LOCUS       sequence-436845-        7652 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Expresses the presequence of Neurospora crassa Fo-ATPase subunit 9 
            (residues 1–69) with HaloTag and SNAP tag for measuring mitophagy 
            activity by Halo processing assay..
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 7652)
  AUTHORS   Yang Z, Yoshii SR, Sakai Y, Zhang J, Chino H, Knorr RL, Mizushima N
  TITLE     Autophagy adaptors mediate Parkin-dependent mitophagy by forming 
            sheet-like liquid condensates.
  JOURNAL   EMBO J. 2024 Nov;43(22):5613-5634. doi: 10.1038/s44318-024-00272-5. 
            Epub 2024 Oct 17.
  PUBMED    39420095
REFERENCE   2  (bases 1 to 7652)
  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"; doi: 
            "10.1038/s44318-024-00272-5"; journalName: "EMBO J"; date: 
            "2024-11"; volume: "43"; issue: "22"; pages: "5613-5634"
COMMENT     Sequence Label: pMRX-IBU-pSu9-HaloTag7-SNAP
FEATURES             Location/Qualifiers
     source          1..7652
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     180..202
                     /label=pLXSN 5'
                     /note="Murine stem cell virus, forward primer. Also called 
                     MSCV"
     primer_bind     218..234
                     /label=pBABE 5'
                     /note="Psi packaging signal, forward primer for pBABE 
                     vectors"
     misc_feature    260..634
                     /label=pol region
                     /note="Moloney murine leukemia virus (MMLV) pol region 
                     containing the splice acceptor site"
     primer_bind     572..588
                     /label=pBMN 5'
                     /note="MMLV sequence, for inserts in pBMN retroviral 
                     vector"
     CDS             922..1806
                     /codon_start=1
                     /product="modified bacterial dehalogenase that forms 
                     covalent bonds with chloroalkane derivatives"
                     /label=HaloTag(R)
                     /translation="EIGTGFPFDPHYVEVLGERMHYVDVGPRDGTPVLFLHGNPTSSYV
                     WRNIIPHVAPTHRCIAPDLIGMGKSDKPDLGYFFDDHVRFMDAFIEALGLEEVVLVIHD
                     WGSALGFHWAKRNPERVKGIAFMEFIRPIPTWDEWPEFARETFQAFRTTDVGRKLIIDQ
                     NVFIEGTLPMGVVRPLTEVEMDHYREPFLNPVDREPLWRFPNELPIAGEPANIVALVEE
                     YMDWLHQSPVPKLLFWGTPGVLIPPAEAARLAKSLPNCKAVDIGPGLNLLQEDNPDLIG
                     SEIARWLSTLEISG"
     CDS             1831..2373
                     /codon_start=1
                     /product="human O6-alkylguanine-DNA-alkyltransferase 
                     variant that forms covalent bonds with benzylguanine 
                     derivatives"
                     /label=SNAP-tag(R)
                     /note="fast-reacting variant, optimized for mammalian 
                     expression"
                     /translation="DKDCEMKRTTLDSPLGKLELSGCEQGLHRIIFLGKGTSAADAVEV
                     PAPAAVLGGPEPLMQATAWLNAYFHQPEAIEEFPVPALHHPVFQQESFTRQVLWKLLKV
                     VKFGEVISYSHLAALAGNPAATAAVKTALSGNPVPILIPCHRVVQGDLDVGGYEGGLAV
                     KEWLLAHEGHRLGKPGLG"
     misc_feature    2407..2973
                     /label=IRES
                     /note="internal ribosome entry site (IRES) of the 
                     encephalomyocarditis virus (EMCV)"
     misc_feature    2423..2975
                     /label=IRES
                     /note="internal ribosome entry site (IRES) of the 
                     encephalomyocarditis virus (EMCV)"
     primer_bind     complement(2567..2584)
                     /label=IRES reverse
                     /note="IRES internal ribosome entry site, reverse primer. 
                     Also called pCDH-rev"
     primer_bind     2794..2813
                     /label=IRES-F
                     /note="IRES internal ribosome entry site, forward primer"
     LTR             3651..4120
                     /note="long terminal repeat from Moloney murine leukemia 
                     virus"
     primer_bind     complement(4250..4267)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(4421..5009)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(4501..4520)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(5173..5964,5965..6033))
                     /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     5796..5815
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(6034..6138)
                     /gene="bla"
                     /label=AmpR promoter
     primer_bind     6206..6224
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     enhancer        6326..6705
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
     misc_feature    7064..7421
                     /label=MMLV Psi
                     /note="packaging signal of Moloney murine leukemia virus 
                     (MMLV)"
     CDS             join(7486..7652,1..250)
                     /label=gag (truncated)
                     /note="truncated Moloney murine leukemia virus (MMLV) gag 
                     gene lacking the start codon"
ORIGIN
        1 ccgagacctc atcacccagg ttaagatcaa ggtcttttca cctggcccgc atggacaccc
       61 agaccaggtc ccctacatcg tgacctggga agccttggct tttgaccccc ctccctgggt
      121 caagcccttt gtacacccta agcctccgcc tcctcttcct ccatccgccc cgtctctccc
      181 ccttgaacct cctcgttcga ccccgcctcg atcctccctt tatccagccc tcactccttc
      241 tctaggcgcc cccatatggc catatgagat cttatatggg gcacccccgc cccttgtaaa
      301 cttccctgac cctgacatga caagagttac taacagcccc tctctccaag ctcacttaca
      361 ggctctctac ttagtccagc acgaagtctg gagacctctg gcggcagcct accaagaaca
      421 actggaccga ccggtggtac ctcaccctta ccgagtcggc gacacagtgt gggtccgccg
      481 acaccagact aagaacctag aacctcgctg gaaaggacct tacacagtcc tgctgaccac
      541 ccccaccgcc ctcaaagtag acggcatcgc agcttggata cacgccgccc acgtgaaggc
      601 tgccgacccc gggggtggac catcctctag actgccggat ctgccatcga tataaagctt
      661 aaaggatcca tggcctccac tcgtgtcctc gcctctcgcc tggcctccca gatggctgct
      721 tccgccaagg ttgcccgccc tgctgtccgc gttgctcagg tcagcaagcg caccatccag
      781 actggctccc ccctccagac cctcaagcgc acccagatga cctccatcgt caacgccacc
      841 acccgccagg ctttccagaa gcgcgcctac tcttctgatc caccggtcgc caccggtagc
      901 ggaagcggca gcggcagcgg cgaaatcggt actggctttc cattcgaccc ccattatgtg
      961 gaagtcctgg gcgagcgcat gcactacgtc gatgttggtc cgcgcgatgg cacccctgtg
     1021 ctgttcctgc acggtaaccc gacctcctcc tacgtgtggc gcaacatcat cccgcatgtt
     1081 gcaccgaccc atcgctgcat tgctccagac ctgatcggta tgggcaaatc cgacaaacca
     1141 gacctgggtt atttcttcga cgaccacgtc cgcttcatgg atgccttcat cgaagccctg
     1201 ggtctggaag aggtcgtcct ggtcattcac gactggggct ccgctctggg tttccactgg
     1261 gccaagcgca atccagagcg cgtcaaaggt attgcattta tggagttcat ccgccctatc
     1321 ccgacctggg acgaatggcc agaatttgcc cgcgagacct tccaggcctt ccgcaccacc
     1381 gacgtcggcc gcaagctgat catcgatcag aacgttttta tcgagggtac gctgccgatg
     1441 ggtgtcgtcc gcccgctgac tgaagtcgag atggaccatt accgcgagcc gttcctgaat
     1501 cctgttgacc gcgagccact gtggcgcttc ccaaacgagc tgccaatcgc cggtgagcca
     1561 gcgaacatcg tcgcgctggt cgaagaatac atggactggc tgcaccagtc ccctgtcccg
     1621 aagctgctgt tctggggcac cccaggcgtt ctgatcccac cggccgaagc cgctcgcctg
     1681 gccaaaagcc tgcctaactg caaggctgtg gacatcggcc caggtctgaa tctgctgcaa
     1741 gaagacaacc cggacctgat cggcagcgag atcgcgcgct ggctgtctac tctggagatt
     1801 tccggtggga attcgggctc tggttcaggc gacaaagact gcgaaatgaa gcgcaccacc
     1861 ctggatagcc ctctgggcaa gctggaactg tctgggtgcg aacagggcct gcaccgtatc
     1921 atcttcctgg gcaaaggaac atctgccgcc gacgccgtgg aagtgcctgc cccagccgcc
     1981 gtgctgggcg gaccagagcc actgatgcag gccaccgcct ggctcaacgc ctactttcac
     2041 cagcctgagg ccatcgagga gttccctgtg ccagccctgc accacccagt gttccagcag
     2101 gagagcttta cccgccaggt gctgtggaaa ctgctgaaag tggtgaagtt cggagaggtc
     2161 atcagctaca gccacctggc cgccctggcc ggcaatcccg ccgccaccgc cgccgtgaaa
     2221 accgccctga gcggaaatcc cgtgcccatt ctgatcccct gccaccgggt ggtgcagggc
     2281 gacctggacg tggggggcta cgagggcggg ctcgccgtga aagagtggct gctggcccac
     2341 gagggccaca gactgggcaa gcctgggctg ggttagtgat ttctcgagag cggccgctac
     2401 gtaaattccg cccccccccc taacgttact ggccgaagcc gcttggaata aggccggtgt
     2461 gcgtttgtct atatgttatt ttccaccata ttgccgtctt ttggcaatgt gagggcccgg
     2521 aaacctggcc ctgtcttctt gacgagcatt cctaggggtc tttcccctct cgccaaagga
     2581 atgcaaggtc tgttgaatgt cgtgaaggaa gcagttcctc tggaagtttc ttgaagacaa
     2641 acaacgtctg tagcgaccct ttgcaggcag cggaaccccc cacctggcga caggtgcctc
     2701 tgcggccaaa agccacgtgt ataagataca cctgcaaagg cggcacaacc ccagtgccac
     2761 gttgtgagtt ggatagttgt ggaaagagtc aaatggctct cctcaagcgt attcaacaag
     2821 gggctgaagg atgcccagaa ggtaccccat tgtatgggat ctgatctggg gcctcggtgc
     2881 acatgcttta catgtgttta gtcgaggtta aaaaaacgtc taggcccccc gaaccacggg
     2941 gacgtggttt tcctttgaaa aacacgatga taatatggcc acaaccgtcg acatggtcat
     3001 gaaaacattt aacatttctc aacaagatct agaattagta gaagtagcga cagagaagat
     3061 tacaatgctt tatgaggata ataaacatca tgtgggagcg gcaattcgta cgaaaacagg
     3121 agaaatcatt tcggcagtac atattgaagc gtatatagga cgagtaactg tttgtgcaga
     3181 agccattgcg attggtagtg cagtttcgaa tggacaaaag gattttgaca cgattgtagc
     3241 tgttagacac ccttattctg acgaagtaga tagaagtatt cgagtggtaa gtccttgtgg
     3301 tatgtgtagg gagttgattt cagactatgc accagattgt tttgtgttaa tagaaatgaa
     3361 tggcaagtta gtcaaaacta cgattgaaga actcattcca ctcaaatata cccgaaatta
     3421 agtcgaccgc ccgccccacg acccgcagcg cccgaccgaa aggagcgcac gaccccatgg
     3481 ctcgacgata aaataaaaga ttttatttag tctccagaaa aaggggggaa tgaaagaccc
     3541 cacctgtagg tttggcaagc tagcttaagt aacgccattt tgcaaggcat ggaaaaatac
     3601 ataactgaga atagagaagt tcagatcaag gtcaggaaca gatggaacag ctgaatatgg
     3661 gccaaacagg atatctgtgg taagcagttc ctgccccggc tcagggccaa gaacagatgg
     3721 aacagctgaa tatgggccaa acaggatatc tgtggtaagc agttcctgcc ccggctcagg
     3781 gccaagaaca gatggtcccc agatgcggtc cagccctcag cagtttctag agaaccatca
     3841 gatgtttcca gggtgcccca aggacctgaa atgaccctgt gccttatttg aactaaccaa
     3901 tcagttcgct tctcgcttct gttcgcgcgc ttctgctccc cgagctcaat aaaagagccc
     3961 acaacccctc actcggcgcg ccagtcctcc gattgactga gtcgcccggg tacccgtgta
     4021 tccaataaac cctcttgcag ttgcatccga cttgtggtct cgctgttcct tgggagggtc
     4081 tcctctgagt gattgactac ccgtcagcgg gggtctttca catgcagcat gtatcaaaat
     4141 taatttggtt ttttttctta agtatttaca ttaaatggcc atagttgcat taatgaatcg
     4201 gccaacgcgc ggggagaggc ggtttgcgta ttgggcgctc ttccgcttcc tcgctcactg
     4261 actcgctgcg ctcggtcgtt cggctgcggc gagcggtatc agctcactca aaggcggtaa
     4321 tacggttatc cacagaatca ggggataacg caggaaagaa catgtgagca aaaggccagc
     4381 aaaaggccag gaaccgtaaa aaggccgcgt tgctggcgtt tttccatagg ctccgccccc
     4441 ctgacgagca tcacaaaaat cgacgctcaa gtcagaggtg gcgaaacccg acaggactat
     4501 aaagatacca ggcgtttccc cctggaagct ccctcgtgcg ctctcctgtt ccgaccctgc
     4561 cgcttaccgg atacctgtcc gcctttctcc cttcgggaag cgtggcgctt tctcatagct
     4621 cacgctgtag gtatctcagt tcggtgtagg tcgttcgctc caagctgggc tgtgtgcacg
     4681 aaccccccgt tcagcccgac cgctgcgcct tatccggtaa ctatcgtctt gagtccaacc
     4741 cggtaagaca cgacttatcg ccactggcag cagccactgg taacaggatt agcagagcga
     4801 ggtatgtagg cggtgctaca gagttcttga agtggtggcc taactacggc tacactagaa
     4861 gaacagtatt tggtatctgc gctctgctga agccagttac cttcggaaaa agagttggta
     4921 gctcttgatc cggcaaacaa accaccgctg gtagcggtgg tttttttgtt tgcaagcagc
     4981 agattacgcg cagaaaaaaa ggatctcaag aagatccttt gatcttttct acggggtctg
     5041 acgctcagtg gaacgaaaac tcacgttaag ggattttggt catgagatta tcaaaaagga
     5101 tcttcaccta gatccttttg cggccggccg caaatcaatc taaagtatat atgagtaaac
     5161 ttggtctgac agttaccaat gcttaatcag tgaggcacct atctcagcga tctgtctatt
     5221 tcgttcatcc atagttgcct gactccccgt cgtgtagata actacgatac gggagggctt
     5281 accatctggc cccagtgctg caatgatacc gcgagaccca cgctcaccgg ctccagattt
     5341 atcagcaata aaccagccag ccggaagggc cgagcgcaga agtggtcctg caactttatc
     5401 cgcctccatc cagtctatta attgttgccg ggaagctaga gtaagtagtt cgccagttaa
     5461 tagtttgcgc aacgttgttg ccattgctac aggcatcgtg gtgtcacgct cgtcgtttgg
     5521 tatggcttca ttcagctccg gttcccaacg atcaaggcga gttacatgat cccccatgtt
     5581 gtgcaaaaaa gcggttagct ccttcggtcc tccgatcgtt gtcagaagta agttggccgc
     5641 agtgttatca ctcatggtta tggcagcact gcataattct cttactgtca tgccatccgt
     5701 aagatgcttt tctgtgactg gtgagtactc aaccaagtca ttctgagaat agtgtatgcg
     5761 gcgaccgagt tgctcttgcc cggcgtcaac acgggataat accgcgccac atagcagaac
     5821 tttaaaagtg ctcatcattg gaaaacgttc ttcggggcga aaactctcaa ggatcttacc
     5881 gctgttgaga tccagttcga tgtaacccac tcgtgcaccc aactgatctt cagcatcttt
     5941 tactttcacc agcgtttctg ggtgagcaaa aacaggaagg caaaatgccg caaaaaaggg
     6001 aataagggcg acacggaaat gttgaatact catactcttc ctttttcaat attattgaag
     6061 catttatcag ggttattgtc tcatgagcgg atacatattt gaatgtattt agaaaaataa
     6121 acaaataggg gttccgcgca catttccccg aaaagtgcca cctgacgtct aagaaaccat
     6181 tattatcatg acattaacct ataaaaatag gcgtatcacg aggccctttc gtcttcacta
     6241 taggagcttg gcccattgca tacgttgtat ccatatcata atatgtacat ttatattggc
     6301 tcatgtccaa cattaccgcc atgttgacat tgattattga ctagttatta atagtaatca
     6361 attacggggt cattagttca tagcccatat atggagttcc gcgttacata acttacggta
     6421 aatggcccgc ctggctgacc gcccaacgac ccccgcccat tgacgtcaat aatgacgtat
     6481 gttcccatag taacgccaat agggactttc cattgacgtc aatgggtgga gtatttacgg
     6541 taaactgccc acttggcagt acatcaagtg tatcatatgc caagtacgcc ccctattgac
     6601 gtcaatgacg gtaaatggcc cgcctggcat tatgcccagt acatgacctt atgggacttt
     6661 cctacttggc agtacatcta cgtattagtc atcgctatta ccatgctaga gaaccatcag
     6721 atgtttccag ggtgccccaa ggacctgaaa tgaccctgtg ccttatttga actaaccaat
     6781 cagttcgctt ctcgcttctg ttcgcgcgct tctgctcccc gagctcaata aaagagccca
     6841 caacccctca ctcggcgcgc cagtcctccg attgactgag tcgcccgggt acccgtgtat
     6901 ccaataaacc ctcttgcagt tgcatccgac ttgtggtctc gctgttcctt gggagggtct
     6961 cctctgagtg attgactacc cgtcagcggg ggtctttcat ttgggggctc gtccgggatc
     7021 gggagacccc tgcccaggga ccaccgaccc accaccggga ggtaagctgg ccagcaactt
     7081 atctgtgtct gtccgattgt ctagtgtcta tgactgattt tatgcgcctg cgtcggtact
     7141 agttagctaa ctagctctgt atctggcgga cccgtggtgg aactgacgag ttcggaacac
     7201 ccggccgcaa ccctgggaga cgtcccaggg acttcggggg ccgtttttgt ggcccgacct
     7261 gagtccaaaa atcccgatcg ttttggactc tttggtgcac cccccttaga ggagggatat
     7321 gtggttctgg taggagacga gaacctaaaa cagttcccgc ctccgtctga atttttgctt
     7381 tcggtttggg accgaagccg cgccgcgcgt cttgtctgct gcagcatcgt tctgtgttgt
     7441 ctctgtctga ctgtgtttct gtatttgtct gaaaatatgg gcccgggcca gactgttacc
     7501 actcccttaa gtttgacctt aggtcactgg aaagatgtcg agcggatcgc tcacaaccag
     7561 tcggtagatg tcaagaagag acgttgggtt accttctgct ctgcagaatg gccaaccttt
     7621 aacgtcggat ggccgcgaga cggcaccttt aa
//
