LOCUS       sequence-431709-        7604 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Probe to evaluate lysophagic activity.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 7604)
  AUTHORS   Shima T, Ogura M, Matsuda R, Nakamura S, Jin N, Yoshimori T, Kuma A
  TITLE     The TMEM192-mKeima probe specifically assays lysophagy and reveals 
            its initial steps.
  JOURNAL   J Cell Biol. 2023 Dec 4;222(12):e202204048. doi: 
            10.1083/jcb.202204048. Epub 2023 Oct 6.
  PUBMED    37801070
REFERENCE   2  (bases 1 to 7604)
  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 Cell 
            Biol."; date: "2023-12-4"; volume: "222(12):e202204048. doi"; pages:
            " 10.1083/jcb.202204048. Epub 2023 Oct 6"
COMMENT     Sequence Label: pMRX-IP-TMEM192-mKeima
FEATURES             Location/Qualifiers
     source          1..7604
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     62..80
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     enhancer        182..561
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
     misc_feature    920..1277
                     /label=MMLV Psi
                     /note="packaging signal of Moloney murine leukemia virus 
                     (MMLV)"
     CDS             1342..1758
                     /label=gag (truncated)
                     /note="truncated Moloney murine leukemia virus (MMLV) gag 
                     gene lacking the start codon"
     primer_bind     1688..1710
                     /label=pLXSN 5'
                     /note="Murine stem cell virus, forward primer. Also called 
                     MSCV"
     primer_bind     1726..1742
                     /label=pBABE 5'
                     /note="Psi packaging signal, forward primer for pBABE 
                     vectors"
     misc_feature    1768..2142
                     /label=pol region
                     /note="Moloney murine leukemia virus (MMLV) pol region 
                     containing the splice acceptor site"
     primer_bind     2080..2096
                     /label=pBMN 5'
                     /note="MMLV sequence, for inserts in pBMN retroviral 
                     vector"
     regulatory      3030..3039
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     CDS             join(3036..3038,3039..3041,3042..3704)
                     /codon_start=1
                     /product="monomeric Keima-Red fluorescent protein, also 
                     known as mKeima"
                     /label=mKeima-Red
                     /translation="MVSVIAKQMTYKVYMSGTVNGHYFEVEGDGKGKPYEGEQTVKLTV
                     TKGGPLPFAWDILSPQLQYGSIPFTKYPEDIPDYFKQSFPEGYTWERSMNFEDGAVCTV
                     SNDSSIQGNCFIYNVKISGENFPPNGPVMQKKTQGWEPSTERLFARDGMLIGNDYMALK
                     LEGGGHYLCEFKSTYKAKKPVRMPGRHEIDRKLDVTSHNRDYTSVEQCEIAIARHSLLG
                     "
     misc_feature    3720..4286
                     /label=IRES
                     /note="internal ribosome entry site (IRES) of the 
                     encephalomyocarditis virus (EMCV)"
     misc_feature    3736..4288
                     /label=IRES
                     /note="internal ribosome entry site (IRES) of the 
                     encephalomyocarditis virus (EMCV)"
     primer_bind     complement(3880..3897)
                     /label=IRES reverse
                     /note="IRES internal ribosome entry site, reverse primer. 
                     Also called pCDH-rev"
     primer_bind     4107..4126
                     /label=IRES-F
                     /note="IRES internal ribosome entry site, forward primer"
     CDS             4300..4899
                     /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(4300..4319)
                     /label=Puro-R
                     /note="Puromycin resistance gene, reverse primer. Also 
                     called puro-variant-R"
     primer_bind     4796..4816
                     /label=Puro-F
                     /note="Puromycin resistance gene, forward primer"
     LTR             5123..5592
                     /note="long terminal repeat from Moloney murine leukemia 
                     virus"
     primer_bind     complement(5722..5739)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(5893..6481)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(5973..5992)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(6633..7424,7425..7493))
                     /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     7256..7275
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(7494..7598)
                     /gene="bla"
                     /label=AmpR promoter
ORIGIN
        1 tccccgaaaa gtgccacctg acgtctaaga aaccattatt atcatgacat taacctataa
       61 aaataggcgt atcacgaggc cctttcgtct tcactcgagg agcttggccc attgcatacg
      121 ttgtatccat atcataatat gtacatttat attggctcat gtccaacatt accgccatgt
      181 tgacattgat tattgactag ttattaatag taatcaatta cggggtcatt agttcatagc
      241 ccatatatgg agttccgcgt tacataactt acggtaaatg gcccgcctgg ctgaccgccc
      301 aacgaccccc gcccattgac gtcaataatg acgtatgttc ccatagtaac gccaataggg
      361 actttccatt gacgtcaatg ggtggagtat ttacggtaaa ctgcccactt ggcagtacat
      421 caagtgtatc atatgccaag tacgccccct attgacgtca atgacggtaa atggcccgcc
      481 tggcattatg cccagtacat gaccttatgg gactttccta cttggcagta catctacgta
      541 ttagtcatcg ctattaccat gctagagaac catcagatgt ttccagggtg ccccaaggac
      601 ctgaaatgac cctgtgcctt atttgaacta accaatcagt tcgcttctcg cttctgttcg
      661 cgcgcttctg ctccccgagc tcaataaaag agcccacaac ccctcactcg gcgcgccagt
      721 cctccgattg actgagtcgc ccgggtaccc gtgtatccaa taaaccctct tgcagttgca
      781 tccgacttgt ggtctcgctg ttccttggga gggtctcctc tgagtgattg actacccgtc
      841 agcgggggtc tttcatttgg gggctcgtcc gggatcggga gacccctgcc cagggaccac
      901 cgacccacca ccgggaggta agctggccag caacttatct gtgtctgtcc gattgtctag
      961 tgtctatgac tgattttatg cgcctgcgtc ggtactagtt agctaactag ctctgtatct
     1021 ggcggacccg tggtggaact gacgagttcg gaacacccgg ccgcaaccct gggagacgtc
     1081 ccagggactt cgggggccgt ttttgtggcc cgacctgagt ccaaaaatcc cgatcgtttt
     1141 ggactctttg gtgcaccccc cttagaggag ggatatgtgg ttctggtagg agacgagaac
     1201 ctaaaacagt tcccgcctcc gtctgaattt ttgctttcgg tttgggaccg aagccgcgcc
     1261 gcgcgtcttg tctgctgcag catcgttctg tgttgtctct gtctgactgt gtttctgtat
     1321 ttgtctgaaa atatgggccc gggccagact gttaccactc ccttaagttt gaccttaggt
     1381 cactggaaag atgtcgagcg gatcgctcac aaccagtcgg tagatgtcaa gaagagacgt
     1441 tgggttacct tctgctctgc agaatggcca acctttaacg tcggatggcc gcgagacggc
     1501 acctttaacc gagacctcat cacccaggtt aagatcaagg tcttttcacc tggcccgcat
     1561 ggacacccag accaggtccc ctacatcgtg acctgggaag ccttggcttt tgacccccct
     1621 ccctgggtca agccctttgt acaccctaag cctccgcctc ctcttcctcc atccgccccg
     1681 tctctccccc ttgaacctcc tcgttcgacc ccgcctcgat cctcccttta tccagccctc
     1741 actccttctc taggcgcccc catatggcca tatgagatct tatatggggc acccccgccc
     1801 cttgtaaact tccctgaccc tgacatgaca agagttacta acagcccctc tctccaagct
     1861 cacttacagg ctctctactt agtccagcac gaagtctgga gacctctggc ggcagcctac
     1921 caagaacaac tggaccgacc ggtggtacct cacccttacc gagtcggcga cacagtgtgg
     1981 gtccgccgac accagactaa gaacctagaa cctcgctgga aaggacctta cacagtcctg
     2041 ctgaccaccc ccaccgccct caaagtagac ggcatcgcag cttggataca cgccgcccac
     2101 gtgaaggctg ccgaccccgg gggtggacca tcctctagac tgccggatcc cagtgtggtg
     2161 gtacgggaat tatggcggcg gggggcagga tggaggacgg ttccttggat atcacccaga
     2221 gtattgaaga cgacccactt ctggatgccc agcttctccc acaccactca ttacaagctc
     2281 actttagacc ccgattccat cctcttccta cagtcatcat agtgaatctt ctgtggttta
     2341 ttcatctcgt gtttgttgtt ttagcatttt taacaggtgt gctttgttct tatcctaatc
     2401 caaatgagga caagtgccca ggaaattaca caaacccatt gaaagttcag acggttataa
     2461 tccttgggaa agttattttg tggattctcc atttactcct tgaatgctac atccagtatc
     2521 accacagcaa aatcagaaac cgaggctata acttgatcta ccgatcaaca aggcatctca
     2581 agagacttgc gttgatgata cagtcctctg gcaacacagt gcttctcctc atactgtgca
     2641 tgcagcactc cttcccagag cctggcagat tgtatcttga cctcattctg gccatcttgg
     2701 cactggaact catctgttcc ctgatatgtc tcctcattta cacagtgaaa atccggagat
     2761 ttaataaagc taaaccagag cctgatatac ttgaagaaga aaaaatctat gcttacccca
     2821 gcaatattac ctcggagact ggattcagaa ctatttcaag cctagaagaa attgttgaaa
     2881 agcaaggaga caccattgaa tacctgaagc gacacaatgc gctgctgagt aagcgattgt
     2941 tggctctcac ttcctcagac ctgggctgtc agccaagtag aacgggaatt ctgcagtcga
     3001 cggtaccgcg ggcccgggat ccaccggtcg ccaccatggt gagtgtgatc gctaaacaaa
     3061 tgacctacaa ggtttatatg tcaggcacgg tcaatggaca ctactttgag gtcgaaggcg
     3121 atggaaaagg aaagccttac gagggagagc agacagtaaa gctcactgtc accaagggtg
     3181 gacctctgcc atttgcttgg gatattttat caccacagct tcagtacgga agcataccat
     3241 tcaccaagta ccctgaagac atccctgatt atttcaagca gtcattccct gagggatata
     3301 catgggagag gagcatgaac tttgaagatg gtgcagtgtg tactgtcagc aatgattcca
     3361 gcatccaagg caactgtttc atctacaatg tcaaaatctc tggtgagaac tttcctccca
     3421 atggacctgt tatgcagaag aagacacagg gctgggaacc cagcactgag cgtctctttg
     3481 cacgagatgg aatgctgata ggaaacgatt atatggctct gaagttggaa ggaggtggtc
     3541 actatttgtg tgaatttaaa tctacttaca aggcaaagaa gcctgtgagg atgccagggc
     3601 gccacgagat tgaccgcaaa ctggatgtaa ccagtcacaa cagggattac acatctgttg
     3661 agcagtgtga aatagccatt gcacgccact ctttgctcgg ttaaggccgc tacgtaaatt
     3721 ccgccccccc ccctaacgtt actggccgaa gccgcttgga ataaggccgg tgtgcgtttg
     3781 tctatatgtt attttccacc atattgccgt cttttggcaa tgtgagggcc cggaaacctg
     3841 gccctgtctt cttgacgagc attcctaggg gtctttcccc tctcgccaaa ggaatgcaag
     3901 gtctgttgaa tgtcgtgaag gaagcagttc ctctggaagc ttcttgaaga caaacaacgt
     3961 ctgtagcgac cctttgcagg cagcggaacc ccccacctgg cgacaggtgc ctctgcggcc
     4021 aaaagccacg tgtataagat acacctgcaa aggcggcaca accccagtgc cacgttgtga
     4081 gttggatagt tgtggaaaga gtcaaatggc tctcctcaag cgtattcaac aaggggctga
     4141 aggatgccca gaaggtaccc cattgtatgg gatctgatct ggggcctcgg tgcacatgct
     4201 ttacatgtgt ttagtcgagg ttaaaaaaac gtctaggccc cccgaaccac ggggacgtgg
     4261 ttttcctttg aaaaacacga tgataatatg gccacaacca tgaccgagta caagcccacg
     4321 gtgcgcctcg ccacccgcga cgacgtcccc cgggccgtac gcaccctcgc cgccgcgttc
     4381 gccgactacc ccgccacgcg ccacaccgtc gacccggacc gccacatcga gcgggtcacc
     4441 gagctgcaag aactcttcct cacgcgcgtc gggctcgaca tcggcaaggt gtgggtcgcg
     4501 gacgacggcg ccgcggtggc ggtctggacc acgccggaga gcgtcgaagc gggggcggtg
     4561 ttcgccgaga tcggcccgcg catggccgag ttgagcggtt cccggctggc cgcgcagcaa
     4621 cagatggaag gcctcctggc gccgcaccgg cccaaggagc ccgcgtggtt cctggccacc
     4681 gtcggcgtct cgcccgacca ccagggcaag ggtctgggca gcgccgtcgt gctccccgga
     4741 gtggaggcgg ccgagcgcgc cggggtgccc gccttcctgg agacctccgc gccccgcaac
     4801 ctccccttct acgagcggct cggcttcacc gtcaccgccg acgtcgaggt gcccgaagga
     4861 ccgcgcacct ggtgcatgac ccgcaagccc ggtgcctgac gcccgcccca cgacccgcag
     4921 cgcccgaccg aaaggagcgc acgaccccat ggctcgacga taaaataaaa gattttattt
     4981 agtctccaga aaaagggggg aatgaaagac cccacctgta ggtttggcaa gctagcttaa
     5041 gtaacgccat tttgcaaggc atggaaaaat acataactga gaatagagaa gttcagatca
     5101 aggtcaggaa cagatggaac agctgaatat gggccaaaca ggatatctgt ggtaagcagt
     5161 tcctgccccg gctcagggcc aagaacagat ggaacagctg aatatgggcc aaacaggata
     5221 tctgtggtaa gcagttcctg ccccggctca gggccaagaa cagatggtcc ccagatgcgg
     5281 tccagccctc agcagtttct agagaaccat cagatgtttc cagggtgccc caaggacctg
     5341 aaatgaccct gtgccttatt tgaactaacc aatcagttcg cttctcgctt ctgttcgcgc
     5401 gcttctgctc cccgagctca ataaaagagc ccacaacccc tcactcgggg cgccagtcct
     5461 ccgattgact gagtcgcccg ggtacccgtg tatccaataa accctcttgc agttgcatcc
     5521 gacttgtggt ctcgctgttc cttgggaggg tctcctctga gtgattgact acccgtcagc
     5581 gggggtcttt cacatgcagc atgtatcaaa attaatttgg ttttttttct taagtattta
     5641 cattaaatgg ccatagttgc attaatgaat cggccaacgc gcggggagag gcggtttgcg
     5701 tattgggcgc tcttccgctt cctcgctcac tgactcgctg cgctcggtcg ttcggctgcg
     5761 gcgagcggta tcagctcact caaaggcggt aatacggtta tccacagaat caggggataa
     5821 cgcaggaaag aacatgtgag caaaaggcca gcaaaaggcc aggaaccgta aaaaggccgc
     5881 gttgctggcg tttttccata ggctccgccc ccctgacgag catcacaaaa atcgacgctc
     5941 aagtcagagg tggcgaaacc cgacaggact ataaagatac caggcgtttc cccctggaag
     6001 ctccctcgtg cgctctcctg ttccgaccct gccgcttacc ggatacctgt ccgcctttct
     6061 cccttcggga agcgtggcgc tttctcatag ctcacgctgt aggtatctca gttcggtgta
     6121 ggtcgttcgc tccaagctgg gctgtgtgca cgaacccccc gttcagcccg accgctgcgc
     6181 cttatccggt aactatcgtc ttgagtccaa cccggtaaga cacgacttat cgccactggc
     6241 agcagccact ggtaacagga ttagcagagc gaggtatgta ggcggtgcta cagagttctt
     6301 gaagtggtgg cctaactacg gctacactag aagaacagta tttggtatct gcgctctgct
     6361 gaagccagtt accttcggaa aaagagttgg tagctcttga tccggcaaac aaaccaccgc
     6421 tggtagcggt ggtttttttg tttgcaagca gcagattacg cgcagaaaaa aaggatctca
     6481 agaagatcct ttgatctttt ctacggggtc tgacgctcag tggaacgaaa actcacgtta
     6541 agggattttg gtcatgagat tatcaaaaag gatcttcacc tagatccttt taaatcaatc
     6601 taaagtatat atgagtaaac ttggtctgac agttaccaat gcttaatcag tgaggcacct
     6661 atctcagcga tctgtctatt tcgttcatcc atagttgcct gactccccgt cgtgtagata
     6721 actacgatac gggagggctt accatctggc cccagtgctg caatgatacc gcgagaccca
     6781 cgctcaccgg ctccagattt atcagcaata aaccagccag ccggaagggc cgagcgcaga
     6841 agtggtcctg caactttatc cgcctccatc cagtctatta attgttgccg ggaagctaga
     6901 gtaagtagtt cgccagttaa tagtttgcgc aacgttgttg ccattgctac aggcatcgtg
     6961 gtgtcacgct cgtcgtttgg tatggcttca ttcagctccg gttcccaacg atcaaggcga
     7021 gttacatgat cccccatgtt gtgcaaaaaa gcggttagct ccttcggtcc tccgatcgtt
     7081 gtcagaagta agttggccgc agtgttatca ctcatggtta tggcagcact gcataattct
     7141 cttactgtca tgccatccgt aagatgcttt tctgtgactg gtgagtactc aaccaagtca
     7201 ttctgagaat agtgtatgcg gcgaccgagt tgctcttgcc cggcgtcaac acgggataat
     7261 accgcgccac atagcagaac tttaaaagtg ctcatcattg gaaaacgttc ttcggggcga
     7321 aaactctcaa ggatcttacc gctgttgaga tccagttcga tgtaacccac tcgtgcaccc
     7381 aactgatctt cagcatcttt tactttcacc agcgtttctg ggtgagcaaa aacaggaagg
     7441 caaaatgccg caaaaaaggg aataagggcg acacggaaat gttgaatact catactcttc
     7501 ctttttcaat attattgaag catttatcag ggttattgtc tcatgagcgg atacatattt
     7561 gaatgtattt agaaaaataa acaaataggg gttccgcgca catt
//
