LOCUS       sequence-469408-        7826 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Stable expression of EGFP-LOV2(G528A, N538E)-TMEM106B 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 7826)
  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 7826)
  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-TMEM106B
FEATURES             Location/Qualifiers
     source          1..7826
                     /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    3737..4303
                     /label=IRES
                     /note="internal ribosome entry site (IRES) of the 
                     encephalomyocarditis virus (EMCV)"
     misc_feature    3753..4305
                     /label=IRES
                     /note="internal ribosome entry site (IRES) of the 
                     encephalomyocarditis virus (EMCV)"
     primer_bind     complement(3897..3914)
                     /label=IRES reverse
                     /note="IRES internal ribosome entry site, reverse primer. 
                     Also called pCDH-rev"
     primer_bind     4124..4143
                     /label=IRES-F
                     /note="IRES internal ribosome entry site, forward primer"
     CDS             4323..4922
                     /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(4323..4342)
                     /label=Puro-R
                     /note="Puromycin resistance gene, reverse primer. Also 
                     called puro-variant-R"
     primer_bind     4819..4839
                     /label=Puro-F
                     /note="Puromycin resistance gene, forward primer"
     LTR             5152..5621
                     /note="long terminal repeat from Moloney murine leukemia 
                     virus"
     primer_bind     complement(5751..5768)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(5922..6510)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(6002..6021)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(6674..7465,7466..7534))
                     /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     7297..7316
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(7535..7639)
                     /gene="bla"
                     /label=AmpR promoter
     primer_bind     7707..7725
                     /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 acttaggcca agaagaacaa agctgtatgt
     3181 gatggcttct gtgtttgtct gtctactcct ttctggattg gctgtgtttt tccttttccc
     3241 tcgctctatc gacgtgaaat acattggtgt aaaatcagcc tatgtcagtt atgatgttca
     3301 gaagcgtaca atttatttaa atatcacaaa cacactaaat ataacaaaca ataactatta
     3361 ctctgtcgaa gttgaaaaca tcactgccca agttcaattt tcaaaaacag ttattggaaa
     3421 ggcacgctta aacaacataa gcattattgg tccacttgat atgaaacaaa ttgattacac
     3481 agtacctacc gttatagcag aggaaatgag ttatatgtat gatttctgta ctctgatatc
     3541 catcaaagtg cataacatag tactcatgat gcaagttact gtgacaacaa catactttgg
     3601 ccactctgaa cagatatccc aggagaggta tcagtatgtc gactgtggaa gaaacacaac
     3661 ttatcagttg gggcagtctg aatatttaaa tgtacttcag ccacaacagt aactcgagag
     3721 cggccgctac gtaaattccg cccccccccc taacgttact ggccgaagcc gcttggaata
     3781 aggccggtgt gcgtttgtct atatgttatt ttccaccata ttgccgtctt ttggcaatgt
     3841 gagggcccgg aaacctggcc ctgtcttctt gacgagcatt cctaggggtc tttcccctct
     3901 cgccaaagga atgcaaggtc tgttgaatgt cgtgaaggaa gcagttcctc tggaagtttc
     3961 ttgaagacaa acaacgtctg tagcgaccct ttgcaggcag cggaaccccc cacctggcga
     4021 caggtgcctc tgcggccaaa agccacgtgt ataagataca cctgcaaagg cggcacaacc
     4081 ccagtgccac gttgtgagtt ggatagttgt ggaaagagtc aaatggctct cctcaagcgt
     4141 attcaacaag gggctgaagg atgcccagaa ggtaccccat tgtatgggat ctgatctggg
     4201 gcctcggtgc acatgcttta catgtgttta gtcgaggtta aaaaaacgtc taggcccccc
     4261 gaaccacggg gacgtggttt tcctttgaaa aacacgatga taatatggcc acaaccgtcg
     4321 acatgaccga gtacaagccc acggtgcgcc tcgccacccg cgacgacgtc ccccgggccg
     4381 tacgcaccct cgccgccgcg ttcgccgact accccgccac gcgccacacc gtcgacccgg
     4441 accgccacat cgagcgggtc accgagctgc aagaactctt cctcacgcgc gtcgggctcg
     4501 acatcggcaa ggtgtgggtc gcggacgacg gcgccgcggt ggcggtctgg accacgccgg
     4561 agagcgtcga agcgggggcg gtgttcgccg agatcggccc gcgcatggcc gagttgagcg
     4621 gttcccggct ggccgcgcag caacagatgg aaggcctcct ggcgccgcac cggcccaagg
     4681 agcccgcgtg gttcctggcc accgtcggcg tctcgcccga ccaccagggc aagggtctgg
     4741 gcagcgccgt cgtgctcccc ggagtggagg cggccgagcg cgccggggtg cccgccttcc
     4801 tggagacctc cgcgccccgc aacctcccct tctacgagcg gctcggcttc accgtcaccg
     4861 ccgacgtcga ggtgcccgaa ggaccgcgca cctggtgcat gacccgcaag cccggtgcct
     4921 gagtcgaccg cccgccccac gacccgcagc gcccgaccga aaggagcgca cgaccccatg
     4981 gctcgacgat aaaataaaag attttattta gtctccagaa aaagggggga atgaaagacc
     5041 ccacctgtag gtttggcaag ctagcttaag taacgccatt ttgcaaggca tggaaaaata
     5101 cataactgag aatagagaag ttcagatcaa ggtcaggaac agatggaaca gctgaatatg
     5161 ggccaaacag gatatctgtg gtaagcagtt cctgccccgg ctcagggcca agaacagatg
     5221 gaacagctga atatgggcca aacaggatat ctgtggtaag cagttcctgc cccggctcag
     5281 ggccaagaac agatggtccc cagatgcggt ccagccctca gcagtttcta gagaaccatc
     5341 agatgtttcc agggtgcccc aaggacctga aatgaccctg tgccttattt gaactaacca
     5401 atcagttcgc ttctcgcttc tgttcgcgcg cttctgctcc ccgagctcaa taaaagagcc
     5461 cacaacccct cactcggcgc gccagtcctc cgattgactg agtcgcccgg gtacccgtgt
     5521 atccaataaa ccctcttgca gttgcatccg acttgtggtc tcgctgttcc ttgggagggt
     5581 ctcctctgag tgattgacta cccgtcagcg ggggtctttc acatgcagca tgtatcaaaa
     5641 ttaatttggt tttttttctt aagtatttac attaaatggc catagttgca ttaatgaatc
     5701 ggccaacgcg cggggagagg cggtttgcgt attgggcgct cttccgcttc ctcgctcact
     5761 gactcgctgc gctcggtcgt tcggctgcgg cgagcggtat cagctcactc aaaggcggta
     5821 atacggttat ccacagaatc aggggataac gcaggaaaga acatgtgagc aaaaggccag
     5881 caaaaggcca ggaaccgtaa aaaggccgcg ttgctggcgt ttttccatag gctccgcccc
     5941 cctgacgagc atcacaaaaa tcgacgctca agtcagaggt ggcgaaaccc gacaggacta
     6001 taaagatacc aggcgtttcc ccctggaagc tccctcgtgc gctctcctgt tccgaccctg
     6061 ccgcttaccg gatacctgtc cgcctttctc ccttcgggaa gcgtggcgct ttctcatagc
     6121 tcacgctgta ggtatctcag ttcggtgtag gtcgttcgct ccaagctggg ctgtgtgcac
     6181 gaaccccccg ttcagcccga ccgctgcgcc ttatccggta actatcgtct tgagtccaac
     6241 ccggtaagac acgacttatc gccactggca gcagccactg gtaacaggat tagcagagcg
     6301 aggtatgtag gcggtgctac agagttcttg aagtggtggc ctaactacgg ctacactaga
     6361 agaacagtat ttggtatctg cgctctgctg aagccagtta ccttcggaaa aagagttggt
     6421 agctcttgat ccggcaaaca aaccaccgct ggtagcggtg gtttttttgt ttgcaagcag
     6481 cagattacgc gcagaaaaaa aggatctcaa gaagatcctt tgatcttttc tacggggtct
     6541 gacgctcagt ggaacgaaaa ctcacgttaa gggattttgg tcatgagatt atcaaaaagg
     6601 atcttcacct agatcctttt gcggccggcc gcaaatcaat ctaaagtata tatgagtaaa
     6661 cttggtctga cagttaccaa tgcttaatca gtgaggcacc tatctcagcg atctgtctat
     6721 ttcgttcatc catagttgcc tgactccccg tcgtgtagat aactacgata cgggagggct
     6781 taccatctgg ccccagtgct gcaatgatac cgcgagaccc acgctcaccg gctccagatt
     6841 tatcagcaat aaaccagcca gccggaaggg ccgagcgcag aagtggtcct gcaactttat
     6901 ccgcctccat ccagtctatt aattgttgcc gggaagctag agtaagtagt tcgccagtta
     6961 atagtttgcg caacgttgtt gccattgcta caggcatcgt ggtgtcacgc tcgtcgtttg
     7021 gtatggcttc attcagctcc ggttcccaac gatcaaggcg agttacatga tcccccatgt
     7081 tgtgcaaaaa agcggttagc tccttcggtc ctccgatcgt tgtcagaagt aagttggccg
     7141 cagtgttatc actcatggtt atggcagcac tgcataattc tcttactgtc atgccatccg
     7201 taagatgctt ttctgtgact ggtgagtact caaccaagtc attctgagaa tagtgtatgc
     7261 ggcgaccgag ttgctcttgc ccggcgtcaa cacgggataa taccgcgcca catagcagaa
     7321 ctttaaaagt gctcatcatt ggaaaacgtt cttcggggcg aaaactctca aggatcttac
     7381 cgctgttgag atccagttcg atgtaaccca ctcgtgcacc caactgatct tcagcatctt
     7441 ttactttcac cagcgtttct gggtgagcaa aaacaggaag gcaaaatgcc gcaaaaaagg
     7501 gaataagggc gacacggaaa tgttgaatac tcatactctt cctttttcaa tattattgaa
     7561 gcatttatca gggttattgt ctcatgagcg gatacatatt tgaatgtatt tagaaaaata
     7621 aacaaatagg ggttccgcgc acatttcccc gaaaagtgcc acctgacgtc taagaaacca
     7681 ttattatcat gacattaacc tataaaaata ggcgtatcac gaggcccttt cgtcttcact
     7741 ataggagctt ggcccattgc atacgttgta tccatatcat aatatgtaca tttatattgg
     7801 ctcatgtcca acattaccgc catgtt
//
