LOCUS       sequence-487475-        7520 bp    DNA     circular SYN 30-SEP-2025
DEFINITION  Stable expression of HaloTag7-mGFP-LC3 in mammalian cells to measure
            autophagic flux.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 7520)
  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 7520)
  AUTHORS   .
  TITLE     Direct Submission
  JOURNAL   Exported Sep 30, 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-No-HaloTag7-mGFP-LC3
FEATURES             Location/Qualifiers
     source          1..7520
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     enhancer        30..409
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
     misc_feature    768..1125
                     /label=MMLV Psi
                     /note="packaging signal of Moloney murine leukemia virus 
                     (MMLV)"
     CDS             1190..1606
                     /label=gag (truncated)
                     /note="truncated Moloney murine leukemia virus (MMLV) gag 
                     gene lacking the start codon"
     primer_bind     1536..1558
                     /label=pLXSN 5'
                     /note="Murine stem cell virus, forward primer. Also called 
                     MSCV"
     primer_bind     1574..1590
                     /label=pBABE 5'
                     /note="Psi packaging signal, forward primer for pBABE 
                     vectors"
     misc_feature    1616..1990
                     /label=pol region
                     /note="Moloney murine leukemia virus (MMLV) pol region 
                     containing the splice acceptor site"
     primer_bind     1928..1944
                     /label=pBMN 5'
                     /note="MMLV sequence, for inserts in pBMN retroviral 
                     vector"
     regulatory      2017..2026
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     CDS             2023..2910
                     /codon_start=1
                     /product="modified bacterial dehalogenase that forms 
                     covalent bonds with chloroalkane derivatives"
                     /label=HaloTag(R)
                     /translation="MEIGTGFPFDPHYVEVLGERMHYVDVGPRDGTPVLFLHGNPTSSY
                     VWRNIIPHVAPTHRCIAPDLIGMGKSDKPDLGYFFDDHVRFMDAFIEALGLEEVVLVIH
                     DWGSALGFHWAKRNPERVKGIAFMEFIRPIPTWDEWPEFARETFQAFRTTDVGRKLIID
                     QNVFIEGTLPMGVVRPLTEVEMDHYREPFLNPVDREPLWRFPNELPIAGEPANIVALVE
                     EYMDWLHQSPVPKLLFWGTPGVLIPPAEAARLAKSLPNCKAVDIGPGLNLLQEDNPDLI
                     GSEIARWLSTLEISG"
     CDS             join(2932..2934,2935..3645)
                     /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(2974..2995)
                     /label=EGFP-N
                     /note="EGFP, reverse primer"
     primer_bind     complement(3235..3254)
                     /label=EXFP-R
                     /note="For distinguishing EGFP variants, reverse primer"
     primer_bind     3582..3603
                     /label=EGFP-C
                     /note="EGFP, forward primer"
     misc_feature    4066..4632
                     /label=IRES
                     /note="internal ribosome entry site (IRES) of the 
                     encephalomyocarditis virus (EMCV)"
     misc_feature    4082..4634
                     /label=IRES
                     /note="internal ribosome entry site (IRES) of the 
                     encephalomyocarditis virus (EMCV)"
     primer_bind     complement(4226..4243)
                     /label=IRES reverse
                     /note="IRES internal ribosome entry site, reverse primer. 
                     Also called pCDH-rev"
     primer_bind     4453..4472
                     /label=IRES-F
                     /note="IRES internal ribosome entry site, forward primer"
     LTR             4875..5344
                     /note="long terminal repeat from Moloney murine leukemia 
                     virus"
     primer_bind     complement(5474..5491)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(5645..6233)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(5725..5744)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(6397..7188,7189..7257))
                     /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     7020..7039
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(7258..7362)
                     /gene="bla"
                     /label=AmpR promoter
     primer_bind     7430..7448
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
ORIGIN
        1 tggctcatgt ccaacattac cgccatgttg acattgatta ttgactagtt attaatagta
       61 atcaattacg gggtcattag ttcatagccc atatatggag ttccgcgtta cataacttac
      121 ggtaaatggc ccgcctggct gaccgcccaa cgacccccgc ccattgacgt caataatgac
      181 gtatgttccc atagtaacgc caatagggac tttccattga cgtcaatggg tggagtattt
      241 acggtaaact gcccacttgg cagtacatca agtgtatcat atgccaagta cgccccctat
      301 tgacgtcaat gacggtaaat ggcccgcctg gcattatgcc cagtacatga ccttatggga
      361 ctttcctact tggcagtaca tctacgtatt agtcatcgct attaccatgc tagagaacca
      421 tcagatgttt ccagggtgcc ccaaggacct gaaatgaccc tgtgccttat ttgaactaac
      481 caatcagttc gcttctcgct tctgttcgcg cgcttctgct ccccgagctc aataaaagag
      541 cccacaaccc ctcactcggc gcgccagtcc tccgattgac tgagtcgccc gggtacccgt
      601 gtatccaata aaccctcttg cagttgcatc cgacttgtgg tctcgctgtt ccttgggagg
      661 gtctcctctg agtgattgac tacccgtcag cgggggtctt tcatttgggg gctcgtccgg
      721 gatcgggaga cccctgccca gggaccaccg acccaccacc gggaggtaag ctggccagca
      781 acttatctgt gtctgtccga ttgtctagtg tctatgactg attttatgcg cctgcgtcgg
      841 tactagttag ctaactagct ctgtatctgg cggacccgtg gtggaactga cgagttcgga
      901 acacccggcc gcaaccctgg gagacgtccc agggacttcg ggggccgttt ttgtggcccg
      961 acctgagtcc aaaaatcccg atcgttttgg actctttggt gcacccccct tagaggaggg
     1021 atatgtggtt ctggtaggag acgagaacct aaaacagttc ccgcctccgt ctgaattttt
     1081 gctttcggtt tgggaccgaa gccgcgccgc gcgtcttgtc tgctgcagca tcgttctgtg
     1141 ttgtctctgt ctgactgtgt ttctgtattt gtctgaaaat atgggcccgg gccagactgt
     1201 taccactccc ttaagtttga ccttaggtca ctggaaagat gtcgagcgga tcgctcacaa
     1261 ccagtcggta gatgtcaaga agagacgttg ggttaccttc tgctctgcag aatggccaac
     1321 ctttaacgtc ggatggccgc gagacggcac ctttaaccga gacctcatca cccaggttaa
     1381 gatcaaggtc ttttcacctg gcccgcatgg acacccagac caggtcccct acatcgtgac
     1441 ctgggaagcc ttggcttttg acccccctcc ctgggtcaag ccctttgtac accctaagcc
     1501 tccgcctcct cttcctccat ccgccccgtc tctccccctt gaacctcctc gttcgacccc
     1561 gcctcgatcc tccctttatc cagccctcac tccttctcta ggcgccccca tatggccata
     1621 tgagatctta tatggggcac ccccgcccct tgtaaacttc cctgaccctg acatgacaag
     1681 agttactaac agcccctctc tccaagctca cttacaggct ctctacttag tccagcacga
     1741 agtctggaga cctctggcgg cagcctacca agaacaactg gaccgaccgg tggtacctca
     1801 cccttaccga gtcggcgaca cagtgtgggt ccgccgacac cagactaaga acctagaacc
     1861 tcgctggaaa ggaccttaca cagtcctgct gaccaccccc accgccctca aagtagacgg
     1921 catcgcagct tggatacacg ccgcccacgt gaaggctgcc gaccccgggg gtggaccatc
     1981 ctctagactg ccggatctgc catcgatata aagcttgcca ccatggaaat cggtactggc
     2041 tttccattcg acccccatta tgtggaagtc ctgggcgagc gcatgcacta cgtcgatgtt
     2101 ggtccgcgcg atggcacccc tgtgctgttc ctgcacggta acccgacctc ctcctacgtg
     2161 tggcgcaaca tcatcccgca tgttgcaccg acccatcgct gcattgctcc agacctgatc
     2221 ggtatgggca aatccgacaa accagacctg ggttatttct tcgacgacca cgtccgcttc
     2281 atggatgcct tcatcgaagc cctgggtctg gaagaggtcg tcctggtcat tcacgactgg
     2341 ggctccgctc tgggtttcca ctgggccaag cgcaatccag agcgcgtcaa aggtattgca
     2401 tttatggagt tcatccgccc tatcccgacc tgggacgaat ggccagaatt tgcccgcgag
     2461 accttccagg ccttccgcac caccgacgtc ggccgcaagc tgatcatcga tcagaacgtt
     2521 tttatcgagg gtacgctgcc gatgggtgtc gtccgcccgc tgactgaagt cgagatggac
     2581 cattaccgcg agccgttcct gaatcctgtt gaccgcgagc cactgtggcg cttcccaaac
     2641 gagctgccaa tcgccggtga gccagcgaac atcgtcgcgc tggtcgaaga atacatggac
     2701 tggctgcacc agtcccctgt cccgaagctg ctgttctggg gcaccccagg cgttctgatc
     2761 ccaccggccg aagccgctcg cctggccaaa agcctgccta actgcaaggc tgtggacatc
     2821 ggcccaggtc tgaatctgct gcaagaagac aacccggacc tgatcggcag cgagatcgcg
     2881 cgctggctgt ctactctgga gatttccggt ggttcaggct cgggatccgg ggtgagcaag
     2941 ggcgaggagc tgttcaccgg ggtggtgccc atcctggtcg agctggacgg cgacgtaaac
     3001 ggccacaagt tcagcgtgtc cggcgagggc gagggcgatg ccacctacgg caagctgacc
     3061 ctgaagttca tctgcaccac cggcaagctg cccgtgccct ggcccaccct cgtgaccacc
     3121 ctgacctacg gcgtgcagtg cttcagccgc taccccgacc acatgaagca gcacgacttc
     3181 ttcaagtccg ccatgcccga aggctacgtc caggagcgca ccatcttctt caaggacgac
     3241 ggcaactaca agacccgcgc cgaggtgaag ttcgagggcg acaccctggt gaaccgcatc
     3301 gagctgaagg gcatcgactt caaggaggac ggcaacatcc tggggcacaa gctggagtac
     3361 aactacaaca gccacaacgt ctatatcatg gccgacaagc agaagaacgg catcaaggtg
     3421 aacttcaaga tccgccacaa catcgaggac ggcagcgtgc agctcgccga ccactaccag
     3481 cagaacaccc ccatcggcga cggccccgtg ctgctgcccg acaaccacta cctgagcacc
     3541 cagtccaaac tgagcaaaga ccccaacgag aagcgcgatc acatggtcct gctggagttc
     3601 gtgaccgccg ccgggatcac tctcggcatg gacgagctgt acaagggttc aggctcggga
     3661 tctgggaatt cgggtccgtc cgagaagacc ttcaaacagc gccggagctt cgaacaaaga
     3721 gtggaagatg tccggctcat ccgggagcag caccccacca agatcccagt gattatagag
     3781 cgatacaagg gtgagaagca gctgcccgtc ctggacaaga ccaagttcct tgtacctgat
     3841 cacgtgaata tgagcgaact catcaagata attagaaggc gcctgcagct caatgctaac
     3901 caagccttct tcctcctggt gaatgggcac agcatggtga gtgtgtccac acccatctct
     3961 gaagtgtacg agagcgagag agatgaagac ggcttcctgt acatggtcta tgcctcccag
     4021 gagacgttcg ggtaatgact cgagtaaagc ggccgctacg taaattccgc ccccccccct
     4081 aacgttactg gccgaagccg cttggaataa ggccggtgtg cgtttgtcta tatgttattt
     4141 tccaccatat tgccgtcttt tggcaatgtg agggcccgga aacctggccc tgtcttcttg
     4201 acgagcattc ctaggggtct ttcccctctc gccaaaggaa tgcaaggtct gttgaatgtc
     4261 gtgaaggaag cagttcctct ggaagtttct tgaagacaaa caacgtctgt agcgaccctt
     4321 tgcaggcagc ggaacccccc acctggcgac aggtgcctct gcggccaaaa gccacgtgta
     4381 taagatacac ctgcaaaggc ggcacaaccc cagtgccacg ttgtgagttg gatagttgtg
     4441 gaaagagtca aatggctctc ctcaagcgta ttcaacaagg ggctgaagga tgcccagaag
     4501 gtaccccatt gtatgggatc tgatctgggg cctcggtgca catgctttac atgtgtttag
     4561 tcgaggttaa aaaaacgtct aggccccccg aaccacgggg acgtggtttt cctttgaaaa
     4621 acacgatgat aatatggcca caaccgtcga ccgcccgccc cacgacccgc agcgcccgac
     4681 cgaaaggagc gcacgacccc atggctcgac gataaaataa aagattttat ttagtctcca
     4741 gaaaaagggg ggaatgaaag accccacctg taggtttggc aagctagctt aagtaacgcc
     4801 attttgcaag gcatggaaaa atacataact gagaatagag aagttcagat caaggtcagg
     4861 aacagatgga acagctgaat atgggccaaa caggatatct gtggtaagca gttcctgccc
     4921 cggctcaggg ccaagaacag atggaacagc tgaatatggg ccaaacagga tatctgtggt
     4981 aagcagttcc tgccccggct cagggccaag aacagatggt ccccagatgc ggtccagccc
     5041 tcagcagttt ctagagaacc atcagatgtt tccagggtgc cccaaggacc tgaaatgacc
     5101 ctgtgcctta tttgaactaa ccaatcagtt cgcttctcgc ttctgttcgc gcgcttctgc
     5161 tccccgagct caataaaaga gcccacaacc cctcactcgg ggcgccagtc ctccgattga
     5221 ctgagtcgcc cgggtacccg tgtatccaat aaaccctctt gcagttgcat ccgacttgtg
     5281 gtctcgctgt tccttgggag ggtctcctct gagtgattga ctacccgtca gcgggggtct
     5341 ttcacatgca gcatgtatca aaattaattt ggtttttttt cttaagtatt tacattaaat
     5401 ggccatagtt gcattaatga atcggccaac gcgcggggag aggcggtttg cgtattgggc
     5461 gctcttccgc ttcctcgctc actgactcgc tgcgctcggt cgttcggctg cggcgagcgg
     5521 tatcagctca ctcaaaggcg gtaatacggt tatccacaga atcaggggat aacgcaggaa
     5581 agaacatgtg agcaaaaggc cagcaaaagg ccaggaaccg taaaaaggcc gcgttgctgg
     5641 cgtttttcca taggctccgc ccccctgacg agcatcacaa aaatcgacgc tcaagtcaga
     5701 ggtggcgaaa cccgacagga ctataaagat accaggcgtt tccccctgga agctccctcg
     5761 tgcgctctcc tgttccgacc ctgccgctta ccggatacct gtccgccttt ctcccttcgg
     5821 gaagcgtggc gctttctcat agctcacgct gtaggtatct cagttcggtg taggtcgttc
     5881 gctccaagct gggctgtgtg cacgaacccc ccgttcagcc cgaccgctgc gccttatccg
     5941 gtaactatcg tcttgagtcc aacccggtaa gacacgactt atcgccactg gcagcagcca
     6001 ctggtaacag gattagcaga gcgaggtatg taggcggtgc tacagagttc ttgaagtggt
     6061 ggcctaacta cggctacact agaagaacag tatttggtat ctgcgctctg ctgaagccag
     6121 ttaccttcgg aaaaagagtt ggtagctctt gatccggcaa acaaaccacc gctggtagcg
     6181 gtggtttttt tgtttgcaag cagcagatta cgcgcagaaa aaaaggatct caagaagatc
     6241 ctttgatctt ttctacgggg tctgacgctc agtggaacga aaactcacgt taagggattt
     6301 tggtcatgag attatcaaaa aggatcttca cctagatcct tttgcggccg gccgcaaatc
     6361 aatctaaagt atatatgagt aaacttggtc tgacagttac caatgcttaa tcagtgaggc
     6421 acctatctca gcgatctgtc tatttcgttc atccatagtt gcctgactcc ccgtcgtgta
     6481 gataactacg atacgggagg gcttaccatc tggccccagt gctgcaatga taccgcgaga
     6541 cccacgctca ccggctccag atttatcagc aataaaccag ccagccggaa gggccgagcg
     6601 cagaagtggt cctgcaactt tatccgcctc catccagtct attaattgtt gccgggaagc
     6661 tagagtaagt agttcgccag ttaatagttt gcgcaacgtt gttgccattg ctacaggcat
     6721 cgtggtgtca cgctcgtcgt ttggtatggc ttcattcagc tccggttccc aacgatcaag
     6781 gcgagttaca tgatccccca tgttgtgcaa aaaagcggtt agctccttcg gtcctccgat
     6841 cgttgtcaga agtaagttgg ccgcagtgtt atcactcatg gttatggcag cactgcataa
     6901 ttctcttact gtcatgccat ccgtaagatg cttttctgtg actggtgagt actcaaccaa
     6961 gtcattctga gaatagtgta tgcggcgacc gagttgctct tgcccggcgt caacacggga
     7021 taataccgcg ccacatagca gaactttaaa agtgctcatc attggaaaac gttcttcggg
     7081 gcgaaaactc tcaaggatct taccgctgtt gagatccagt tcgatgtaac ccactcgtgc
     7141 acccaactga tcttcagcat cttttacttt caccagcgtt tctgggtgag caaaaacagg
     7201 aaggcaaaat gccgcaaaaa agggaataag ggcgacacgg aaatgttgaa tactcatact
     7261 cttccttttt caatattatt gaagcattta tcagggttat tgtctcatga gcggatacat
     7321 atttgaatgt atttagaaaa ataaacaaat aggggttccg cgcacatttc cccgaaaagt
     7381 gccacctgac gtctaagaaa ccattattat catgacatta acctataaaa ataggcgtat
     7441 cacgaggccc tttcgtcttc actataggag cttggcccat tgcatacgtt gtatccatat
     7501 cataatatgt acatttatat
//
