt gccgaatatc
     4201 atggtggaaa atggccgctt ttctggattc atcgactgtg gccggctggg tgtggcggac
     4261 cgctatcagg acatagcgtt ggctacccgt gatattgctg aagagcttgg cggcgaatgg
     4321 gctgaccgct tcctcgtgct ttacggtatc gccgctcccg attcgcagcg catcgccttc
     4381 tatcgccttc ttgacgagtt cttctgagcg ggactctggg gttcgaaatg accgaccaag
     4441 cgacgcccaa cctgccatca cgagatttcg attccaccgc cgccttctat gaaaggttgg
     4501 gcttcggaat cgttttccgg gacgccggct ggatgatcct ccagcgcggg gatctcatgc
     4561 tggagttctt cgcccaccct agggggaggc taactgaaac acggaaggag acaataccgg
     4621 aaggaacccg cgctatgacg gcaataaaaa gacagaataa aacgcacggt gttgggtcgt
     4681 ttgttcataa acgcggggtt cggtcccagg gctggcactc tgtcgatacc ccaccgagac
     4741 cccattgggg ccaatacgcc cgcgtttctt ccttttcccc accccacccc ccaagttcgg
     4801 gtgaaggccc agggctcgca gccaacgtcg gggcggcagg ccctgccata gcctcaggtt
     4861 actcatatat actttagatt gatttaaaac ttcattttta atttaaaagg atctaggtga
     4921 agatcctttt tgataatctc atgaccaaaa tcccttaacg tgagttttcg ttccactgag
     4981 cgtcagaccc cgtagaaaag atcaaaggat cttcttgaga tccttttttt ctgcgcgtaa
     5041 tctgctgctt gcaaacaaaa aaaccaccgc taccagcggt ggtttgtttg ccggatcaag
     5101 agctaccaac tctttttccg aaggtaactg gcttcagcag agcgcagata ccaaatactg
     5161 ttcttctagt gtagccgtag ttaggccacc acttcaagaa ctctgtagca ccgcctacat
     5221 acctcgctct gctaatcctg ttaccagtgg ctgctgccag tggcgataag tcgtgtctta
     5281 ccgggttgga ctcaagacga tagttaccgg ataaggcgca gcggtcgggc tgaacggggg
     5341 gttcgtgcac acagcccagc ttggagcgaa cgacctacac cgaactgaga tacctacagc
     5401 gtgagctatg agaaagcgcc acgcttcccg aagggagaaa ggcggacagg tatccggtaa
     5461 gcggcagggt cggaacagga gagcgcacga gggagcttcc agggggaaac gcctggtatc
     5521 tttatagtcc tgtcgggttt cgccacctct gacttgagcg tcgatttttg tgatgctcgt
     5581 caggggggcg gagcctatgg aaaaacgcca gcaacgcggc ctttttacgg ttcctggcct
     5641 tttgctggcc ttttgctcac atgttctttc ctgcgttatc ccctgattct gtggataacc
     5701 gtattaccgc catgcat
//
