LOCUS       sequence-469412-        7892 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Stable expression of EGFP-LOV2(N538E)-BAX(G108V)-OMP25 in mammalian 
            cells. The G108V mutation suppresses pore-forming activity of BAX..
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 7892)
  AUTHORS   Nagashima Y, Eguchi T, Koyama-Honda I, Mizushima N
  TITLE     Optogenetic tools for inducing organelle membrane rupture.
  JOURNAL   J Biol Chem. 2025 Mar 18;301(4):108421. doi: 
            10.1016/j.jbc.2025.108421.
  PUBMED    40113041
REFERENCE   2  (bases 1 to 7892)
  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 Biol 
            Chem."; date: "2025-03-18"; volume: "301(4):108421. doi"; pages: " 
            10.1016/j.jbc.2025.108421"
COMMENT     Sequence Label: pMRX-IP-EGFP-PA-BAX(mito, G108V)
FEATURES             Location/Qualifiers
     source          1..7892
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     enhancer        1..380
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
     misc_feature    739..1096
                     /label=MMLV Psi
                     /note="packaging signal of Moloney murine leukemia virus 
                     (MMLV)"
     CDS             1161..1577
                     /label=gag (truncated)
                     /note="truncated Moloney murine leukemia virus (MMLV) gag 
                     gene lacking the start codon"
     primer_bind     1507..1529
                     /label=pLXSN 5'
                     /note="Murine stem cell virus, forward primer. Also called 
                     MSCV"
     primer_bind     1545..1561
                     /label=pBABE 5'
                     /note="Psi packaging signal, forward primer for pBABE 
                     vectors"
     misc_feature    1587..1961
                     /label=pol region
                     /note="Moloney murine leukemia virus (MMLV) pol region 
                     containing the splice acceptor site"
     primer_bind     1899..1915
                     /label=pBMN 5'
                     /note="MMLV sequence, for inserts in pBMN retroviral 
                     vector"
     CDS             join(1979..1981,1982..1984,1985..2695)
                     /codon_start=1
                     /product="enhanced GFP"
                     /label=EGFP
                     /translation="MVSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTL
                     KFICTTGKLPVPWPTLVTTLTYGVQCFSRYPDHMKQHDFFKSAMPEGYVQERTIFFKDD
                     GNYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIK
                     VNFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSALSKDPNEKRDHMVLL
                     EFVTAAGITLGMDELYK"
     CDS             join(1979..1981,1982..1984,1985..2695)
                     /codon_start=1
                     /product="the original enhanced GFP (Yang et al., 1996)"
                     /label=EGFP
                     /note="mammalian codon-optimized"
                     /translation="MVSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTL
                     KFICTTGKLPVPWPTLVTTLTYGVQCFSRYPDHMKQHDFFKSAMPEGYVQERTIFFKDD
                     GNYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIK
                     VNFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSALSKDPNEKRDHMVLL
                     EFVTAAGITLGMDELYK"
     primer_bind     complement(2024..2045)
                     /label=EGFP-N
                     /note="EGFP, reverse primer"
     primer_bind     complement(2285..2304)
                     /label=EXFP-R
                     /note="For distinguishing EGFP variants, reverse primer"
     primer_bind     2632..2653
                     /label=EGFP-C
                     /note="EGFP, forward primer"
     CDS             2726..3154
                     /codon_start=1
                     /gene="NPH1 from Avena sativa"
                     /product="light, oxygen, or voltage domain from phototropin
                     1 of Avena sativa (Herrou and Crosson, 2011)"
                     /label=LOV2
                     /note="photoswitch that undergoes a light-inducible 
                     conformational change"
                     /translation="LATTLERIEKNFVITDPRLPDNPIIFASDSFLQLTEYSREEILGR
                     NCRFLQGPETDRATVRKIRDAIDNQTEVTVQLINYTKSGKKFWNLFHLQPMRDQKGDVQ
                     YFIGVQLDGTEHVRDAAEREGVMLIKKTAEEIDEAAKEL"
     misc_feature    3803..4369
                     /label=IRES
                     /note="internal ribosome entry site (IRES) of the 
                     encephalomyocarditis virus (EMCV)"
     misc_feature    3819..4371
                     /label=IRES
                     /note="internal ribosome entry site (IRES) of the 
                     encephalomyocarditis virus (EMCV)"
     primer_bind     complement(3963..3980)
                     /label=IRES reverse
                     /note="IRES internal ribosome entry site, reverse primer. 
                     Also called pCDH-rev"
     primer_bind     4190..4209
                     /label=IRES-F
                     /note="IRES internal ribosome entry site, forward primer"
     CDS             4389..4988
                     /codon_start=1
                     /gene="pac from Streptomyces alboniger"
                     /product="puromycin N-acetyltransferase"
                     /label=PuroR
                     /note="confers resistance to puromycin"
                     /translation="MTEYKPTVRLATRDDVPRAVRTLAAAFADYPATRHTVDPDRHIER
                     VTELQELFLTRVGLDIGKVWVADDGAAVAVWTTPESVEAGAVFAEIGPRMAELSGSRLA
                     AQQQMEGLLAPHRPKEPAWFLATVGVSPDHQGKGLGSAVVLPGVEAAERAGVPAFLETS
                     APRNLPFYERLGFTVTADVEVPEGPRTWCMTRKPGA"
     primer_bind     complement(4389..4408)
                     /label=Puro-R
                     /note="Puromycin resistance gene, reverse primer. Also 
                     called puro-variant-R"
     primer_bind     4885..4905
                     /label=Puro-F
                     /note="Puromycin resistance gene, forward primer"
     LTR             5218..5687
                     /note="long terminal repeat from Moloney murine leukemia 
                     virus"
     primer_bind     complement(5817..5834)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(5988..6576)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(6068..6087)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(6740..7531,7532..7600))
                     /codon_start=1
                     /gene="bla"
                     /product="beta-lactamase"
                     /label=AmpR
                     /note="confers resistance to ampicillin, carbenicillin, and
                     related antibiotics"
                     /translation="MSIQHFRVALIPFFAAFCLPVFAHPETLVKVKDAEDQLGARVGYI
                     ELDLNSGKILESFRPEERFPMMSTFKVLLCGAVLSRVDAGQEQLGRRIHYSQNDLVEYS
                     PVTEKHLTDGMTVRELCSAAITMSDNTAANLLLTTIGGPKELTAFLHNMGDHVTRLDRW
                     EPELNEAIPNDERDTTMPVAMATTLRKLLTGELLTLASRQQLIDWMEADKVAGPLLRSA
                     LPAGWFIADKSGAGERGSRGIIAALGPDGKPSRIVVIYTTGSQATMDERNRQIAEIGAS
                     LIKHW"
     primer_bind     7363..7382
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(7601..7705)
                     /gene="bla"
                     /label=AmpR promoter
     primer_bind     7773..7791
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
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 ctagagaacc atcagatgtt tccagggtgc cccaaggacc
      421 tgaaatgacc ctgtgcctta tttgaactaa ccaatcagtt cgcttctcgc ttctgttcgc
      481 gcgcttctgc tccccgagct caataaaaga gcccacaacc cctcactcgg cgcgccagtc
      541 ctccgattga ctgagtcgcc cgggtacccg tgtatccaat aaaccctctt gcagttgcat
      601 ccgacttgtg gtctcgctgt tccttgggag ggtctcctct gagtgattga ctacccgtca
      661 gcgggggtct ttcatttggg ggctcgtccg ggatcgggag acccctgccc agggaccacc
      721 gacccaccac cgggaggtaa gctggccagc aacttatctg tgtctgtccg attgtctagt
      781 gtctatgact gattttatgc gcctgcgtcg gtactagtta gctaactagc tctgtatctg
      841 gcggacccgt ggtggaactg acgagttcgg aacacccggc cgcaaccctg ggagacgtcc
      901 cagggacttc gggggccgtt tttgtggccc gacctgagtc caaaaatccc gatcgttttg
      961 gactctttgg tgcacccccc ttagaggagg gatatgtggt tctggtagga gacgagaacc
     1021 taaaacagtt cccgcctccg tctgaatttt tgctttcggt ttgggaccga agccgcgccg
     1081 cgcgtcttgt ctgctgcagc atcgttctgt gttgtctctg tctgactgtg tttctgtatt
     1141 tgtctgaaaa tatgggcccg ggccagactg ttaccactcc cttaagtttg accttaggtc
     1201 actggaaaga tgtcgagcgg atcgctcaca accagtcggt agatgtcaag aagagacgtt
     1261 gggttacctt ctgctctgca gaatggccaa cctttaacgt cggatggccg cgagacggca
     1321 cctttaaccg agacctcatc acccaggtta agatcaaggt cttttcacct ggcccgcatg
     1381 gacacccaga ccaggtcccc tacatcgtga cctgggaagc cttggctttt gacccccctc
     1441 cctgggtcaa gccctttgta caccctaagc ctccgcctcc tcttcctcca tccgccccgt
     1501 ctctccccct tgaacctcct cgttcgaccc cgcctcgatc ctccctttat ccagccctca
     1561 ctccttctct aggcgccccc atatggccat atgagatctt atatggggca cccccgcccc
     1621 ttgtaaactt ccctgaccct gacatgacaa gagttactaa cagcccctct ctccaagctc
     1681 acttacaggc tctctactta gtccagcacg aagtctggag acctctggcg gcagcctacc
     1741 aagaacaact ggaccgaccg gtggtacctc acccttaccg agtcggcgac acagtgtggg
     1801 tccgccgaca ccagactaag aacctagaac ctcgctggaa aggaccttac acagtcctgc
     1861 tgaccacccc caccgccctc aaagtagacg gcatcgcagc ttggatacac gccgcccacg
     1921 tgaaggctgc cgaccccggg ggtggaccat cctctagact gccggatctg ccatcgatat
     1981 ggtgagcaag ggcgaggagc tgttcaccgg ggtggtgccc atcctggtcg agctggacgg
     2041 cgacgtaaac ggccacaagt tcagcgtgtc cggcgagggc gagggcgatg ccacctacgg
     2101 caagctgacc ctgaagttca tctgcaccac cggcaagctg cccgtgccct ggcccaccct
     2161 cgtgaccacc ctgacctacg gcgtgcagtg cttcagccgc taccccgacc acatgaagca
     2221 gcacgacttc ttcaagtccg ccatgcccga aggctacgtc caggagcgca ccatcttctt
     2281 caaggacgac ggcaactaca agacccgcgc cgaggtgaag ttcgagggcg acaccctggt
     2341 gaaccgcatc gagctgaagg gcatcgactt caaggaggac ggcaacatcc tggggcacaa
     2401 gctggagtac aactacaaca gccacaacgt ctatatcatg gccgacaagc agaagaacgg
     2461 catcaaggtg aacttcaaga tccgccacaa catcgaggac ggcagcgtgc agctcgccga
     2521 ccactaccag cagaacaccc ccatcggcga cggccccgtg ctgctgcccg acaaccacta
     2581 cctgagcacc cagtccgccc tgagcaaaga ccccaacgag aagcgcgatc acatggtcct
     2641 gctggagttc gtgaccgccg ccgggatcac tctcggcatg gacgagctgt acaagggttc
     2701 aggctcggga ggcatgggga ctagtttggc tactacactt gaacgtattg agaagaactt
     2761 tgtcattact gacccaagat tgccagataa tcccattata ttcgcgtccg atagtttctt
     2821 gcagttgaca gaatatagcc gtgaagaaat tttgggaaga aactgcaggt ttctacaagg
     2881 tcctgaaact gatcgcgcga cagtgagaaa aattagagat gccatagata accaaacaga
     2941 ggtcactgtt cagctgatta attatacaaa gagtggtaaa aagttctgga acctctttca
     3001 cttgcagcct atgcgagatc agaagggaga tgtccagtac tttattgggg ttcagttgga
     3061 tggaactgag catgtccgag atgctgccga gagagaggga gtcatgctga ttaagaaaac
     3121 tgcagaagaa attgatgagg cggcaaaaga acttgggtcc ggggagcagc ccagaggcgg
     3181 ggggcccacc agctctgagc agatcatgaa gacaggggcc cttttgcttc agggtttcat
     3241 ccaggatcga gcagggcgaa tgggggggga ggcacccgag ctggccctgg acccggtgcc
     3301 tcaggatgcg tccaccaaga agctgagcga gtgtctcaag cgcatcgggg acgaactgga
     3361 cagtaacatg gagctgcaga ggatgattgc cgccgtggac acagactccc cccgagaggt
     3421 ctttttccga gtggcagctg acatgttttc tgacggcaac ttcaactggg ttcgggttgt
     3481 cgcccttttc tactttgcca gcaaactggt gctcaaggcc ctgtgcacca aggtgccgga
     3541 actgatcaga accatcatgg gctggacatt ggacttcctc cgggagcggc tgttgggctg
     3601 gatccaagac cagggtggtt gggacggcct cctctcctac tttgggacgc ccacgtggca
     3661 gagacatcga ggcgacggag agcctagtgg agttcctgta gctgtggtgc tgctgccagt
     3721 gtttgccctt accctggtag cagtttgggc cttcgtgaga taccgaaagc agctctgact
     3781 cgagagcggc cgctacgtaa attccgcccc cccccctaac gttactggcc gaagccgctt
     3841 ggaataaggc cggtgtgcgt ttgtctatat gttattttcc accatattgc cgtcttttgg
     3901 caatgtgagg gcccggaaac ctggccctgt cttcttgacg agcattccta ggggtctttc
     3961 ccctctcgcc aaaggaatgc aaggtctgtt gaatgtcgtg aaggaagcag ttcctctgga
     4021 agtttcttga agacaaacaa cgtctgtagc gaccctttgc aggcagcgga accccccacc
     4081 tggcgacagg tgcctctgcg gccaaaagcc acgtgtataa gatacacctg caaaggcggc
     4141 acaaccccag tgccacgttg tgagttggat agttgtggaa agagtcaaat ggctctcctc
     4201 aagcgtattc aacaaggggc tgaaggatgc ccagaaggta ccccattgta tgggatctga
     4261 tctggggcct cggtgcacat gctttacatg tgtttagtcg aggttaaaaa aacgtctagg
     4321 ccccccgaac cacggggacg tggttttcct ttgaaaaaca cgatgataat atggccacaa
     4381 ccgtcgacat gaccgagtac aagcccacgg tgcgcctcgc cacccgcgac gacgtccccc
     4441 gggccgtacg caccctcgcc gccgcgttcg ccgactaccc cgccacgcgc cacaccgtcg
     4501 acccggaccg ccacatcgag cgggtcaccg agctgcaaga actcttcctc acgcgcgtcg
     4561 ggctcgacat cggcaaggtg tgggtcgcgg acgacggcgc cgcggtggcg gtctggacca
     4621 cgccggagag cgtcgaagcg ggggcggtgt tcgccgagat cggcccgcgc atggccgagt
     4681 tgagcggttc ccggctggcc gcgcagcaac agatggaagg cctcctggcg ccgcaccggc
     4741 ccaaggagcc cgcgtggttc ctggccaccg tcggcgtctc gcccgaccac cagggcaagg
     4801 gtctgggcag cgccgtcgtg ctccccggag tggaggcggc cgagcgcgcc ggggtgcccg
     4861 ccttcctgga gacctccgcg ccccgcaacc tccccttcta cgagcggctc ggcttcaccg
     4921 tcaccgccga cgtcgaggtg cccgaaggac cgcgcacctg gtgcatgacc cgcaagcccg
     4981 gtgcctgagt cgaccgcccg ccccacgacc cgcagcgccc gaccgaaagg agcgcacgac
     5041 cccatggctc gacgataaaa taaaagattt tatttagtct ccagaaaaag gggggaatga
     5101 aagaccccac ctgtaggttt ggcaagctag cttaagtaac gccattttgc aaggcatgga
     5161 aaaatacata actgagaata gagaagttca gatcaaggtc aggaacagat ggaacagctg
     5221 aatatgggcc aaacaggata tctgtggtaa gcagttcctg ccccggctca gggccaagaa
     5281 cagatggaac agctgaatat gggccaaaca ggatatctgt ggtaagcagt tcctgccccg
     5341 gctcagggcc aagaacagat ggtccccaga tgcggtccag ccctcagcag tttctagaga
     5401 accatcagat gtttccaggg tgccccaagg acctgaaatg accctgtgcc ttatttgaac
     5461 taaccaatca gttcgcttct cgcttctgtt cgcgcgcttc tgctccccga gctcaataaa
     5521 agagcccaca acccctcact cggcgcgcca gtcctccgat tgactgagtc gcccgggtac
     5581 ccgtgtatcc aataaaccct cttgcagttg catccgactt gtggtctcgc tgttccttgg
     5641 gagggtctcc tctgagtgat tgactacccg tcagcggggg tctttcacat gcagcatgta
     5701 tcaaaattaa tttggttttt tttcttaagt atttacatta aatggccata gttgcattaa
     5761 tgaatcggcc aacgcgcggg gagaggcggt ttgcgtattg ggcgctcttc cgcttcctcg
     5821 ctcactgact cgctgcgctc ggtcgttcgg ctgcggcgag cggtatcagc tcactcaaag
     5881 gcggtaatac ggttatccac agaatcaggg gataacgcag gaaagaacat gtgagcaaaa
     5941 ggccagcaaa aggccaggaa ccgtaaaaag gccgcgttgc tggcgttttt ccataggctc
     6001 cgcccccctg acgagcatca caaaaatcga cgctcaagtc agaggtggcg aaacccgaca
     6061 ggactataaa gataccaggc gtttccccct ggaagctccc tcgtgcgctc tcctgttccg
     6121 accctgccgc ttaccggata cctgtccgcc tttctccctt cgggaagcgt ggcgctttct
     6181 catagctcac gctgtaggta tctcagttcg gtgtaggtcg ttcgctccaa gctgggctgt
     6241 gtgcacgaac cccccgttca gcccgaccgc tgcgccttat ccggtaacta tcgtcttgag
     6301 tccaacccgg taagacacga cttatcgcca ctggcagcag ccactggtaa caggattagc
     6361 agagcgaggt atgtaggcgg tgctacagag ttcttgaagt ggtggcctaa ctacggctac
     6421 actagaagaa cagtatttgg tatctgcgct ctgctgaagc cagttacctt cggaaaaaga
     6481 gttggtagct cttgatccgg caaacaaacc accgctggta gcggtggttt ttttgtttgc
     6541 aagcagcaga ttacgcgcag aaaaaaagga tctcaagaag atcctttgat cttttctacg
     6601 gggtctgacg ctcagtggaa cgaaaactca cgttaaggga ttttggtcat gagattatca
     6661 aaaaggatct tcacctagat ccttttgcgg ccggccgcaa atcaatctaa agtatatatg
     6721 agtaaacttg gtctgacagt taccaatgct taatcagtga ggcacctatc tcagcgatct
     6781 gtctatttcg ttcatccata gttgcctgac tccccgtcgt gtagataact acgatacggg
     6841 agggcttacc atctggcccc agtgctgcaa tgataccgcg agacccacgc tcaccggctc
     6901 cagatttatc agcaataaac cagccagccg gaagggccga gcgcagaagt ggtcctgcaa
     6961 ctttatccgc ctccatccag tctattaatt gttgccggga agctagagta agtagttcgc
     7021 cagttaatag tttgcgcaac gttgttgcca ttgctacagg catcgtggtg tcacgctcgt
     7081 cgtttggtat ggcttcattc agctccggtt cccaacgatc aaggcgagtt acatgatccc
     7141 ccatgttgtg caaaaaagcg gttagctcct tcggtcctcc gatcgttgtc agaagtaagt
     7201 tggccgcagt gttatcactc atggttatgg cagcactgca taattctctt actgtcatgc
     7261 catccgtaag atgcttttct gtgactggtg agtactcaac caagtcattc tgagaatagt
     7321 gtatgcggcg accgagttgc tcttgcccgg cgtcaacacg ggataatacc gcgccacata
     7381 gcagaacttt aaaagtgctc atcattggaa aacgttcttc ggggcgaaaa ctctcaagga
     7441 tcttaccgct gttgagatcc agttcgatgt aacccactcg tgcacccaac tgatcttcag
     7501 catcttttac tttcaccagc gtttctgggt gagcaaaaac aggaaggcaa aatgccgcaa
     7561 aaaagggaat aagggcgaca cggaaatgtt gaatactcat actcttcctt tttcaatatt
     7621 attgaagcat ttatcagggt tattgtctca tgagcggata catatttgaa tgtatttaga
     7681 aaaataaaca aataggggtt ccgcgcacat ttccccgaaa agtgccacct gacgtctaag
     7741 aaaccattat tatcatgaca ttaacctata aaaataggcg tatcacgagg ccctttcgtc
     7801 ttcactatag gagcttggcc cattgcatac gttgtatcca tatcataata tgtacattta
     7861 tattggctca tgtccaacat taccgccatg tt
//
