LOCUS       sequence-364106-        7802 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Stable expression of pSu9-HaloTag7-mGFP in mammalian cells to 
            measure mitophagy activity.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 7802)
  AUTHORS   Yim WW, Yamamoto H, Mizushima N
  TITLE     A pulse-chasable reporter processing assay for mammalian autophagic 
            flux with HaloTag.
  JOURNAL   Elife. 2022 Aug 8;11. pii: 78923. doi: 10.7554/eLife.78923.
  PUBMED    35938926
REFERENCE   2  (bases 1 to 7802)
  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: "Elife."; 
            date: "2022-08-8"; volume: "11. pii: 78923. doi"; pages: " 
            10.7554/eLife.78923"
COMMENT     Sequence Label: pMRX-IB-pSu9-HaloTag7-mGFP
FEATURES             Location/Qualifiers
     source          1..7802
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     enhancer        42..421
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
     misc_feature    780..1137
                     /label=MMLV Psi
                     /note="packaging signal of Moloney murine leukemia virus 
                     (MMLV)"
     CDS             1202..1618
                     /label=gag (truncated)
                     /note="truncated Moloney murine leukemia virus (MMLV) gag 
                     gene lacking the start codon"
     primer_bind     1548..1570
                     /label=pLXSN 5'
                     /note="Murine stem cell virus, forward primer. Also called 
                     MSCV"
     primer_bind     1586..1602
                     /label=pBABE 5'
                     /note="Psi packaging signal, forward primer for pBABE 
                     vectors"
     misc_feature    1628..2002
                     /label=pol region
                     /note="Moloney murine leukemia virus (MMLV) pol region 
                     containing the splice acceptor site"
     primer_bind     1940..1956
                     /label=pBMN 5'
                     /note="MMLV sequence, for inserts in pBMN retroviral 
                     vector"
     regulatory      2032..2041
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     CDS             2293..3177
                     /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             join(3184..3186,3187..3900)
                     /codon_start=1
                     /product="enhanced GFP with monomerizing A206K mutation 
                     (Zacharias et al., 2002)"
                     /label=mEGFP
                     /note="mammalian codon-optimized"
                     /translation="VSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTLK
                     FICTTGKLPVPWPTLVTTLTYGVQCFSRYPDHMKQHDFFKSAMPEGYVQERTIFFKDDG
                     NYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIKV
                     NFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSKLSKDPNEKRDHMVLLE
                     FVTAAGITLGMDELYK"
     primer_bind     complement(3226..3247)
                     /label=EGFP-N
                     /note="EGFP, reverse primer"
     primer_bind     complement(3487..3506)
                     /label=EXFP-R
                     /note="For distinguishing EGFP variants, reverse primer"
     primer_bind     3834..3855
                     /label=EGFP-C
                     /note="EGFP, forward primer"
     misc_feature    3925..4491
                     /label=IRES
                     /note="internal ribosome entry site (IRES) of the 
                     encephalomyocarditis virus (EMCV)"
     misc_feature    3941..4493
                     /label=IRES
                     /note="internal ribosome entry site (IRES) of the 
                     encephalomyocarditis virus (EMCV)"
     primer_bind     complement(4085..4102)
                     /label=IRES reverse
                     /note="IRES internal ribosome entry site, reverse primer. 
                     Also called pCDH-rev"
     primer_bind     4312..4331
                     /label=IRES-F
                     /note="IRES internal ribosome entry site, forward primer"
     LTR             5169..5638
                     /note="long terminal repeat from Moloney murine leukemia 
                     virus"
     primer_bind     complement(5768..5785)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(5939..6527)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(6019..6038)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(6691..7482,7483..7551))
                     /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     7314..7333
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(7552..7656)
                     /gene="bla"
                     /label=AmpR promoter
     primer_bind     7724..7742
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
ORIGIN
        1 gtacatttat attggctcat gtccaacatt accgccatgt tgacattgat tattgactag
       61 ttattaatag taatcaatta cggggtcatt agttcatagc ccatatatgg agttccgcgt
      121 tacataactt acggtaaatg gcccgcctgg ctgaccgccc aacgaccccc gcccattgac
      181 gtcaataatg acgtatgttc ccatagtaac gccaataggg actttccatt gacgtcaatg
      241 ggtggagtat ttacggtaaa ctgcccactt ggcagtacat caagtgtatc atatgccaag
      301 tacgccccct attgacgtca atgacggtaa atggcccgcc tggcattatg cccagtacat
      361 gaccttatgg gactttccta cttggcagta catctacgta ttagtcatcg ctattaccat
      421 gctagagaac catcagatgt ttccagggtg ccccaaggac ctgaaatgac cctgtgcctt
      481 atttgaacta accaatcagt tcgcttctcg cttctgttcg cgcgcttctg ctccccgagc
      541 tcaataaaag agcccacaac ccctcactcg gcgcgccagt cctccgattg actgagtcgc
      601 ccgggtaccc gtgtatccaa taaaccctct tgcagttgca tccgacttgt ggtctcgctg
      661 ttccttggga gggtctcctc tgagtgattg actacccgtc agcgggggtc tttcatttgg
      721 gggctcgtcc gggatcggga gacccctgcc cagggaccac cgacccacca ccgggaggta
      781 agctggccag caacttatct gtgtctgtcc gattgtctag tgtctatgac tgattttatg
      841 cgcctgcgtc ggtactagtt agctaactag ctctgtatct ggcggacccg tggtggaact
      901 gacgagttcg gaacacccgg ccgcaaccct gggagacgtc ccagggactt cgggggccgt
      961 ttttgtggcc cgacctgagt ccaaaaatcc cgatcgtttt ggactctttg gtgcaccccc
     1021 cttagaggag ggatatgtgg ttctggtagg agacgagaac ctaaaacagt tcccgcctcc
     1081 gtctgaattt ttgctttcgg tttgggaccg aagccgcgcc gcgcgtcttg tctgctgcag
     1141 catcgttctg tgttgtctct gtctgactgt gtttctgtat ttgtctgaaa atatgggccc
     1201 gggccagact gttaccactc ccttaagttt gaccttaggt cactggaaag atgtcgagcg
     1261 gatcgctcac aaccagtcgg tagatgtcaa gaagagacgt tgggttacct tctgctctgc
     1321 agaatggcca acctttaacg tcggatggcc gcgagacggc acctttaacc gagacctcat
     1381 cacccaggtt aagatcaagg tcttttcacc tggcccgcat ggacacccag accaggtccc
     1441 ctacatcgtg acctgggaag ccttggcttt tgacccccct ccctgggtca agccctttgt
     1501 acaccctaag cctccgcctc ctcttcctcc atccgccccg tctctccccc ttgaacctcc
     1561 tcgttcgacc ccgcctcgat cctcccttta tccagccctc actccttctc taggcgcccc
     1621 catatggcca tatgagatct tatatggggc acccccgccc cttgtaaact tccctgaccc
     1681 tgacatgaca agagttacta acagcccctc tctccaagct cacttacagg ctctctactt
     1741 agtccagcac gaagtctgga gacctctggc ggcagcctac caagaacaac tggaccgacc
     1801 ggtggtacct cacccttacc gagtcggcga cacagtgtgg gtccgccgac accagactaa
     1861 gaacctagaa cctcgctgga aaggacctta cacagtcctg ctgaccaccc ccaccgccct
     1921 caaagtagac ggcatcgcag cttggataca cgccgcccac gtgaaggctg ccgaccccgg
     1981 gggtggacca tcctctagac tgccggatct gccatcgata taaagcttgc cgccaccatg
     2041 gcctccactc gtgtcctcgc ctctcgcctg gcctcccaga tggctgcttc cgccaaggtt
     2101 gcccgccctg ctgtccgcgt tgctcaggtc agcaagcgca ccatccagac tggctccccc
     2161 ctccagaccc tcaagcgcac ccagatgacc tccatcgtca acgccaccac ccgccaggct
     2221 ttccagaagc gcgcctactc ttctgatcca ccggtcgcca ccggatccgg gaattcgggc
     2281 tctggttcag gcgaaatcgg tactggcttt ccattcgacc cccattatgt ggaagtcctg
     2341 ggcgagcgca tgcactacgt cgatgttggt ccgcgcgatg gcacccctgt gctgttcctg
     2401 cacggtaacc cgacctcctc ctacgtgtgg cgcaacatca tcccgcatgt tgcaccgacc
     2461 catcgctgca ttgctccaga cctgatcggt atgggcaaat ccgacaaacc agacctgggt
     2521 tatttcttcg acgaccacgt ccgcttcatg gatgccttca tcgaagccct gggtctggaa
     2581 gaggtcgtcc tggtcattca cgactggggc tccgctctgg gtttccactg ggccaagcgc
     2641 aatccagagc gcgtcaaagg tattgcattt atggagttca tccgccctat cccgacctgg
     2701 gacgaatggc cagaatttgc ccgcgagacc ttccaggcct tccgcaccac cgacgtcggc
     2761 cgcaagctga tcatcgatca gaacgttttt atcgagggta cgctgccgat gggtgtcgtc
     2821 cgcccgctga ctgaagtcga gatggaccat taccgcgagc cgttcctgaa tcctgttgac
     2881 cgcgagccac tgtggcgctt cccaaacgag ctgccaatcg ccggtgagcc agcgaacatc
     2941 gtcgcgctgg tcgaagaata catggactgg ctgcaccagt cccctgtccc gaagctgctg
     3001 ttctggggca ccccaggcgt tctgatccca ccggccgaag ccgctcgcct ggccaaaagc
     3061 ctgcctaact gcaaggctgt ggacatcggc ccaggtctga atctgctgca agaagacaac
     3121 ccggacctga tcggcagcga gatcgcgcgc tggctgtcta ctctggagat ttccggtctc
     3181 gaggtgagca agggcgagga gctgttcacc ggggtggtgc ccatcctggt cgagctggac
     3241 ggcgacgtaa acggccacaa gttcagcgtg tccggcgagg gcgagggcga tgccacctac
     3301 ggcaagctga ccctgaagtt catctgcacc accggcaagc tgcccgtgcc ctggcccacc
     3361 ctcgtgacca ccctgaccta cggcgtgcag tgcttcagcc gctaccccga ccacatgaag
     3421 cagcacgact tcttcaagtc cgccatgccc gaaggctacg tccaggagcg caccatcttc
     3481 ttcaaggacg acggcaacta caagacccgc gccgaggtga agttcgaggg cgacaccctg
     3541 gtgaaccgca tcgagctgaa gggcatcgac ttcaaggagg acggcaacat cctggggcac
     3601 aagctggagt acaactacaa cagccacaac gtctatatca tggccgacaa gcagaagaac
     3661 ggcatcaagg tgaacttcaa gatccgccac aacatcgagg acggcagcgt gcagctcgcc
     3721 gaccactacc agcagaacac ccccatcggc gacggccccg tgctgctgcc cgacaaccac
     3781 tacctgagca cccagtccaa actgagcaaa gaccccaacg agaagcgcga tcacatggtc
     3841 ctgctggagt tcgtgaccgc cgccgggatc actctcggca tggacgagct gtacaagtaa
     3901 ctcgagagcg gccgctacgt aaattccgcc ccccccccta acgttactgg ccgaagccgc
     3961 ttggaataag gccggtgtgc gtttgtctat atgttatttt ccaccatatt gccgtctttt
     4021 ggcaatgtga gggcccggaa acctggccct gtcttcttga cgagcattcc taggggtctt
     4081 tcccctctcg ccaaaggaat gcaaggtctg ttgaatgtcg tgaaggaagc agttcctctg
     4141 gaagtttctt gaagacaaac aacgtctgta gcgacccttt gcaggcagcg gaacccccca
     4201 cctggcgaca ggtgcctctg cggccaaaag ccacgtgtat aagatacacc tgcaaaggcg
     4261 gcacaacccc agtgccacgt tgtgagttgg atagttgtgg aaagagtcaa atggctctcc
     4321 tcaagcgtat tcaacaaggg gctgaaggat gcccagaagg taccccattg tatgggatct
     4381 gatctggggc ctcggtgcac atgctttaca tgtgtttagt cgaggttaaa aaaacgtcta
     4441 ggccccccga accacgggga cgtggttttc ctttgaaaaa cacgatgata atatggccac
     4501 aaccgtcgac atggtcatga aaacatttaa catttctcaa caagatctag aattagtaga
     4561 agtagcgaca gagaagatta caatgcttta tgaggataat aaacatcatg tgggagcggc
     4621 aattcgtacg aaaacaggag aaatcatttc ggcagtacat attgaagcgt atataggacg
     4681 agtaactgtt tgtgcagaag ccattgcgat tggtagtgca gtttcgaatg gacaaaagga
     4741 ttttgacacg attgtagctg ttagacaccc ttattctgac gaagtagata gaagtattcg
     4801 agtggtaagt ccttgtggta tgtgtaggga gttgatttca gactatgcac cagattgttt
     4861 tgtgttaata gaaatgaatg gcaagttagt caaaactacg attgaagaac tcattccact
     4921 caaatatacc cgaaattaag tcgaccgccc gccccacgac ccgcagcgcc cgaccgaaag
     4981 gagcgcacga ccccatggct cgacgataaa ataaaagatt ttatttagtc tccagaaaaa
     5041 ggggggaatg aaagacccca cctgtaggtt tggcaagcta gcttaagtaa cgccattttg
     5101 caaggcatgg aaaaatacat aactgagaat agagaagttc agatcaaggt caggaacaga
     5161 tggaacagct gaatatgggc caaacaggat atctgtggta agcagttcct gccccggctc
     5221 agggccaaga acagatggaa cagctgaata tgggccaaac aggatatctg tggtaagcag
     5281 ttcctgcccc ggctcagggc caagaacaga tggtccccag atgcggtcca gccctcagca
     5341 gtttctagag aaccatcaga tgtttccagg gtgccccaag gacctgaaat gaccctgtgc
     5401 cttatttgaa ctaaccaatc agttcgcttc tcgcttctgt tcgcgcgctt ctgctccccg
     5461 agctcaataa aagagcccac aacccctcac tcggggcgcc agtcctccga ttgactgagt
     5521 cgcccgggta cccgtgtatc caataaaccc tcttgcagtt gcatccgact tgtggtctcg
     5581 ctgttccttg ggagggtctc ctctgagtga ttgactaccc gtcagcgggg gtctttcaca
     5641 tgcagcatgt atcaaaatta atttggtttt ttttcttaag tatttacatt aaatggccat
     5701 agttgcatta atgaatcggc caacgcgcgg ggagaggcgg tttgcgtatt gggcgctctt
     5761 ccgcttcctc gctcactgac tcgctgcgct cggtcgttcg gctgcggcga gcggtatcag
     5821 ctcactcaaa ggcggtaata cggttatcca cagaatcagg ggataacgca ggaaagaaca
     5881 tgtgagcaaa aggccagcaa aaggccagga accgtaaaaa ggccgcgttg ctggcgtttt
     5941 tccataggct ccgcccccct gacgagcatc acaaaaatcg acgctcaagt cagaggtggc
     6001 gaaacccgac aggactataa agataccagg cgtttccccc tggaagctcc ctcgtgcgct
     6061 ctcctgttcc gaccctgccg cttaccggat acctgtccgc ctttctccct tcgggaagcg
     6121 tggcgctttc tcatagctca cgctgtaggt atctcagttc ggtgtaggtc gttcgctcca
     6181 agctgggctg tgtgcacgaa ccccccgttc agcccgaccg ctgcgcctta tccggtaact
     6241 atcgtcttga gtccaacccg gtaagacacg acttatcgcc actggcagca gccactggta
     6301 acaggattag cagagcgagg tatgtaggcg gtgctacaga gttcttgaag tggtggccta
     6361 actacggcta cactagaaga acagtatttg gtatctgcgc tctgctgaag ccagttacct
     6421 tcggaaaaag agttggtagc tcttgatccg gcaaacaaac caccgctggt agcggtggtt
     6481 tttttgtttg caagcagcag attacgcgca gaaaaaaagg atctcaagaa gatcctttga
     6541 tcttttctac ggggtctgac gctcagtgga acgaaaactc acgttaaggg attttggtca
     6601 tgagattatc aaaaaggatc ttcacctaga tccttttgcg gccggccgca aatcaatcta
     6661 aagtatatat gagtaaactt ggtctgacag ttaccaatgc ttaatcagtg aggcacctat
     6721 ctcagcgatc tgtctatttc gttcatccat agttgcctga ctccccgtcg tgtagataac
     6781 tacgatacgg gagggcttac catctggccc cagtgctgca atgataccgc gagacccacg
     6841 ctcaccggct ccagatttat cagcaataaa ccagccagcc ggaagggccg agcgcagaag
     6901 tggtcctgca actttatccg cctccatcca gtctattaat tgttgccggg aagctagagt
     6961 aagtagttcg ccagttaata gtttgcgcaa cgttgttgcc attgctacag gcatcgtggt
     7021 gtcacgctcg tcgtttggta tggcttcatt cagctccggt tcccaacgat caaggcgagt
     7081 tacatgatcc cccatgttgt gcaaaaaagc ggttagctcc ttcggtcctc cgatcgttgt
     7141 cagaagtaag ttggccgcag tgttatcact catggttatg gcagcactgc ataattctct
     7201 tactgtcatg ccatccgtaa gatgcttttc tgtgactggt gagtactcaa ccaagtcatt
     7261 ctgagaatag tgtatgcggc gaccgagttg ctcttgcccg gcgtcaacac gggataatac
     7321 cgcgccacat agcagaactt taaaagtgct catcattgga aaacgttctt cggggcgaaa
     7381 actctcaagg atcttaccgc tgttgagatc cagttcgatg taacccactc gtgcacccaa
     7441 ctgatcttca gcatctttta ctttcaccag cgtttctggg tgagcaaaaa caggaaggca
     7501 aaatgccgca aaaaagggaa taagggcgac acggaaatgt tgaatactca tactcttcct
     7561 ttttcaatat tattgaagca tttatcaggg ttattgtctc atgagcggat acatatttga
     7621 atgtatttag aaaaataaac aaataggggt tccgcgcaca tttccccgaa aagtgccacc
     7681 tgacgtctaa gaaaccatta ttatcatgac attaacctat aaaaataggc gtatcacgag
     7741 gccctttcgt cttcactata ggagcttggc ccattgcata cgttgtatcc atatcataat
     7801 at
//
