LOCUS       sequence-466824-        7391 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Stable expression of EGFP-LOV2(N538E)-Cyb5a 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 7391)
  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 7391)
  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-Cyb5a
FEATURES             Location/Qualifiers
     source          1..7391
                     /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    3302..3868
                     /label=IRES
                     /note="internal ribosome entry site (IRES) of the 
                     encephalomyocarditis virus (EMCV)"
     misc_feature    3318..3870
                     /label=IRES
                     /note="internal ribosome entry site (IRES) of the 
                     encephalomyocarditis virus (EMCV)"
     primer_bind     complement(3462..3479)
                     /label=IRES reverse
                     /note="IRES internal ribosome entry site, reverse primer. 
                     Also called pCDH-rev"
     primer_bind     3689..3708
                     /label=IRES-F
                     /note="IRES internal ribosome entry site, forward primer"
     CDS             3888..4487
                     /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(3888..3907)
                     /label=Puro-R
                     /note="Puromycin resistance gene, reverse primer. Also 
                     called puro-variant-R"
     primer_bind     4384..4404
                     /label=Puro-F
                     /note="Puromycin resistance gene, forward primer"
     LTR             4717..5186
                     /note="long terminal repeat from Moloney murine leukemia 
                     virus"
     primer_bind     complement(5316..5333)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(5487..6075)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(5567..5586)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(6239..7030,7031..7099))
                     /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     6862..6881
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(7100..7204)
                     /gene="bla"
                     /label=AmpR promoter
     primer_bind     7272..7290
                     /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 acttccttcg gaaaccctta tcactactgt
     3181 cgagtctaat tccagttggt ggaccaactg ggtgatccca gccatctcag ccctggtggt
     3241 agctctgatg tatcgcctct acatggcaga agattaactc gagagcggcc gctacgtaaa
     3301 ttccgccccc ccccctaacg ttactggccg aagccgcttg gaataaggcc ggtgtgcgtt
     3361 tgtctatatg ttattttcca ccatattgcc gtcttttggc aatgtgaggg cccggaaacc
     3421 tggccctgtc ttcttgacga gcattcctag gggtctttcc cctctcgcca aaggaatgca
     3481 aggtctgttg aatgtcgtga aggaagcagt tcctctggaa gtttcttgaa gacaaacaac
     3541 gtctgtagcg accctttgca ggcagcggaa ccccccacct ggcgacaggt gcctctgcgg
     3601 ccaaaagcca cgtgtataag atacacctgc aaaggcggca caaccccagt gccacgttgt
     3661 gagttggata gttgtggaaa gagtcaaatg gctctcctca agcgtattca acaaggggct
     3721 gaaggatgcc cagaaggtac cccattgtat gggatctgat ctggggcctc ggtgcacatg
     3781 ctttacatgt gtttagtcga ggttaaaaaa acgtctaggc cccccgaacc acggggacgt
     3841 ggttttcctt tgaaaaacac gatgataata tggccacaac cgtcgacatg accgagtaca
     3901 agcccacggt gcgcctcgcc acccgcgacg acgtcccccg ggccgtacgc accctcgccg
     3961 ccgcgttcgc cgactacccc gccacgcgcc acaccgtcga cccggaccgc cacatcgagc
     4021 gggtcaccga gctgcaagaa ctcttcctca cgcgcgtcgg gctcgacatc ggcaaggtgt
     4081 gggtcgcgga cgacggcgcc gcggtggcgg tctggaccac gccggagagc gtcgaagcgg
     4141 gggcggtgtt cgccgagatc ggcccgcgca tggccgagtt gagcggttcc cggctggccg
     4201 cgcagcaaca gatggaaggc ctcctggcgc cgcaccggcc caaggagccc gcgtggttcc
     4261 tggccaccgt cggcgtctcg cccgaccacc agggcaaggg tctgggcagc gccgtcgtgc
     4321 tccccggagt ggaggcggcc gagcgcgccg gggtgcccgc cttcctggag acctccgcgc
     4381 cccgcaacct ccccttctac gagcggctcg gcttcaccgt caccgccgac gtcgaggtgc
     4441 ccgaaggacc gcgcacctgg tgcatgaccc gcaagcccgg tgcctgagtc gaccgcccgc
     4501 cccacgaccc gcagcgcccg accgaaagga gcgcacgacc ccatggctcg acgataaaat
     4561 aaaagatttt atttagtctc cagaaaaagg ggggaatgaa agaccccacc tgtaggtttg
     4621 gcaagctagc ttaagtaacg ccattttgca aggcatggaa aaatacataa ctgagaatag
     4681 agaagttcag atcaaggtca ggaacagatg gaacagctga atatgggcca aacaggatat
     4741 ctgtggtaag cagttcctgc cccggctcag ggccaagaac agatggaaca gctgaatatg
     4801 ggccaaacag gatatctgtg gtaagcagtt cctgccccgg ctcagggcca agaacagatg
     4861 gtccccagat gcggtccagc cctcagcagt ttctagagaa ccatcagatg tttccagggt
     4921 gccccaagga cctgaaatga ccctgtgcct tatttgaact aaccaatcag ttcgcttctc
     4981 gcttctgttc gcgcgcttct gctccccgag ctcaataaaa gagcccacaa cccctcactc
     5041 ggggcgccag tcctccgatt gactgagtcg cccgggtacc cgtgtatcca ataaaccctc
     5101 ttgcagttgc atccgacttg tggtctcgct gttccttggg agggtctcct ctgagtgatt
     5161 gactacccgt cagcgggggt ctttcacatg cagcatgtat caaaattaat ttggtttttt
     5221 ttcttaagta tttacattaa atggccatag ttgcattaat gaatcggcca acgcgcgggg
     5281 agaggcggtt tgcgtattgg gcgctcttcc gcttcctcgc tcactgactc gctgcgctcg
     5341 gtcgttcggc tgcggcgagc ggtatcagct cactcaaagg cggtaatacg gttatccaca
     5401 gaatcagggg ataacgcagg aaagaacatg tgagcaaaag gccagcaaaa ggccaggaac
     5461 cgtaaaaagg ccgcgttgct ggcgtttttc cataggctcc gcccccctga cgagcatcac
     5521 aaaaatcgac gctcaagtca gaggtggcga aacccgacag gactataaag ataccaggcg
     5581 tttccccctg gaagctccct cgtgcgctct cctgttccga ccctgccgct taccggatac
     5641 ctgtccgcct ttctcccttc gggaagcgtg gcgctttctc atagctcacg ctgtaggtat
     5701 ctcagttcgg tgtaggtcgt tcgctccaag ctgggctgtg tgcacgaacc ccccgttcag
     5761 cccgaccgct gcgccttatc cggtaactat cgtcttgagt ccaacccggt aagacacgac
     5821 ttatcgccac tggcagcagc cactggtaac aggattagca gagcgaggta tgtaggcggt
     5881 gctacagagt tcttgaagtg gtggcctaac tacggctaca ctagaagaac agtatttggt
     5941 atctgcgctc tgctgaagcc agttaccttc ggaaaaagag ttggtagctc ttgatccggc
     6001 aaacaaacca ccgctggtag cggtggtttt tttgtttgca agcagcagat tacgcgcaga
     6061 aaaaaaggat ctcaagaaga tcctttgatc ttttctacgg ggtctgacgc tcagtggaac
     6121 gaaaactcac gttaagggat tttggtcatg agattatcaa aaaggatctt cacctagatc
     6181 cttttgcggc cggccgcaaa tcaatctaaa gtatatatga gtaaacttgg tctgacagtt
     6241 accaatgctt aatcagtgag gcacctatct cagcgatctg tctatttcgt tcatccatag
     6301 ttgcctgact ccccgtcgtg tagataacta cgatacggga gggcttacca tctggcccca
     6361 gtgctgcaat gataccgcga gacccacgct caccggctcc agatttatca gcaataaacc
     6421 agccagccgg aagggccgag cgcagaagtg gtcctgcaac tttatccgcc tccatccagt
     6481 ctattaattg ttgccgggaa gctagagtaa gtagttcgcc agttaatagt ttgcgcaacg
     6541 ttgttgccat tgctacaggc atcgtggtgt cacgctcgtc gtttggtatg gcttcattca
     6601 gctccggttc ccaacgatca aggcgagtta catgatcccc catgttgtgc aaaaaagcgg
     6661 ttagctcctt cggtcctccg atcgttgtca gaagtaagtt ggccgcagtg ttatcactca
     6721 tggttatggc agcactgcat aattctctta ctgtcatgcc atccgtaaga tgcttttctg
     6781 tgactggtga gtactcaacc aagtcattct gagaatagtg tatgcggcga ccgagttgct
     6841 cttgcccggc gtcaacacgg gataataccg cgccacatag cagaacttta aaagtgctca
     6901 tcattggaaa acgttcttcg gggcgaaaac tctcaaggat cttaccgctg ttgagatcca
     6961 gttcgatgta acccactcgt gcacccaact gatcttcagc atcttttact ttcaccagcg
     7021 tttctgggtg agcaaaaaca ggaaggcaaa atgccgcaaa aaagggaata agggcgacac
     7081 ggaaatgttg aatactcata ctcttccttt ttcaatatta ttgaagcatt tatcagggtt
     7141 attgtctcat gagcggatac atatttgaat gtatttagaa aaataaacaa ataggggttc
     7201 cgcgcacatt tccccgaaaa gtgccacctg acgtctaaga aaccattatt atcatgacat
     7261 taacctataa aaataggcgt atcacgaggc cctttcgtct tcactatagg agcttggccc
     7321 attgcatacg ttgtatccat atcataatat gtacatttat attggctcat gtccaacatt
     7381 accgccatgt t
//
