LOCUS       sequence-469416-        8333 bp    DNA     circular SYN 17-APR-2025
DEFINITION  Stable expression of EGFP-LOV2(G528A, N538E)-BAX-TMEM106B in 
            mammalian cells. Photoactivatable BAX localizes on lysosomal 
            membrane and induces lysosomal rupture upon blue light stimulation..
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 8333)
  AUTHORS   Nagashima Y, Eguchi T, Koyama-Honda I, Mizushima N
  TITLE     Optogenetic tools for inducing organelle membrane rupture
  JOURNAL   bioRxiv 2024.08.13.607738
REFERENCE   2  (bases 1 to 8333)
  AUTHORS   .
  TITLE     Direct Submission
  JOURNAL   Exported Apr 17, 2025 from SnapGene Server 8.0.1
            https://www.snapgene.com
COMMENT     SGRef: number: 1; type: "Journal Article"; journalName: "bioRxiv 
            2024.08.13.607738"
COMMENT     Sequence Label: pMRX-IP-EGFP-PA-BAX(lyso)
FEATURES             Location/Qualifiers
     source          1..8333
                     /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"
     misc_feature    4244..4810
                     /label=IRES
                     /note="internal ribosome entry site (IRES) of the 
                     encephalomyocarditis virus (EMCV)"
     misc_feature    4260..4812
                     /label=IRES
                     /note="internal ribosome entry site (IRES) of the 
                     encephalomyocarditis virus (EMCV)"
     primer_bind     complement(4404..4421)
                     /label=IRES reverse
                     /note="IRES internal ribosome entry site, reverse primer. 
                     Also called pCDH-rev"
     primer_bind     4631..4650
                     /label=IRES-F
                     /note="IRES internal ribosome entry site, forward primer"
     CDS             4830..5429
                     /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(4830..4849)
                     /label=Puro-R
                     /note="Puromycin resistance gene, reverse primer. Also 
                     called puro-variant-R"
     primer_bind     5326..5346
                     /label=Puro-F
                     /note="Puromycin resistance gene, forward primer"
     LTR             5659..6128
                     /note="long terminal repeat from Moloney murine leukemia 
                     virus"
     primer_bind     complement(6258..6275)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(6429..7017)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(6509..6528)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(7181..7972,7973..8041))
                     /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     7804..7823
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(8042..8146)
                     /gene="bla"
                     /label=AmpR promoter
     primer_bind     8214..8232
                     /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 gagagaggca 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 gccgggttgt
     3481 cgcccttttc tactttgcca gcaaactggt gctcaaggcc ctgtgcacca aggtgccgga
     3541 actgatcaga accatcatgg gctggacatt ggacttcctc cgggagcggc tgttgggctg
     3601 gatccaagac cagggtggtt gggacggcct cctctcctac tttgggacgc ccacgtggca
     3661 gaggccaaga agaacaaagc tgtatgtgat ggcttctgtg tttgtctgtc tactcctttc
     3721 tggattggct gtgtttttcc ttttccctcg ctctatcgac gtgaaataca ttggtgtaaa
     3781 atcagcctat gtcagttatg atgttcagaa gcgtacaatt tatttaaata tcacaaacac
     3841 actaaatata acaaacaata actattactc tgtcgaagtt gaaaacatca ctgcccaagt
     3901 tcaattttca aaaacagtta ttggaaaggc acgcttaaac aacataagca ttattggtcc
     3961 acttgatatg aaacaaattg attacacagt acctaccgtt atagcagagg aaatgagtta
     4021 tatgtatgat ttctgtactc tgatatccat caaagtgcat aacatagtac tcatgatgca
     4081 agttactgtg acaacaacat actttggcca ctctgaacag atatcccagg agaggtatca
     4141 gtatgtcgac tgtggaagaa acacaactta tcagttgggg cagtctgaat atttaaatgt
     4201 acttcagcca caacagtaac tcgagagcgg ccgctacgta aattccgccc ccccccctaa
     4261 cgttactggc cgaagccgct tggaataagg ccggtgtgcg tttgtctata tgttattttc
     4321 caccatattg ccgtcttttg gcaatgtgag ggcccggaaa cctggccctg tcttcttgac
     4381 gagcattcct aggggtcttt cccctctcgc caaaggaatg caaggtctgt tgaatgtcgt
     4441 gaaggaagca gttcctctgg aagtttcttg aagacaaaca acgtctgtag cgaccctttg
     4501 caggcagcgg aaccccccac ctggcgacag gtgcctctgc ggccaaaagc cacgtgtata
     4561 agatacacct gcaaaggcgg cacaacccca gtgccacgtt gtgagttgga tagttgtgga
     4621 aagagtcaaa tggctctcct caagcgtatt caacaagggg ctgaaggatg cccagaaggt
     4681 accccattgt atgggatctg atctggggcc tcggtgcaca tgctttacat gtgtttagtc
     4741 gaggttaaaa aaacgtctag gccccccgaa ccacggggac gtggttttcc tttgaaaaac
     4801 acgatgataa tatggccaca accgtcgaca tgaccgagta caagcccacg gtgcgcctcg
     4861 ccacccgcga cgacgtcccc cgggccgtac gcaccctcgc cgccgcgttc gccgactacc
     4921 ccgccacgcg ccacaccgtc gacccggacc gccacatcga gcgggtcacc gagctgcaag
     4981 aactcttcct cacgcgcgtc gggctcgaca tcggcaaggt gtgggtcgcg gacgacggcg
     5041 ccgcggtggc ggtctggacc acgccggaga gcgtcgaagc gggggcggtg ttcgccgaga
     5101 tcggcccgcg catggccgag ttgagcggtt cccggctggc cgcgcagcaa cagatggaag
     5161 gcctcctggc gccgcaccgg cccaaggagc ccgcgtggtt cctggccacc gtcggcgtct
     5221 cgcccgacca ccagggcaag ggtctgggca gcgccgtcgt gctccccgga gtggaggcgg
     5281 ccgagcgcgc cggggtgccc gccttcctgg agacctccgc gccccgcaac ctccccttct
     5341 acgagcggct cggcttcacc gtcaccgccg acgtcgaggt gcccgaagga ccgcgcacct
     5401 ggtgcatgac ccgcaagccc ggtgcctgag tcgaccgccc gccccacgac ccgcagcgcc
     5461 cgaccgaaag gagcgcacga ccccatggct cgacgataaa ataaaagatt ttatttagtc
     5521 tccagaaaaa ggggggaatg aaagacccca cctgtaggtt tggcaagcta gcttaagtaa
     5581 cgccattttg caaggcatgg aaaaatacat aactgagaat agagaagttc agatcaaggt
     5641 caggaacaga tggaacagct gaatatgggc caaacaggat atctgtggta agcagttcct
     5701 gccccggctc agggccaaga acagatggaa cagctgaata tgggccaaac aggatatctg
     5761 tggtaagcag ttcctgcccc ggctcagggc caagaacaga tggtccccag atgcggtcca
     5821 gccctcagca gtttctagag aaccatcaga tgtttccagg gtgccccaag gacctgaaat
     5881 gaccctgtgc cttatttgaa ctaaccaatc agttcgcttc tcgcttctgt tcgcgcgctt
     5941 ctgctccccg agctcaataa aagagcccac aacccctcac tcggcgcgcc agtcctccga
     6001 ttgactgagt cgcccgggta cccgtgtatc caataaaccc tcttgcagtt gcatccgact
     6061 tgtggtctcg ctgttccttg ggagggtctc ctctgagtga ttgactaccc gtcagcgggg
     6121 gtctttcaca tgcagcatgt atcaaaatta atttggtttt ttttcttaag tatttacatt
     6181 aaatggccat agttgcatta atgaatcggc caacgcgcgg ggagaggcgg tttgcgtatt
     6241 gggcgctctt ccgcttcctc gctcactgac tcgctgcgct cggtcgttcg gctgcggcga
     6301 gcggtatcag ctcactcaaa ggcggtaata cggttatcca cagaatcagg ggataacgca
     6361 ggaaagaaca tgtgagcaaa aggccagcaa aaggccagga accgtaaaaa ggccgcgttg
     6421 ctggcgtttt tccataggct ccgcccccct gacgagcatc acaaaaatcg acgctcaagt
     6481 cagaggtggc gaaacccgac aggactataa agataccagg cgtttccccc tggaagctcc
     6541 ctcgtgcgct ctcctgttcc gaccctgccg cttaccggat acctgtccgc ctttctccct
     6601 tcgggaagcg tggcgctttc tcatagctca cgctgtaggt atctcagttc ggtgtaggtc
     6661 gttcgctcca agctgggctg tgtgcacgaa ccccccgttc agcccgaccg ctgcgcctta
     6721 tccggtaact atcgtcttga gtccaacccg gtaagacacg acttatcgcc actggcagca
     6781 gccactggta acaggattag cagagcgagg tatgtaggcg gtgctacaga gttcttgaag
     6841 tggtggccta actacggcta cactagaaga acagtatttg gtatctgcgc tctgctgaag
     6901 ccagttacct tcggaaaaag agttggtagc tcttgatccg gcaaacaaac caccgctggt
     6961 agcggtggtt tttttgtttg caagcagcag attacgcgca gaaaaaaagg atctcaagaa
     7021 gatcctttga tcttttctac ggggtctgac gctcagtgga acgaaaactc acgttaaggg
     7081 attttggtca tgagattatc aaaaaggatc ttcacctaga tccttttgcg gccggccgca
     7141 aatcaatcta aagtatatat gagtaaactt ggtctgacag ttaccaatgc ttaatcagtg
     7201 aggcacctat ctcagcgatc tgtctatttc gttcatccat agttgcctga ctccccgtcg
     7261 tgtagataac tacgatacgg gagggcttac catctggccc cagtgctgca atgataccgc
     7321 gagacccacg ctcaccggct ccagatttat cagcaataaa ccagccagcc ggaagggccg
     7381 agcgcagaag tggtcctgca actttatccg cctccatcca gtctattaat tgttgccggg
     7441 aagctagagt aagtagttcg ccagttaata gtttgcgcaa cgttgttgcc attgctacag
     7501 gcatcgtggt gtcacgctcg tcgtttggta tggcttcatt cagctccggt tcccaacgat
     7561 caaggcgagt tacatgatcc cccatgttgt gcaaaaaagc ggttagctcc ttcggtcctc
     7621 cgatcgttgt cagaagtaag ttggccgcag tgttatcact catggttatg gcagcactgc
     7681 ataattctct tactgtcatg ccatccgtaa gatgcttttc tgtgactggt gagtactcaa
     7741 ccaagtcatt ctgagaatag tgtatgcggc gaccgagttg ctcttgcccg gcgtcaacac
     7801 gggataatac cgcgccacat agcagaactt taaaagtgct catcattgga aaacgttctt
     7861 cggggcgaaa actctcaagg atcttaccgc tgttgagatc cagttcgatg taacccactc
     7921 gtgcacccaa ctgatcttca gcatctttta ctttcaccag cgtttctggg tgagcaaaaa
     7981 caggaaggca aaatgccgca aaaaagggaa taagggcgac acggaaatgt tgaatactca
     8041 tactcttcct ttttcaatat tattgaagca tttatcaggg ttattgtctc atgagcggat
     8101 acatatttga atgtatttag aaaaataaac aaataggggt tccgcgcaca tttccccgaa
     8161 aagtgccacc tgacgtctaa gaaaccatta ttatcatgac attaacctat aaaaataggc
     8221 gtatcacgag gccctttcgt cttcactata ggagcttggc ccattgcata cgttgtatcc
     8281 atatcataat atgtacattt atattggctc atgtccaaca ttaccgccat gtt
//
