LOCUS       sequence-469932-        7859 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Lentivirus plasmid for the stable expression of mitochondrial 
            matrix-localized BFP in mammalian cells.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 7859)
  AUTHORS   Rose K, Herrmann E, Kakudji E, Lizarrondo J, Celebi AY, Wilfling F, 
            Lewis SC, Hurley JH
  TITLE     In situ cryo-ET visualization of mitochondrial depolarization and 
            mitophagic engulfment.
  JOURNAL   Proc Natl Acad Sci U S A. 2025 Aug 5;122(31):e2511890122. doi: 
            10.1073/pnas.2511890122. Epub 2025 Jul 31.
  PUBMED    40743392
REFERENCE   2  (bases 1 to 7859)
  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: "Proc Natl 
            Acad Sci U S A."; date: "2025-08-5"; volume: "122(31):e2511890122. 
            doi"; pages: " 10.1073/pnas.2511890122. Epub 2025 Jul 31"
COMMENT     Sequence Label: pLV-BFP-Mito
FEATURES             Location/Qualifiers
     source          1..7859
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     80..99
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     promoter        174..278
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(279..347,348..1139)
                     /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     complement(497..516)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      1310..1898
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     1799..1818
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     LTR             1960..2140
                     /label=5' LTR (truncated)
                     /note="truncated 5' long terminal repeat (LTR) from HIV-1"
     primer_bind     2649..2665
                     /label=pBluescriptKS
                     /note="For pBluescript vector"
     primer_bind     2650..2666
                     /label=KS primer
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     misc_feature    2669..3257
                     /label=WPRE
                     /note="woodchuck hepatitis virus posttranscriptional 
                     regulatory element"
     CDS             2775..2786
                     /codon_start=1
                     /product="Factor Xa recognition and cleavage site"
                     /label=Factor Xa site
                     /translation="IEGR"
     primer_bind     3184..3204
                     /label=WPRE-R
                     /note="WPRE, reverse primer"
     CDS             complement(join(3348..4061,4062..4064,4065..4067))
                     /codon_start=1
                     /product="enhanced blue variant of GFP (Ai et al., 2007)"
                     /label=EBFP2
                     /note="mammalian codon-optimized"
                     /translation="MVSKGEELFTGVVPILVELDGDVNGHKFSVRGEGEGDATNGKLTL
                     KFICTTGKLPVPWPTLVTTLSHGVQCFARYPDHMKQHDFFKSAMPEGYVQERTIFFKDD
                     GTYKTRAEVKFEGDTLVNRIELKGVDFKEDGNILGHKLEYNFNSHNIYIMAVKQKNGIK
                     VNFKIRHNVEDGSVQLADHYQQNTPIGDGPVLLPDSHYLSTQSVLSKDPNEKRDHMVLL
                     EFRTAAGITLGMDELYK"
     primer_bind     4001..4022
                     /label=EGFP-N
                     /note="EGFP, reverse primer"
     regulatory      4064..4073
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     CDS             complement(4101..4175)
                     /codon_start=1
                     /product="mitochondrial presequence of human cytochrome c 
                     oxidase subunit VIII (Rizutto et al., 1989)"
                     /label=COX8 presequence
                     /translation="MSVLTPLLLRGLTGSARRLPVPRAK"
     intron          4191..5129
                     /label=EF-1-alpha intron A
                     /note="intron upstream of the start codon of human 
                     EF-1-alpha"
     primer_bind     complement(4214..4234)
                     /label=EF1a-F
                     /note="Human elongation factor-1a promoter, forward primer"
     promoter        complement(5130..5341)
                     /label=EF-1-alpha core promoter
                     /note="core promoter for human elongation factor 
                     EF-1-alpha"
     misc_feature    5444..5561
                     /label=cPPT/CTS
                     /note="central polypurine tract and central termination 
                     sequence of HIV-1"
     CDS             complement(5859..5903)
                     /codon_start=1
                     /product="antigenic peptide corresponding to amino acids 
                     655 to 669 of the HIV envelope protein gp41 (Lutje Hulsik 
                     et al., 2013)"
                     /label=gp41 peptide
                     /note="recognized by the 2H10 single-chain llama nanobody"
                     /translation="KNEQELLELDKWASL"
     misc_feature    6088..6321
                     /label=RRE
                     /note="The Rev response element (RRE) of HIV-1 allows for 
                     Rev-dependent mRNA export from the nucleus to the 
                     cytoplasm."
     misc_feature    6818..6943
                     /label=HIV-1 Psi
                     /note="packaging signal of human immunodeficiency virus 
                     type 1"
     LTR             6990..7170
                     /label=5' LTR (truncated)
                     /note="truncated 5' long terminal repeat (LTR) from HIV-1"
     promoter        complement(7188..7386)
                     /label=CMV promoter
                     /note="human cytomegalovirus (CMV) immediate early 
                     promoter"
     primer_bind     complement(7213..7233)
                     /label=CMV-F
                     /note="Human CMV immediate early promoter, forward primer"
     enhancer        7388..7767
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
ORIGIN
        1 gttgtagctt aaattttgct cgcgcactac tcagcgacct ccaacacaca agcagggagc
       61 agatactggc ttaactatgc ggcatcagag cagattgtac tgagagtgca ccatagggga
      121 tcgggagatc tcccgatccg tcgacgtcag gtggcacttt tcggggaaat gtgcgcggaa
      181 cccctatttg tttatttttc taaatacatt caaatatgta tccgctcatg agacaataac
      241 cctgataaat gcttcaataa tattgaaaaa ggaagagtat gagtattcaa catttccgtg
      301 tcgcccttat tccctttttt gcggcatttt gccttcctgt ttttgctcac ccagaaacgc
      361 tggtgaaagt aaaagatgct gaagatcagt tgggtgcacg agtgggttac atcgaactgg
      421 atctcaacag cggtaagatc cttgagagtt ttcgccccga agaacgtttt ccaatgatga
      481 gcacttttaa agttctgcta tgtggcgcgg tattatcccg tattgacgcc gggcaagagc
      541 aactcggtcg ccgcatacac tattctcaga atgacttggt tgagtactca ccagtcacag
      601 aaaagcatct tacggatggc atgacagtaa gagaattatg cagtgctgcc ataaccatga
      661 gtgataacac tgcggccaac ttacttctga caacgatcgg aggaccgaag gagctaaccg
      721 cttttttgca caacatgggg gatcatgtaa ctcgccttga tcgttgggaa ccggagctga
      781 atgaagccat accaaacgac gagcgtgaca ccacgatgcc tgtagcaatg gcaacaacgt
      841 tgcgcaaact attaactggc gaactactta ctctagcttc ccggcaacaa ttaatagact
      901 ggatggaggc ggataaagtt gcaggaccac ttctgcgctc ggcccttccg gctggctggt
      961 ttattgctga taaatctgga gccggtgagc gtgggtctcg cggtatcatt gcagcactgg
     1021 ggccagatgg taagccctcc cgtatcgtag ttatctacac gacggggagt caggcaacta
     1081 tggatgaacg aaatagacag atcgctgaga taggtgcctc actgattaag cattggtaac
     1141 tgtcagacca agtttactca tatatacttt agattgattt aaaacttcat ttttaattta
     1201 aaaggatcta ggtgaagatc ctttttgata atctcatgac caaaatccct taacgtgagt
     1261 tttcgttcca ctgagcgtca gaccccgtag aaaagatcaa aggatcttct tgagatcctt
     1321 tttttctgcg cgtaatctgc tgcttgcaaa caaaaaaacc accgctacca gcggtggttt
     1381 gtttgccgga tcaagagcta ccaactcttt ttccgaaggt aactggcttc agcagagcgc
     1441 agataccaaa tactgttctt ctagtgtagc cgtagttagg ccaccacttc aagaactctg
     1501 tagcaccgcc tacatacctc gctctgctaa tcctgttacc agtggctgct gccagtggcg
     1561 ataagtcgtg tcttaccggg ttggactcaa gacgatagtt accggataag gcgcagcggt
     1621 cgggctgaac ggggggttcg tgcacacagc ccagcttgga gcgaacgacc tacaccgaac
     1681 tgagatacct acagcgtgag ctatgagaaa gcgccacgct tcccgaaggg agaaaggcgg
     1741 acaggtatcc ggtaagcggc agggtcggaa caggagagcg cacgagggag cttccagggg
     1801 gaaacgcctg gtatctttat agtcctgtcg ggtttcgcca cctctgactt gagcgtcgat
     1861 ttttgtgatg ctcgtcaggg gggcggagcc tatggaaaaa cgccagcaac gcggcctttt
     1921 tacggttcct ggccttttgc tggccttttg ctcacatgct gctagagatt ttccacactg
     1981 actaaaaggg tctgagggat ctctagttac cagagtcaca caacagacgg gcacacacta
     2041 cttgaagcac tcaaggcaag ctttattgag gcttaagcag tgggttccct agttagccag
     2101 agagctccca ggctcagatc tggtctaacc agagagaccc agtacagtcc ggatgcagct
     2161 ctcgggccat gtgatgaaat gctaggcggc tgtcaaacct ccactctaat acttctctct
     2221 ccgggtcatc catcccatgc aggctcacag ggtgtaacaa gcgggtgttc tctccttcat
     2281 tggcttcttc taccttctct tgctcaactg gtactagctt gtagcaccat ccaaaggtca
     2341 gtggatatct gatccctggc cctggtgtgt agttctgcca atcagggaag tagccttgtg
     2401 tgtggtagat ccacagatca aggatatctt gtcttcgttg ggagtgaatt agcccttcca
     2461 gtcccccctt ttcttttaaa aagtggctaa gatctacagc tgccttgtaa gtcattggtc
     2521 ttaaaggtac ctgaggtgtg actggaaaac ccacctcctc ctcctcttgt gcttctagcc
     2581 aggcacaatc agcattggta gctgctgtat tgctacttgt gattgctcca tgtttttcta
     2641 ggtctcgatc gaggtcgacg gtatcgatgc ggggaggcgg cccaaaggga gatccgactc
     2701 gtctgagggc gaaggcgaag acgcggaaga ggccgcagag ccggcagcag gccgcgggaa
     2761 ggaaggtccg ctggattgag ggccgaaggg acgtagcaga aggacgtccc gcgcagaatc
     2821 caggtggcaa cacaggcgag cagccaagga aaggacgatg atttccccga caacaccacg
     2881 gaattgtcag tgcccaacag ccgagcccct gtccagcagc gggcaaggca ggcggcgatg
     2941 agttccgccg tggcaatagg gagggggaaa gcgaaagtcc cggaaaggag ctgacaggtg
     3001 gtggcaatgc cccaaccagt gggggttgcg tcagcaaaca cagtgcacac cacgccacgt
     3061 tgcctgacaa cgggccacaa ctcctcataa agagacagca accaggattt atacaaggag
     3121 gagaaaatga aagccatacg ggaagcaata gcatgataca aaggcattaa agcagcgtat
     3181 ccacatagcg taaaaggagc aacatagtta agaataccag tcaatctttc acaaattttg
     3241 taatccagag gttgattatc gataagcttg atatcgaatt gtacctagtg gaaccggaac
     3301 ccttaaacat gtctcgacct ggcttactag tcggttcaac tctaggttta cttgtacagc
     3361 tcgtccatgc cgagagtgat cccggcggcg gtgcggaact ccagcaggac catgtgatcg
     3421 cgcttctcgt tggggtcttt gctcagcacg gactgggtgc tcaggtagtg gctgtcgggc
     3481 agcagcacgg ggccgtcgcc gatgggggtg ttctgctggt agtggtcggc gagctgcacg
     3541 ctgccgtcct ccacgttgtg gcggatcttg aagttcacct tgatgccgtt cttctgcttg
     3601 acggccatga tatagatgtt gtggctgttg aagttgtact ccagcttgtg ccccaggatg
     3661 ttgccgtcct ccttgaagtc gacgcccttc agctcgatgc ggttcaccag ggtgtcgccc
     3721 tcgaacttca cctcggcgcg ggtcttgtag gtgccgtcgt ccttgaagaa gatggtgcgc
     3781 tcctggacgt agccttcggg catggcggac ttgaagaagt cgtgctgctt catgtggtcg
     3841 gggtagcggg cgaagcactg cacgccgtgg ctcagggtgg tcacgagggt gggccagggc
     3901 acgggcagct tgccggtggt gcagatgaac ttcagggtca gcttgccgtt ggtggcatcg
     3961 ccctcgccct cgcccctcac gctgaacttg tggccgttta cgtcgccgtc cagctcgacc
     4021 aggatgggca ccaccccggt gaacagctcc tcgcccttgc tcaccatggt ggcgaccggt
     4081 ggatccccca acgaatggat cttggcgcgc ggcactggga gccgccgggc cgagcctgtc
     4141 aagccccgca gcagcagcgg cgtcaggacg gacatgctag ctcacgacac ctgaaatgga
     4201 agaaaaaaac tttgaaccac tgtctgaggc ttgagaatga accaagatcc aaactcaaaa
     4261 agggcaaatt ccaaggagaa ttacatcaag tgccaagctg gcctaacttc agtctccacc
     4321 cactcagtgt ggggaaactc catcgcataa aacccctccc cccaacctaa agacgacgta
     4381 ctccaaaagc tcgagaacta atcgaggtgc ctggacggcg cccggtactc cgtggagtca
     4441 catgaagcga cggctgagga cggaaaggcc cttttccttt gtgtgggtga ctcacccgcc
     4501 cgctctcccg agcgccgcgt cctccatttt gagctccctg cagcagggcc gggaagcggc
     4561 catctttccg ctcacgcaac tggtgccgac cgggccagcc ttgccgccca gggcggggcg
     4621 atacacggcg gcgcgaggcc aggcaccaga gcaggccggc cagcttgaga ctacccccgt
     4681 ccgattctcg gtggccgcgc tcgcaggccc cgcctcgccg aacatgtgcg ctgggacgca
     4741 cgggccccgt cgccgcccgc ggccccaaaa accgaaatac cagtgtgcag atcttggccc
     4801 gcatttacaa gactatcttg ccagaaaaaa agcgtcgcag caggtcatca aaaattttaa
     4861 atggctagag acttatcgaa agcagcgaga caggcgcgaa ggtgccacca gattcgcacg
     4921 cggcggcccc agcgcccagg ccaggcctca actcaagcac gaggcgaagg ggctccttaa
     4981 gcgcaaggcc tcgaactctc ccacccactt ccaacccgaa gctcgggatc aagaatcacg
     5041 tactgcagcc aggtggaagt aattcaaggc acgcaagggc cataacccgt aaagaggcca
     5101 ggcccgcggg aaccacacac ggcacttacc tgtgttctgg cggcaaaccc gttgcgaaaa
     5161 agaacgttca cggcgactac tgcacttata tacggttctc ccccaccctc gggaaaaagg
     5221 cggagccagt acacgacatc actttcccag tttaccccgc gccaccttct ctaggcaccg
     5281 gttcaattgc cgacccctcc ccccaacttc tcggggactg tgggcgatgt gcgctctgcc
     5341 cactgacggg cacatgcatg gcggtaatac ggttatccac gcggccctag agcgggcccg
     5401 gtactaacca aactggatct ctgctgtccc tgtaataaac ccgaaaattt tgaatttttg
     5461 taatttgttt ttgtaattct ttagtttgta tgtctgttgc tattatgtct actattcttt
     5521 cccctgcact gtacccccca atcccccctt ttcttttaaa attgtggatg aatactgcca
     5581 tttgtctgca gaattggcgc acgcagtgcc gatccgttca ctaatcgaat ggatctgtct
     5641 ctgtctctct ctccaccttc ttcttctatt ccttcgggcc tgtcgggtcc cctcggggtt
     5701 gggaggtggg tctgaaacga taatggtgaa tatccctgcc taactctatt cactatagaa
     5761 agtacagcaa aaactattct taaacctacc aagcctccta ctatcattat gaataatttt
     5821 atataccaca gccaatttgt tatgttaaac caattccaca aacttgccca tttatctaat
     5881 tccaataatt cttgttcatt cttttcttgc tggttttgcg attcttcaat taaggagtgt
     5941 attaagcttg tgtaattgtt aatttctctg tcccactcca tccaggtcgt gtgattccaa
     6001 atctgttcca gagatttatt actccaacta gcattccaag gcacagcagt ggtgcaaatg
     6061 agttttccag agcaacccca aatccccagg agctgttgat cctttaggta tctttccaca
     6121 gccaggattc ttgcctggag ctgcttgatg ccccagactg tgagttgcaa cagatgctgt
     6181 tgcgcctcaa tagccctcag caaattgttc tgctgctgca ctataccaga caataattgt
     6241 ctggcctgta ccgtcagcgt cattgacgct gcgcccatag tgcttcctgc tgctcccaag
     6301 aacccaagga acaaagctcc tattcccact gctctttttt ctctctgcac cactcttctc
     6361 tttgccttgg tgggtgctac tcctaatggt tcaattttta ctactttata tttatataat
     6421 tcacttctcc aattgtccct catatctcct cctccaggtc tgaagatcag cggccggccg
     6481 cttgctgtgc ggtggtctta cttttgtttt gctcttcctc tatcttgtct aaagcttcct
     6541 tggtgtcttt tatctctatc ctttgatgca cacaatagag ggttgctact gtattatata
     6601 atgatctaag ttcttctgat cctgtctgaa gggatggttg tagctgtccc agtatttgtc
     6661 tacagccttc tgatgtttct aacaggccag gattaactgc gaatcgttct agctccctgc
     6721 ttgcccatac tatatgtttt aatttatatt ttttctttcc ccctggcctt aaccgaattt
     6781 tttcccatcg cgatctaatt ctcccccgct taatactgac gctctcgcac ccatctctct
     6841 ccttctagcc tccgctagtc aaaatttttg gcgtactcac cagtcgccgc ccctcgcctc
     6901 ttgccgtgcg cgcttcagca agccgagtcc tgcgtcgaga gagctcctct ggtttccctt
     6961 tcgctttcaa gtccctgttc gggcgccact gctagagatt ttccacactg actaaaaggg
     7021 tctgagggat ctctagttac cagagtcaca caacagacgg gcacacacta cttgaagcac
     7081 tcaaggcaag ctttattgag gcttaagcag tgggttccct agttagccag agagctccca
     7141 ggctcagatc tggtctaacc agagagaccc agtacaggca aaacgcgctg cttatataga
     7201 cctcccaccg tacacgccta ccgcccattt gcgtcaatgg ggcggagttg ttacgacatt
     7261 ttggaaagtc ccgttgattt tggtgccaaa acaaactccc attgacgtca atggggtgga
     7321 gacttggaaa tccccgtgag tcaaaccgct atccacgccc attgatgtac tgccaaaacc
     7381 gcatcaccat ggtaatagcg atgactaata cgtagatgta ctgccaagta ggaaagtccc
     7441 ataaggtcat gtactgggca taatgccagg cgggccattt accgtcattg acgtcaatag
     7501 ggggcgtact tggcatatga tacacttgat gtactgccaa gtgggcagtt taccgtaaat
     7561 actccaccca ttgacgtcaa tggaaagtcc ctattggcgt tactatggga acatacgtca
     7621 ttattgacgt caatgggcgg gggtcgttgg gcggtcagcc aggcgggcca tttaccgtaa
     7681 gttatgtaac gcggaactcc atatatgggc tatgaactaa tgaccccgta attgattact
     7741 attaataact agtcaataat caatgtcaac gcgtatatct ggcccgtaca tcgcgaagca
     7801 gcgcaaaacg cctaacccta agcagattct tcatgcaatt gtcggtcaag ccttgcctt
//
