LOCUS       sequence-469428-        7385 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Stable expression of EGFP-LOV2(N538E)-OMP25 in mammalian cells. This
            construct lacks BAX domain, and causes no rupture upon 
            photostimulation..
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 7385)
  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 7385)
  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-LOV2-OMP25
FEATURES             Location/Qualifiers
     source          1..7385
                     /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
                     YFIGVQLDGTEHVRDAAEREGVMLIKKTAENIDEAAKEL"
     misc_feature    3296..3862
                     /label=IRES
                     /note="internal ribosome entry site (IRES) of the 
                     encephalomyocarditis virus (EMCV)"
     misc_feature    3312..3864
                     /label=IRES
                     /note="internal ribosome entry site (IRES) of the 
                     encephalomyocarditis virus (EMCV)"
     primer_bind     complement(3456..3473)
                     /label=IRES reverse
                     /note="IRES internal ribosome entry site, reverse primer. 
                     Also called pCDH-rev"
     primer_bind     3683..3702
                     /label=IRES-F
                     /note="IRES internal ribosome entry site, forward primer"
     CDS             3882..4481
                     /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(3882..3901)
                     /label=Puro-R
                     /note="Puromycin resistance gene, reverse primer. Also 
                     called puro-variant-R"
     primer_bind     4378..4398
                     /label=Puro-F
                     /note="Puromycin resistance gene, forward primer"
     LTR             4711..5180
                     /note="long terminal repeat from Moloney murine leukemia 
                     virus"
     primer_bind     complement(5310..5327)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(5481..6069)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(5561..5580)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(6233..7024,7025..7093))
                     /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     6856..6875
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(7094..7198)
                     /gene="bla"
                     /label=AmpR promoter
     primer_bind     7266..7284
                     /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 tgcagaaaat attgatgagg cggcaaaaga acttagacat cgaggcgacg gagagcctag
     3181 tggagttcct gtagctgtgg tgctgctgcc agtgtttgcc cttaccctgg tagcagtttg
     3241 ggccttcgtg agataccgaa agcagctctg actcgagagc ggccgctacg taaattccgc
     3301 ccccccccct aacgttactg gccgaagccg cttggaataa ggccggtgtg cgtttgtcta
     3361 tatgttattt tccaccatat tgccgtcttt tggcaatgtg agggcccgga aacctggccc
     3421 tgtcttcttg acgagcattc ctaggggtct ttcccctctc gccaaaggaa tgcaaggtct
     3481 gttgaatgtc gtgaaggaag cagttcctct ggaagtttct tgaagacaaa caacgtctgt
     3541 agcgaccctt tgcaggcagc ggaacccccc acctggcgac aggtgcctct gcggccaaaa
     3601 gccacgtgta taagatacac ctgcaaaggc ggcacaaccc cagtgccacg ttgtgagttg
     3661 gatagttgtg gaaagagtca aatggctctc ctcaagcgta ttcaacaagg ggctgaagga
     3721 tgcccagaag gtaccccatt gtatgggatc tgatctgggg cctcggtgca catgctttac
     3781 atgtgtttag tcgaggttaa aaaaacgtct aggccccccg aaccacgggg acgtggtttt
     3841 cctttgaaaa acacgatgat aatatggcca caaccgtcga catgaccgag tacaagccca
     3901 cggtgcgcct cgccacccgc gacgacgtcc cccgggccgt acgcaccctc gccgccgcgt
     3961 tcgccgacta ccccgccacg cgccacaccg tcgacccgga ccgccacatc gagcgggtca
     4021 ccgagctgca agaactcttc ctcacgcgcg tcgggctcga catcggcaag gtgtgggtcg
     4081 cggacgacgg cgccgcggtg gcggtctgga ccacgccgga gagcgtcgaa gcgggggcgg
     4141 tgttcgccga gatcggcccg cgcatggccg agttgagcgg ttcccggctg gccgcgcagc
     4201 aacagatgga aggcctcctg gcgccgcacc ggcccaagga gcccgcgtgg ttcctggcca
     4261 ccgtcggcgt ctcgcccgac caccagggca agggtctggg cagcgccgtc gtgctccccg
     4321 gagtggaggc ggccgagcgc gccggggtgc ccgccttcct ggagacctcc gcgccccgca
     4381 acctcccctt ctacgagcgg ctcggcttca ccgtcaccgc cgacgtcgag gtgcccgaag
     4441 gaccgcgcac ctggtgcatg acccgcaagc ccggtgcctg agtcgaccgc ccgccccacg
     4501 acccgcagcg cccgaccgaa aggagcgcac gaccccatgg ctcgacgata aaataaaaga
     4561 ttttatttag tctccagaaa aaggggggaa tgaaagaccc cacctgtagg tttggcaagc
     4621 tagcttaagt aacgccattt tgcaaggcat ggaaaaatac ataactgaga atagagaagt
     4681 tcagatcaag gtcaggaaca gatggaacag ctgaatatgg gccaaacagg atatctgtgg
     4741 taagcagttc ctgccccggc tcagggccaa gaacagatgg aacagctgaa tatgggccaa
     4801 acaggatatc tgtggtaagc agttcctgcc ccggctcagg gccaagaaca gatggtcccc
     4861 agatgcggtc cagccctcag cagtttctag agaaccatca gatgtttcca gggtgcccca
     4921 aggacctgaa atgaccctgt gccttatttg aactaaccaa tcagttcgct tctcgcttct
     4981 gttcgcgcgc ttctgctccc cgagctcaat aaaagagccc acaacccctc actcggcgcg
     5041 ccagtcctcc gattgactga gtcgcccggg tacccgtgta tccaataaac cctcttgcag
     5101 ttgcatccga cttgtggtct cgctgttcct tgggagggtc tcctctgagt gattgactac
     5161 ccgtcagcgg gggtctttca catgcagcat gtatcaaaat taatttggtt ttttttctta
     5221 agtatttaca ttaaatggcc atagttgcat taatgaatcg gccaacgcgc ggggagaggc
     5281 ggtttgcgta ttgggcgctc ttccgcttcc tcgctcactg actcgctgcg ctcggtcgtt
     5341 cggctgcggc gagcggtatc agctcactca aaggcggtaa tacggttatc cacagaatca
     5401 ggggataacg caggaaagaa catgtgagca aaaggccagc aaaaggccag gaaccgtaaa
     5461 aaggccgcgt tgctggcgtt tttccatagg ctccgccccc ctgacgagca tcacaaaaat
     5521 cgacgctcaa gtcagaggtg gcgaaacccg acaggactat aaagatacca ggcgtttccc
     5581 cctggaagct ccctcgtgcg ctctcctgtt ccgaccctgc cgcttaccgg atacctgtcc
     5641 gcctttctcc cttcgggaag cgtggcgctt tctcatagct cacgctgtag gtatctcagt
     5701 tcggtgtagg tcgttcgctc caagctgggc tgtgtgcacg aaccccccgt tcagcccgac
     5761 cgctgcgcct tatccggtaa ctatcgtctt gagtccaacc cggtaagaca cgacttatcg
     5821 ccactggcag cagccactgg taacaggatt agcagagcga ggtatgtagg cggtgctaca
     5881 gagttcttga agtggtggcc taactacggc tacactagaa gaacagtatt tggtatctgc
     5941 gctctgctga agccagttac cttcggaaaa agagttggta gctcttgatc cggcaaacaa
     6001 accaccgctg gtagcggtgg tttttttgtt tgcaagcagc agattacgcg cagaaaaaaa
     6061 ggatctcaag aagatccttt gatcttttct acggggtctg acgctcagtg gaacgaaaac
     6121 tcacgttaag ggattttggt catgagatta tcaaaaagga tcttcaccta gatccttttg
     6181 cggccggccg caaatcaatc taaagtatat atgagtaaac ttggtctgac agttaccaat
     6241 gcttaatcag tgaggcacct atctcagcga tctgtctatt tcgttcatcc atagttgcct
     6301 gactccccgt cgtgtagata actacgatac gggagggctt accatctggc cccagtgctg
     6361 caatgatacc gcgagaccca cgctcaccgg ctccagattt atcagcaata aaccagccag
     6421 ccggaagggc cgagcgcaga agtggtcctg caactttatc cgcctccatc cagtctatta
     6481 attgttgccg ggaagctaga gtaagtagtt cgccagttaa tagtttgcgc aacgttgttg
     6541 ccattgctac aggcatcgtg gtgtcacgct cgtcgtttgg tatggcttca ttcagctccg
     6601 gttcccaacg atcaaggcga gttacatgat cccccatgtt gtgcaaaaaa gcggttagct
     6661 ccttcggtcc tccgatcgtt gtcagaagta agttggccgc agtgttatca ctcatggtta
     6721 tggcagcact gcataattct cttactgtca tgccatccgt aagatgcttt tctgtgactg
     6781 gtgagtactc aaccaagtca ttctgagaat agtgtatgcg gcgaccgagt tgctcttgcc
     6841 cggcgtcaac acgggataat accgcgccac atagcagaac tttaaaagtg ctcatcattg
     6901 gaaaacgttc ttcggggcga aaactctcaa ggatcttacc gctgttgaga tccagttcga
     6961 tgtaacccac tcgtgcaccc aactgatctt cagcatcttt tactttcacc agcgtttctg
     7021 ggtgagcaaa aacaggaagg caaaatgccg caaaaaaggg aataagggcg acacggaaat
     7081 gttgaatact catactcttc ctttttcaat attattgaag catttatcag ggttattgtc
     7141 tcatgagcgg atacatattt gaatgtattt agaaaaataa acaaataggg gttccgcgca
     7201 catttccccg aaaagtgcca cctgacgtct aagaaaccat tattatcatg acattaacct
     7261 ataaaaatag gcgtatcacg aggccctttc gtcttcacta taggagcttg gcccattgca
     7321 tacgttgtat ccatatcata atatgtacat ttatattggc tcatgtccaa cattaccgcc
     7381 atgtt
//
