LOCUS       sequence-368228-        6467 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Localization of LSSmGFP fluorescent protein (ex/em = 400/510 nm) to 
            mitochondria.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 6467)
  AUTHORS   Campbell BC, Paez-Segala MG, Looger LL, Petsko GA, Liu CF
  TITLE     Chemically stable fluorescent proteins for advanced microscopy.
  JOURNAL   Nat Methods. 2022 Nov 7. pii: 10.1038/s41592-022-01660-7. doi: 
            10.1038/s41592-022-01660-7.
  PUBMED    36344833
REFERENCE   2  (bases 1 to 6467)
  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: "Nat 
            Methods. 2022 Nov 7. pii: 10.1038/s41592-022-01660-7. doi: 
            10.1038/s41592-022-01660-7."
COMMENT     Sequence Label: Mito-LSSmGFP
FEATURES             Location/Qualifiers
     source          1..6467
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     enhancer        1..380
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
     promoter        381..584
                     /label=CMV promoter
                     /note="human cytomegalovirus (CMV) immediate early 
                     promoter"
     primer_bind     535..555
                     /label=CMV-F
                     /note="Human CMV immediate early promoter, forward primer"
     primer_bind     629..648
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        629..647
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     CDS             661..735
                     /codon_start=1
                     /product="mitochondrial presequence of human cytochrome c 
                     oxidase subunit VIII (Rizutto et al., 1989)"
                     /label=COX8 presequence
                     /translation="MSVLTPLLLRGLTGSARRLPVPRAK"
     CDS             757..831
                     /codon_start=1
                     /product="mitochondrial presequence of human cytochrome c 
                     oxidase subunit VIII (Rizutto et al., 1989)"
                     /label=COX8 presequence
                     /translation="MSVLTPLLLRGLTGSARRLPVPRAK"
     CDS             853..927
                     /codon_start=1
                     /product="mitochondrial presequence of human cytochrome c 
                     oxidase subunit VIII (Rizutto et al., 1989)"
                     /label=COX8 presequence
                     /translation="MSVLTPLLLRGLTGSARRLPVPRAK"
     CDS             949..1023
                     /codon_start=1
                     /product="mitochondrial presequence of human cytochrome c 
                     oxidase subunit VIII (Rizutto et al., 1989)"
                     /label=COX8 presequence
                     /translation="MSVLTPLLLRGLTGSARRLPVPRAK"
     regulatory      1045..1054
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     primer_bind     complement(1096..1117)
                     /label=EGFP-N
                     /note="EGFP, reverse primer"
     promoter        complement(1786..1804)
                     /label=SP6 promoter
                     /note="promoter for bacteriophage SP6 RNA polymerase"
     primer_bind     complement(1787..1804)
                     /label=SP6
                     /note="SP6 promoter, forward primer"
     primer_bind     complement(1824..1841)
                     /label=BGH-rev
                     /note="Bovine growth hormone terminator, reverse primer. 
                     Also called BGH reverse"
     polyA_signal    1830..2054
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     rep_origin      2100..2528
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(2187..2206)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     2397..2418
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     complement(2537..2557)
                     /label=pBABE 3'
                     /note="SV40 enhancer, reverse primer for pBABE vectors"
     promoter        2542..2871
                     /label=SV40 promoter
                     /note="SV40 enhancer and early promoter"
     rep_origin      2722..2857
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     primer_bind     2784..2803
                     /label=SV40pro-F
                     /note="SV40 promoter/origin, forward primer"
     CDS             2938..3732
                     /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
                     GLAPAELFARLKARMPDGEDLVVTHGDACLPNIMVENGRFSGFIDCGRLGVADRYQDIA
                     LATRDIAEELGGEWADRFLVLYGIAAPDSQRIAFYRLLDEFF"
     primer_bind     complement(2992..3011)
                     /label=Neo-R
                     /note="Neomycin resistance gene, reverse primer"
     primer_bind     3602..3621
                     /label=Neo-F
                     /note="Neomycin resistance gene, forward primer"
     polyA_signal    3906..4027
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(3943..3962)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     3997..4016
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     primer_bind     complement(4076..4092)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(4076..4092)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(4089..4111)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    4100..4116
                     /label=lac operator
                     /bound_moiety="lac repressor encoded by lacI"
                     /note="The lac repressor binds to the lac operator to 
                     inhibit transcription in E. coli. This inhibition can be 
                     relieved by adding lactose or 
                     isopropyl-beta-D-thiogalactopyranoside (IPTG)."
     promoter        complement(join(4124..4130,4131..4148,4149..4154))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    4169..4190
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(4307..4324)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(4478..5066)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(4558..4577)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(5237..6028,6029..6097))
                     /codon_start=1
                     /gene="bla"
                     /product="beta-lactamase"
                     /label=AmpR
                     /note="confers resistance to ampicillin, carbenicillin, and
                     related antibiotics"
                     /translation="MSIQHFRVALIPFFAAFCLPVFAHPETLVKVKDAEDQLGARVGYI
                     ELDLNSGKILESFRPEERFPMMSTFKVLLCGAVLSRIDAGQEQLGRRIHYSQNDLVEYS
                     PVTEKHLTDGMTVRELCSAAITMSDNTAANLLLTTIGGPKELTAFLHNMGDHVTRLDRW
                     EPELNEAIPNDERDTTMPVAMATTLRKLLTGELLTLASRQQLIDWMEADKVAGPLLRSA
                     LPAGWFIADKSGAGERGSRGIIAALGPDGKPSRIVVIYTTGSQATMDERNRQIAEIGAS
                     LIKHW"
     primer_bind     5860..5879
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(6098..6202)
                     /gene="bla"
                     /label=AmpR promoter
     primer_bind     complement(6277..6296)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
ORIGIN
        1 gacattgatt attgactagt tattaatagt aatcaattac ggggtcatta gttcatagcc
       61 catatatgga gttccgcgtt acataactta cggtaaatgg cccgcctggc tgaccgccca
      121 acgacccccg cccattgacg tcaataatga cgtatgttcc catagtaacg ccaataggga
      181 ctttccattg acgtcaatgg gtggagtatt tacggtaaac tgcccacttg gcagtacatc
      241 aagtgtatca tatgccaagt acgcccccta ttgacgtcaa tgacggtaaa tggcccgcct
      301 ggcattatgc ccagtacatg accttatggg actttcctac ttggcagtac atctacgtat
      361 tagtcatcgc tattaccatg gtgatgcggt tttggcagta catcaatggg cgtggatagc
      421 ggtttgactc acggggattt ccaagtctcc accccattga cgtcaatggg agtttgtttt
      481 ggcaccaaaa tcaacgggac tttccaaaat gtcgtaacaa ctccgcccca ttgacgcaaa
      541 tgggcggtag gcgtgtacgg tgggaggtct atataagcag agctctctgg ctaactagag
      601 aacccactgc ttactggctt atcgaaatta atacgactca ctatagggag acccaagctt
      661 atgtccgtcc tgacgccgct gctgctgcgg ggcttgacag gctcggcccg gcggctccca
      721 gtgccgcgcg ccaagatcca ttcgttgggg gatcccatgt ccgtcctgac gccgctgctg
      781 ctgcggggct tgacaggctc ggcccggcgg ctcccagtgc cgcgcgccaa gatccattcg
      841 ttggggaagc ttatgtccgt cctgacgccg ctgctgctgc ggggcttgac aggctcggcc
      901 cggcggctcc cagtgccgcg cgccaagatc cattcgttgg gggatcccat gtccgtcctg
      961 acgccgctgc tgctgcgggg cttgacaggc tcggcccggc ggctcccagt gccgcgcgcc
     1021 aagatccatt cgttggggaa gcttgccacc atggtgagca agggcgagga gctgttcacc
     1081 ggggtggtgc ccatcctggt cgagctggac ggcgacgtaa acggccacaa gttcagcgtc
     1141 cgcggcgagg gcgagggcga tgccaccaac ggcaaactgt ccctgaagct catctccacc
     1201 accggcaagc tgcccgtgcc ctggcccacc ctcgtgacca ccttaagcta cggccagatg
     1261 gtgttcgccc gctaccctga caacatgaag cagcacgact tcttcaagtc cgccatgccc
     1321 gaaggctacg tccaggagcg caccatctct ttcgaggacg acggttacta caagacccgc
     1381 gccgaggtga agttcgaggg cgacaccctg gtgaaccgca tcgtgctgaa gggcatcgac
     1441 ttcaaggagg acggcaacat cctggggcac aacctggagt acaacttcaa cagccacaac
     1501 gtctatatca cggccgacaa gcagaagaac ggcatcaagg ctaacttcaa gatccgccac
     1561 aacgttgagg acggcggcgt gcagctcgcc gaccactacc agcagaacac ccccatcggc
     1621 gacggccccg tgctgctgcc cgacaaccac tacctgagca tccagtccaa gctgagcaaa
     1681 gaccccaacg agaagcgcga tcacatggtc ctgaaggaga gggtgaccgc cgccgggatt
     1741 acacatgaca tgaacgagct gtacaagtaa tctagagggc cctattctat agtgtcacct
     1801 aaatgctaga gctcgctgat cagcctcgac tgtgccttct agttgccagc catctgttgt
     1861 ttgcccctcc cccgtgcctt ccttgaccct ggaaggtgcc actcccactg tcctttccta
     1921 ataaaatgag gaaattgcat cgcattgtct gagtaggtgt cattctattc tggggggtgg
     1981 ggtggggcag gacagcaagg gggaggattg ggaagacaat agcaggcatg ctggggatgc
     2041 ggtgggctct atggcttctg aggcggaaag aaccagctgg ggctctaggg ggtatcccca
     2101 cgcgccctgt agcggcgcat taagcgcggc gggtgtggtg gttacgcgca gcgtgaccgc
     2161 tacacttgcc agcgccctag cgcccgctcc tttcgctttc ttcccttcct ttctcgccac
     2221 gttcgccggc tttccccgtc aagctctaaa tcgggggctc cctttagggt tccgatttag
     2281 tgctttacgg cacctcgacc ccaaaaaact tgattagggt gatggttcac gtagtgggcc
     2341 atcgccctga tagacggttt ttcgcccttt gacgttggag tccacgttct ttaatagtgg
     2401 actcttgttc caaactggaa caacactcaa ccctatctcg gtctattctt ttgatttata
     2461 agggattttg ccgatttcgg cctattggtt aaaaaatgag ctgatttaac aaaaatttaa
     2521 cgcgaattaa ttctgtggaa tgtgtgtcag ttagggtgtg gaaagtcccc aggctcccca
     2581 gcaggcagaa gtatgcaaag catgcatctc aattagtcag caaccaggtg tggaaagtcc
     2641 ccaggctccc cagcaggcag aagtatgcaa agcatgcatc tcaattagtc agcaaccata
     2701 gtcccgcccc taactccgcc catcccgccc ctaactccgc ccagttccgc ccattctccg
     2761 ccccatggct gactaatttt ttttatttat gcagaggccg aggccgcctc tgcctctgag
     2821 ctattccaga agtagtgagg aggctttttt ggaggcctag gcttttgcaa aaagctcccg
     2881 ggagcttgta tatccatttt cggatctgat caagagacag gatgaggatc gtttcgcatg
     2941 attgaacaag atggattgca cgcaggttct ccggccgctt gggtggagag gctattcggc
     3001 tatgactggg cacaacagac aatcggctgc tctgatgccg ccgtgttccg gttgtcagcg
     3061 caggggcgcc cggttctttt tgtcaagacc gacctgtccg gtgccctgaa tgaactgcag
     3121 gacgaggcag cgcggctatc gtggctggcc acgacgggcg ttccttgcgc agctgtgctc
     3181 gacgttgtca ctgaagcggg aagggactgg ctgctattgg gcgaagtgcc ggggcaggat
     3241 ctcctgtcat ctcaccttgc tcctgccgag aaagtatcca tcatggctga tgcaatgcgg
     3301 cggctgcata cgcttgatcc ggctacctgc ccattcgacc accaagcgaa acatcgcatc
     3361 gagcgagcac gtactcggat ggaagccggt cttgtcgatc aggatgatct ggacgaagag
     3421 catcaggggc tcgcgccagc cgaactgttc gccaggctca aggcgcgcat gcccgacggc
     3481 gaggatctcg tcgtgaccca tggcgatgcc tgcttgccga atatcatggt ggaaaatggc
     3541 cgcttttctg gattcatcga ctgtggccgg ctgggtgtgg cggaccgcta tcaggacata
     3601 gcgttggcta cccgtgatat tgctgaagag cttggcggcg aatgggctga ccgcttcctc
     3661 gtgctttacg gtatcgccgc tcccgattcg cagcgcatcg ccttctatcg ccttcttgac
     3721 gagttcttct gagcgggact ctggggttcg aaatgaccga ccaagcgacg cccaacctgc
     3781 catcacgaga tttcgattcc accgccgcct tctatgaaag gttgggcttc ggaatcgttt
     3841 tccgggacgc cggctggatg atcctccagc gcggggatct catgctggag ttcttcgccc
     3901 accccaactt gtttattgca gcttataatg gttacaaata aagcaatagc atcacaaatt
     3961 tcacaaataa agcatttttt tcactgcatt ctagttgtgg tttgtccaaa ctcatcaatg
     4021 tatcttatca tgtctgtata ccgtcgacct ctagctagag cttggcgtaa tcatggtcat
     4081 agctgtttcc tgtgtgaaat tgttatccgc tcacaattcc acacaacata cgagccggaa
     4141 gcataaagtg taaagcctgg ggtgcctaat gagtgagcta actcacatta attgcgttgc
     4201 gctcactgcc cgctttccag tcgggaaacc tgtcgtgcca gctgcattaa tgaatcggcc
     4261 aacgcgcggg gagaggcggt ttgcgtattg ggcgctcttc cgcttcctcg ctcactgact
     4321 cgctgcgctc ggtcgttcgg ctgcggcgag cggtatcagc tcactcaaag gcggtaatac
     4381 ggttatccac agaatcaggg gataacgcag gaaagaacat gtgagcaaaa ggccagcaaa
     4441 aggccaggaa ccgtaaaaag gccgcgttgc tggcgttttt ccataggctc cgcccccctg
     4501 acgagcatca caaaaatcga cgctcaagtc agaggtggcg aaacccgaca ggactataaa
     4561 gataccaggc gtttccccct ggaagctccc tcgtgcgctc tcctgttccg accctgccgc
     4621 ttaccggata cctgtccgcc tttctccctt cgggaagcgt ggcgctttct catagctcac
     4681 gctgtaggta tctcagttcg gtgtaggtcg ttcgctccaa gctgggctgt gtgcacgaac
     4741 cccccgttca gcccgaccgc tgcgccttat ccggtaacta tcgtcttgag tccaacccgg
     4801 taagacacga cttatcgcca ctggcagcag ccactggtaa caggattagc agagcgaggt
     4861 atgtaggcgg tgctacagag ttcttgaagt ggtggcctaa ctacggctac actagaagaa
     4921 cagtatttgg tatctgcgct ctgctgaagc cagttacctt cggaaaaaga gttggtagct
     4981 cttgatccgg caaacaaacc accgctggta gcggtggttt ttttgtttgc aagcagcaga
     5041 ttacgcgcag aaaaaaagga tctcaagaag atcctttgat cttttctacg gggtctgacg
     5101 ctcagtggaa cgaaaactca cgttaaggga ttttggtcat gagattatca aaaaggatct
     5161 tcacctagat ccttttaaat taaaaatgaa gttttaaatc aatctaaagt atatatgagt
     5221 aaacttggtc tgacagttac caatgcttaa tcagtgaggc acctatctca gcgatctgtc
     5281 tatttcgttc atccatagtt gcctgactcc ccgtcgtgta gataactacg atacgggagg
     5341 gcttaccatc tggccccagt gctgcaatga taccgcgaga cccacgctca ccggctccag
     5401 atttatcagc aataaaccag ccagccggaa gggccgagcg cagaagtggt cctgcaactt
     5461 tatccgcctc catccagtct attaattgtt gccgggaagc tagagtaagt agttcgccag
     5521 ttaatagttt gcgcaacgtt gttgccattg ctacaggcat cgtggtgtca cgctcgtcgt
     5581 ttggtatggc ttcattcagc tccggttccc aacgatcaag gcgagttaca tgatccccca
     5641 tgttgtgcaa aaaagcggtt agctccttcg gtcctccgat cgttgtcaga agtaagttgg
     5701 ccgcagtgtt atcactcatg gttatggcag cactgcataa ttctcttact gtcatgccat
     5761 ccgtaagatg cttttctgtg actggtgagt actcaaccaa gtcattctga gaatagtgta
     5821 tgcggcgacc gagttgctct tgcccggcgt caatacggga taataccgcg ccacatagca
     5881 gaactttaaa agtgctcatc attggaaaac gttcttcggg gcgaaaactc tcaaggatct
     5941 taccgctgtt gagatccagt tcgatgtaac ccactcgtgc acccaactga tcttcagcat
     6001 cttttacttt caccagcgtt tctgggtgag caaaaacagg aaggcaaaat gccgcaaaaa
     6061 agggaataag ggcgacacgg aaatgttgaa tactcatact cttccttttt caatattatt
     6121 gaagcattta tcagggttat tgtctcatga gcggatacat atttgaatgt atttagaaaa
     6181 ataaacaaat aggggttccg cgcacatttc cccgaaaagt gccacctgac gtcgacggat
     6241 cgggagatct cccgatcccc tatggtgcac tctcagtaca atctgctctg atgccgcata
     6301 gttaagccag tatctgctcc ctgcttgtgt gttggaggtc gctgagtagt gcgcgagcaa
     6361 aatttaagct acaacaaggc aaggcttgac cgacaattgc atgaagaatc tgcttagggt
     6421 taggcgtttt gcgctgcttc gcgatgtacg ggccagatat acgcgtt
//
