LOCUS       sequence-469427-        7163 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Stable expression of SEC61B-mCherry-FRB in mammalian cells. FRB 
            localizes in the ER lumen..
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 7163)
  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 7163)
  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-IZ-SEC61B-mCherry-FRB
FEATURES             Location/Qualifiers
     source          1..7163
                     /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             1979..2266
                     /codon_start=1
                     /gene="SEC61B"
                     /product="human Sec61-beta subunit of the translocon in the
                     endoplasmic reticulum"
                     /label=Sec61-beta
                     /translation="MPGPTPSGTNVGSSGRSPSKAVAARAAGSTVRQRKNASCGTRSAG
                     RTTSAGTGGMWRFYTEDSPGLKVGPVPVLVMSLLFIASVFMLHIWGKYTRS"
     CDS             2267..2971
                     /codon_start=1
                     /product="monomeric derivative of DsRed fluorescent protein
                     (Shaner et al., 2004)"
                     /label=mCherry
                     /note="mammalian codon-optimized"
                     /translation="VSKGEEDNMAIIKEFMRFKVHMEGSVNGHEFEIEGEGEGRPYEGT
                     QTAKLKVTKGGPLPFAWDILSPQFMYGSKAYVKHPADIPDYLKLSFPEGFKWERVMNFE
                     DGGVVTVTQDSSLQDGEFIYKVKLRGTNFPSDGPVMQKKTMGWEASSERMYPEDGALKG
                     EIKQRLKLKDGGHYDAEVKTTYKAKKPVQLPGAYNVNIKLDITSHNEDYTIVEQYERAE
                     GRHSTGGMDELYK"
     primer_bind     complement(2409..2427)
                     /label=mCherry-R
                     /note="mCherry, reverse primer"
     primer_bind     2677..2696
                     /label=mCherry-F
                     /note="mCherry, forward primer"
     CDS             2993..3271
                     /codon_start=1
                     /product="T2098L mutant of FKBP-rapamycin binding domain of
                     human FRAP"
                     /label=FRB*
                     /note="binds synthetic ligands such as AP21967"
                     /translation="ILWHEMWHEGLEEASRLYFGERNVKGMFEVLEPLHAMMERGPQTL
                     KETSFNQAYGRDLMEAQEWCRKYMKSGNVKDLLQAWDLYYHVFRRISK"
     misc_feature    3299..3865
                     /label=IRES
                     /note="internal ribosome entry site (IRES) of the 
                     encephalomyocarditis virus (EMCV)"
     misc_feature    3315..3867
                     /label=IRES
                     /note="internal ribosome entry site (IRES) of the 
                     encephalomyocarditis virus (EMCV)"
     primer_bind     complement(3459..3476)
                     /label=IRES reverse
                     /note="IRES internal ribosome entry site, reverse primer. 
                     Also called pCDH-rev"
     primer_bind     3686..3705
                     /label=IRES-F
                     /note="IRES internal ribosome entry site, forward primer"
     CDS             3885..4259
                     /codon_start=1
                     /gene="Sh ble from Streptoalloteichus hindustanus"
                     /product="antibiotic-binding protein"
                     /label=BleoR
                     /note="confers resistance to bleomycin, phleomycin, and 
                     Zeocin(TM)"
                     /translation="MAKLTSAVPVLTARDVAGAVEFWTDRLGFSRDFVEDDFAGVVRDD
                     VTLFISAVQDQVVPDNTLAWVWVRGLDELYAEWSEVVSTNFRDASGPAMTEIGEQPWGR
                     EFALRDPAGNCVHFVAEEQD"
     LTR             4489..4958
                     /note="long terminal repeat from Moloney murine leukemia 
                     virus"
     primer_bind     complement(5088..5105)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(5259..5847)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(5339..5358)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(6011..6802,6803..6871))
                     /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     6634..6653
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(6872..6976)
                     /gene="bla"
                     /label=AmpR promoter
     primer_bind     7044..7062
                     /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 gcctggtccg acccccagtg gcactaacgt gggatcctca gggcgctctc ccagcaaagc
     2041 agtggccgcc cgggcggcgg gatccactgt ccggcagagg aaaaatgcca gctgtgggac
     2101 aaggagtgca ggccgcacaa cctcggcagg caccgggggg atgtggcgat tctacacaga
     2161 agattcacct gggctcaaag ttggccctgt tccagtattg gttatgagtc ttctgttcat
     2221 cgcttctgta tttatgttgc acatttgggg caagtacact cgttcggtga gcaagggcga
     2281 ggaggataac atggccatca tcaaggagtt catgcgcttc aaggtgcaca tggagggctc
     2341 cgtgaacggc cacgagttcg agatcgaggg cgagggcgag ggccgcccct acgagggcac
     2401 ccagaccgcc aagctgaagg tgaccaaggg tggccccctg cccttcgcct gggacatcct
     2461 gtcccctcag ttcatgtacg gctccaaggc ctacgtgaag caccccgccg acatccccga
     2521 ctacttgaag ctgtccttcc ccgagggctt caagtgggag cgcgtgatga acttcgagga
     2581 cggcggcgtg gtgaccgtga cccaggactc ctccctgcag gacggcgagt tcatctacaa
     2641 ggtgaagctg cgcggcacca acttcccctc cgacggcccc gtaatgcaga agaagaccat
     2701 gggctgggag gcctcctccg agcggatgta ccccgaggac ggcgccctga agggcgagat
     2761 caagcagagg ctgaagctga aggacggcgg ccactacgac gctgaggtca agaccaccta
     2821 caaggccaag aagcccgtgc agctgcccgg cgcctacaac gtcaacatca agttggacat
     2881 cacctcccac aacgaggact acaccatcgt ggaacagtac gaacgcgccg agggccgcca
     2941 ctccaccggc ggcatggacg agctgtacaa ggcggccgcc ggaggtggcg ggatcctctg
     3001 gcatgagatg tggcatgaag gcctggaaga ggcatctcgt ttgtactttg gggaaaggaa
     3061 cgtgaaaggc atgtttgagg tgctggagcc cttgcatgct atgatggaac ggggccccca
     3121 gactctgaag gaaacatcct ttaatcaggc ctatggtcga gatttaatgg aggcccaaga
     3181 gtggtgcagg aagtacatga aatcagggaa tgtcaaggac ctcctccaag cctgggacct
     3241 ctattatcat gtgttccgac gaatctcaaa gtgactcgag agcggccgct acgtaaattc
     3301 cgcccccccc cctaacgtta ctggccgaag ccgcttggaa taaggccggt gtgcgtttgt
     3361 ctatatgtta ttttccacca tattgccgtc ttttggcaat gtgagggccc ggaaacctgg
     3421 ccctgtcttc ttgacgagca ttcctagggg tctttcccct ctcgccaaag gaatgcaagg
     3481 tctgttgaat gtcgtgaagg aagcagttcc tctggaagtt tcttgaagac aaacaacgtc
     3541 tgtagcgacc ctttgcaggc agcggaaccc cccacctggc gacaggtgcc tctgcggcca
     3601 aaagccacgt gtataagata cacctgcaaa ggcggcacaa ccccagtgcc acgttgtgag
     3661 ttggatagtt gtggaaagag tcaaatggct ctcctcaagc gtattcaaca aggggctgaa
     3721 ggatgcccag aaggtacccc attgtatggg atctgatctg gggcctcggt gcacatgctt
     3781 tacatgtgtt tagtcgaggt taaaaaaacg tctaggcccc ccgaaccacg gggacgtggt
     3841 tttcctttga aaaacacgat gataatatgg ccacaaccgt cgacatggcc aagttgacca
     3901 gtgccgttcc ggtgctcacc gcgcgcgacg tcgccggagc ggtcgagttc tggaccgacc
     3961 ggctcgggtt ctcccgggac ttcgtggagg acgacttcgc cggtgtggtc cgggacgacg
     4021 tgaccctgtt catcagcgcg gtccaggacc aggtggtgcc ggacaacacc ctggcctggg
     4081 tgtgggtgcg cggcctggac gagctgtacg ccgagtggtc ggaggtcgtg tccacgaact
     4141 tccgggacgc ctccgggccg gccatgaccg agatcggcga gcagccgtgg gggcgggagt
     4201 tcgccctgcg cgacccggcc ggcaactgcg tgcacttcgt ggccgaggag caggactgag
     4261 tcgaccgccc gccccacgac ccgcagcgcc cgaccgaaag gagcgcacga ccccatggct
     4321 cgacgataaa ataaaagatt ttatttagtc tccagaaaaa ggggggaatg aaagacccca
     4381 cctgtaggtt tggcaagcta gcttaagtaa cgccattttg caaggcatgg aaaaatacat
     4441 aactgagaat agagaagttc agatcaaggt caggaacaga tggaacagct gaatatgggc
     4501 caaacaggat atctgtggta agcagttcct gccccggctc agggccaaga acagatggaa
     4561 cagctgaata tgggccaaac aggatatctg tggtaagcag ttcctgcccc ggctcagggc
     4621 caagaacaga tggtccccag atgcggtcca gccctcagca gtttctagag aaccatcaga
     4681 tgtttccagg gtgccccaag gacctgaaat gaccctgtgc cttatttgaa ctaaccaatc
     4741 agttcgcttc tcgcttctgt tcgcgcgctt ctgctccccg agctcaataa aagagcccac
     4801 aacccctcac tcggcgcgcc agtcctccga ttgactgagt cgcccgggta cccgtgtatc
     4861 caataaaccc tcttgcagtt gcatccgact tgtggtctcg ctgttccttg ggagggtctc
     4921 ctctgagtga ttgactaccc gtcagcgggg gtctttcaca tgcagcatgt atcaaaatta
     4981 atttggtttt ttttcttaag tatttacatt aaatggccat agttgcatta atgaatcggc
     5041 caacgcgcgg ggagaggcgg tttgcgtatt gggcgctctt ccgcttcctc gctcactgac
     5101 tcgctgcgct cggtcgttcg gctgcggcga gcggtatcag ctcactcaaa ggcggtaata
     5161 cggttatcca cagaatcagg ggataacgca ggaaagaaca tgtgagcaaa aggccagcaa
     5221 aaggccagga accgtaaaaa ggccgcgttg ctggcgtttt tccataggct ccgcccccct
     5281 gacgagcatc acaaaaatcg acgctcaagt cagaggtggc gaaacccgac aggactataa
     5341 agataccagg cgtttccccc tggaagctcc ctcgtgcgct ctcctgttcc gaccctgccg
     5401 cttaccggat acctgtccgc ctttctccct tcgggaagcg tggcgctttc tcatagctca
     5461 cgctgtaggt atctcagttc ggtgtaggtc gttcgctcca agctgggctg tgtgcacgaa
     5521 ccccccgttc agcccgaccg ctgcgcctta tccggtaact atcgtcttga gtccaacccg
     5581 gtaagacacg acttatcgcc actggcagca gccactggta acaggattag cagagcgagg
     5641 tatgtaggcg gtgctacaga gttcttgaag tggtggccta actacggcta cactagaaga
     5701 acagtatttg gtatctgcgc tctgctgaag ccagttacct tcggaaaaag agttggtagc
     5761 tcttgatccg gcaaacaaac caccgctggt agcggtggtt tttttgtttg caagcagcag
     5821 attacgcgca gaaaaaaagg atctcaagaa gatcctttga tcttttctac ggggtctgac
     5881 gctcagtgga acgaaaactc acgttaaggg attttggtca tgagattatc aaaaaggatc
     5941 ttcacctaga tccttttgcg gccggccgca aatcaatcta aagtatatat gagtaaactt
     6001 ggtctgacag ttaccaatgc ttaatcagtg aggcacctat ctcagcgatc tgtctatttc
     6061 gttcatccat agttgcctga ctccccgtcg tgtagataac tacgatacgg gagggcttac
     6121 catctggccc cagtgctgca atgataccgc gagacccacg ctcaccggct ccagatttat
     6181 cagcaataaa ccagccagcc ggaagggccg agcgcagaag tggtcctgca actttatccg
     6241 cctccatcca gtctattaat tgttgccggg aagctagagt aagtagttcg ccagttaata
     6301 gtttgcgcaa cgttgttgcc attgctacag gcatcgtggt gtcacgctcg tcgtttggta
     6361 tggcttcatt cagctccggt tcccaacgat caaggcgagt tacatgatcc cccatgttgt
     6421 gcaaaaaagc ggttagctcc ttcggtcctc cgatcgttgt cagaagtaag ttggccgcag
     6481 tgttatcact catggttatg gcagcactgc ataattctct tactgtcatg ccatccgtaa
     6541 gatgcttttc tgtgactggt gagtactcaa ccaagtcatt ctgagaatag tgtatgcggc
     6601 gaccgagttg ctcttgcccg gcgtcaacac gggataatac cgcgccacat agcagaactt
     6661 taaaagtgct catcattgga aaacgttctt cggggcgaaa actctcaagg atcttaccgc
     6721 tgttgagatc cagttcgatg taacccactc gtgcacccaa ctgatcttca gcatctttta
     6781 ctttcaccag cgtttctggg tgagcaaaaa caggaaggca aaatgccgca aaaaagggaa
     6841 taagggcgac acggaaatgt tgaatactca tactcttcct ttttcaatat tattgaagca
     6901 tttatcaggg ttattgtctc atgagcggat acatatttga atgtatttag aaaaataaac
     6961 aaataggggt tccgcgcaca tttccccgaa aagtgccacc tgacgtctaa gaaaccatta
     7021 ttatcatgac attaacctat aaaaataggc gtatcacgag gccctttcgt cttcactata
     7081 ggagcttggc ccattgcata cgttgtatcc atatcataat atgtacattt atattggctc
     7141 atgtccaaca ttaccgccat gtt
//
