LOCUS       sequence-474143-        7747 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Stable expression of HaloTag7-LC3 in mammalian cells to measure 
            autophagic flux.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 7747)
  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 7747)
  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-IP-HaloTag7-LC3
FEATURES             Location/Qualifiers
     source          1..7747
                     /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"
     regulatory      1988..1997
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     CDS             1994..2881
                     /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             2912..3334
                     /codon_start=1
                     /gene="Map1lc3b"
                     /product="rat LC3 protein"
                     /label=LC3
                     /note="Atg8 family member found on autophagosomes"
                     /translation="PSEKTFKQRRSFEQRVEDVRLIREQHPTKIPVIIERYKGEKQLPV
                     LDKTKFLVPDHVNMSELIKIIRRRLQLNANQAFFLLVNGHSMVSVSTPISEVYESERDE
                     DGFLYMVYASQETFGTALAVTYMSALKATATGREPCL"
     misc_feature    3658..4224
                     /label=IRES
                     /note="internal ribosome entry site (IRES) of the 
                     encephalomyocarditis virus (EMCV)"
     misc_feature    3674..4226
                     /label=IRES
                     /note="internal ribosome entry site (IRES) of the 
                     encephalomyocarditis virus (EMCV)"
     primer_bind     complement(3818..3835)
                     /label=IRES reverse
                     /note="IRES internal ribosome entry site, reverse primer. 
                     Also called pCDH-rev"
     primer_bind     4045..4064
                     /label=IRES-F
                     /note="IRES internal ribosome entry site, forward primer"
     CDS             4244..4843
                     /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(4244..4263)
                     /label=Puro-R
                     /note="Puromycin resistance gene, reverse primer. Also 
                     called puro-variant-R"
     primer_bind     4740..4760
                     /label=Puro-F
                     /note="Puromycin resistance gene, forward primer"
     LTR             5073..5542
                     /note="long terminal repeat from Moloney murine leukemia 
                     virus"
     primer_bind     complement(5672..5689)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(5843..6431)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(5923..5942)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(6595..7386,7387..7455))
                     /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     7218..7237
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(7456..7560)
                     /gene="bla"
                     /label=AmpR promoter
     primer_bind     7628..7646
                     /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 aaagcttgcc accatggaaa tcggtactgg ctttccattc gacccccatt atgtggaagt
     2041 cctgggcgag cgcatgcact acgtcgatgt tggtccgcgc gatggcaccc ctgtgctgtt
     2101 cctgcacggt aacccgacct cctcctacgt gtggcgcaac atcatcccgc atgttgcacc
     2161 gacccatcgc tgcattgctc cagacctgat cggtatgggc aaatccgaca aaccagacct
     2221 gggttatttc ttcgacgacc acgtccgctt catggatgcc ttcatcgaag ccctgggtct
     2281 ggaagaggtc gtcctggtca ttcacgactg gggctccgct ctgggtttcc actgggccaa
     2341 gcgcaatcca gagcgcgtca aaggtattgc atttatggag ttcatccgcc ctatcccgac
     2401 ctgggacgaa tggccagaat ttgcccgcga gaccttccag gccttccgca ccaccgacgt
     2461 cggccgcaag ctgatcatcg atcagaacgt ttttatcgag ggtacgctgc cgatgggtgt
     2521 cgtccgcccg ctgactgaag tcgagatgga ccattaccgc gagccgttcc tgaatcctgt
     2581 tgaccgcgag ccactgtggc gcttcccaaa cgagctgcca atcgccggtg agccagcgaa
     2641 catcgtcgcg ctggtcgaag aatacatgga ctggctgcac cagtcccctg tcccgaagct
     2701 gctgttctgg ggcaccccag gcgttctgat cccaccggcc gaagccgctc gcctggccaa
     2761 aagcctgcct aactgcaagg ctgtggacat cggcccaggt ctgaatctgc tgcaagaaga
     2821 caacccggac ctgatcggca gcgagatcgc gcgctggctg tctactctgg agatttccgg
     2881 tggttcaggc tcgggatccg ggaattcggg tccgtccgag aagaccttca aacagcgccg
     2941 gagcttcgaa caaagagtgg aagatgtccg gctcatccgg gagcagcacc ccaccaagat
     3001 cccagtgatt atagagcgat acaagggtga gaagcagctg cccgtcctgg acaagaccaa
     3061 gttccttgta cctgatcacg tgaatatgag cgaactcatc aagataatta gaaggcgcct
     3121 gcagctcaat gctaaccaag ccttcttcct cctggtgaat gggcacagca tggtgagtgt
     3181 gtccacaccc atctctgaag tgtacgagag cgagagagat gaagacggct tcctgtacat
     3241 ggtctatgcc tcccaggaga cgttcgggac agcactggct gttacataca tgtcagctct
     3301 gaaggcaaca gcaacaggaa gagagccatg cttgtgacag acatacagcc acttccaact
     3361 aaagcaagcc tctgctccct gctacctgca tggagcccac tgtgacactc agaccatccc
     3421 cggtcactca ctcactcgtg tctgagaatc tcagtgagag ctgcctctgt cacggacgga
     3481 agccaacaca gccacctctc gacctggctc cccacagcac ccaccctccc tgcatgcagc
     3541 tgtccctgct aacccccaat gttatgttac actgtgtaag tccccactgc tgccgtgtgt
     3601 gggttgtgta cgtcgtcatg tccctggggt ctcgagtaaa gcggccgcta cgtaaattcc
     3661 gccccccccc ctaacgttac tggccgaagc cgcttggaat aaggccggtg tgcgtttgtc
     3721 tatatgttat tttccaccat attgccgtct tttggcaatg tgagggcccg gaaacctggc
     3781 cctgtcttct tgacgagcat tcctaggggt ctttcccctc tcgccaaagg aatgcaaggt
     3841 ctgttgaatg tcgtgaagga agcagttcct ctggaagttt cttgaagaca aacaacgtct
     3901 gtagcgaccc tttgcaggca gcggaacccc ccacctggcg acaggtgcct ctgcggccaa
     3961 aagccacgtg tataagatac acctgcaaag gcggcacaac cccagtgcca cgttgtgagt
     4021 tggatagttg tggaaagagt caaatggctc tcctcaagcg tattcaacaa ggggctgaag
     4081 gatgcccaga aggtacccca ttgtatggga tctgatctgg ggcctcggtg cacatgcttt
     4141 acatgtgttt agtcgaggtt aaaaaaacgt ctaggccccc cgaaccacgg ggacgtggtt
     4201 ttcctttgaa aaacacgatg ataatatggc cacaaccgtc gacatgaccg agtacaagcc
     4261 cacggtgcgc ctcgccaccc gcgacgacgt cccccgggcc gtacgcaccc tcgccgccgc
     4321 gttcgccgac taccccgcca cgcgccacac cgtcgacccg gaccgccaca tcgagcgggt
     4381 caccgagctg caagaactct tcctcacgcg cgtcgggctc gacatcggca aggtgtgggt
     4441 cgcggacgac ggcgccgcgg tggcggtctg gaccacgccg gagagcgtcg aagcgggggc
     4501 ggtgttcgcc gagatcggcc cgcgcatggc cgagttgagc ggttcccggc tggccgcgca
     4561 gcaacagatg gaaggcctcc tggcgccgca ccggcccaag gagcccgcgt ggttcctggc
     4621 caccgtcggc gtctcgcccg accaccaggg caagggtctg ggcagcgccg tcgtgctccc
     4681 cggagtggag gcggccgagc gcgccggggt gcccgccttc ctggagacct ccgcgccccg
     4741 caacctcccc ttctacgagc ggctcggctt caccgtcacc gccgacgtcg aggtgcccga
     4801 aggaccgcgc acctggtgca tgacccgcaa gcccggtgcc tgagtcgacc gcccgcccca
     4861 cgacccgcag cgcccgaccg aaaggagcgc acgaccccat ggctcgacga taaaataaaa
     4921 gattttattt agtctccaga aaaagggggg aatgaaagac cccacctgta ggtttggcaa
     4981 gctagcttaa gtaacgccat tttgcaaggc atggaaaaat acataactga gaatagagaa
     5041 gttcagatca aggtcaggaa cagatggaac agctgaatat gggccaaaca ggatatctgt
     5101 ggtaagcagt tcctgccccg gctcagggcc aagaacagat ggaacagctg aatatgggcc
     5161 aaacaggata tctgtggtaa gcagttcctg ccccggctca gggccaagaa cagatggtcc
     5221 ccagatgcgg tccagccctc agcagtttct agagaaccat cagatgtttc cagggtgccc
     5281 caaggacctg aaatgaccct gtgccttatt tgaactaacc aatcagttcg cttctcgctt
     5341 ctgttcgcgc gcttctgctc cccgagctca ataaaagagc ccacaacccc tcactcggcg
     5401 cgccagtcct ccgattgact gagtcgcccg ggtacccgtg tatccaataa accctcttgc
     5461 agttgcatcc gacttgtggt ctcgctgttc cttgggaggg tctcctctga gtgattgact
     5521 acccgtcagc gggggtcttt cacatgcagc atgtatcaaa attaatttgg ttttttttct
     5581 taagtattta cattaaatgg ccatagttgc attaatgaat cggccaacgc gcggggagag
     5641 gcggtttgcg tattgggcgc tcttccgctt cctcgctcac tgactcgctg cgctcggtcg
     5701 ttcggctgcg gcgagcggta tcagctcact caaaggcggt aatacggtta tccacagaat
     5761 caggggataa cgcaggaaag aacatgtgag caaaaggcca gcaaaaggcc aggaaccgta
     5821 aaaaggccgc gttgctggcg tttttccata ggctccgccc ccctgacgag catcacaaaa
     5881 atcgacgctc aagtcagagg tggcgaaacc cgacaggact ataaagatac caggcgtttc
     5941 cccctggaag ctccctcgtg cgctctcctg ttccgaccct gccgcttacc ggatacctgt
     6001 ccgcctttct cccttcggga agcgtggcgc tttctcatag ctcacgctgt aggtatctca
     6061 gttcggtgta ggtcgttcgc tccaagctgg gctgtgtgca cgaacccccc gttcagcccg
     6121 accgctgcgc cttatccggt aactatcgtc ttgagtccaa cccggtaaga cacgacttat
     6181 cgccactggc agcagccact ggtaacagga ttagcagagc gaggtatgta ggcggtgcta
     6241 cagagttctt gaagtggtgg cctaactacg gctacactag aagaacagta tttggtatct
     6301 gcgctctgct gaagccagtt accttcggaa aaagagttgg tagctcttga tccggcaaac
     6361 aaaccaccgc tggtagcggt ggtttttttg tttgcaagca gcagattacg cgcagaaaaa
     6421 aaggatctca agaagatcct ttgatctttt ctacggggtc tgacgctcag tggaacgaaa
     6481 actcacgtta agggattttg gtcatgagat tatcaaaaag gatcttcacc tagatccttt
     6541 tgcggccggc cgcaaatcaa tctaaagtat atatgagtaa acttggtctg acagttacca
     6601 atgcttaatc agtgaggcac ctatctcagc gatctgtcta tttcgttcat ccatagttgc
     6661 ctgactcccc gtcgtgtaga taactacgat acgggagggc ttaccatctg gccccagtgc
     6721 tgcaatgata ccgcgagacc cacgctcacc ggctccagat ttatcagcaa taaaccagcc
     6781 agccggaagg gccgagcgca gaagtggtcc tgcaacttta tccgcctcca tccagtctat
     6841 taattgttgc cgggaagcta gagtaagtag ttcgccagtt aatagtttgc gcaacgttgt
     6901 tgccattgct acaggcatcg tggtgtcacg ctcgtcgttt ggtatggctt cattcagctc
     6961 cggttcccaa cgatcaaggc gagttacatg atcccccatg ttgtgcaaaa aagcggttag
     7021 ctccttcggt cctccgatcg ttgtcagaag taagttggcc gcagtgttat cactcatggt
     7081 tatggcagca ctgcataatt ctcttactgt catgccatcc gtaagatgct tttctgtgac
     7141 tggtgagtac tcaaccaagt cattctgaga atagtgtatg cggcgaccga gttgctcttg
     7201 cccggcgtca acacgggata ataccgcgcc acatagcaga actttaaaag tgctcatcat
     7261 tggaaaacgt tcttcggggc gaaaactctc aaggatctta ccgctgttga gatccagttc
     7321 gatgtaaccc actcgtgcac ccaactgatc ttcagcatct tttactttca ccagcgtttc
     7381 tgggtgagca aaaacaggaa ggcaaaatgc cgcaaaaaag ggaataaggg cgacacggaa
     7441 atgttgaata ctcatactct tcctttttca atattattga agcatttatc agggttattg
     7501 tctcatgagc ggatacatat ttgaatgtat ttagaaaaat aaacaaatag gggttccgcg
     7561 cacatttccc cgaaaagtgc cacctgacgt ctaagaaacc attattatca tgacattaac
     7621 ctataaaaat aggcgtatca cgaggccctt tcgtcttcac tataggagct tggcccattg
     7681 catacgttgt atccatatca taatatgtac atttatattg gctcatgtcc aacattaccg
     7741 ccatgtt
//
