LOCUS       sequence-364102-        7141 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 7141)
  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 7141)
  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-No-HaloTag7-LC3
FEATURES             Location/Qualifiers
     source          1..7141
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     enhancer        32..411
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
     misc_feature    770..1127
                     /label=MMLV Psi
                     /note="packaging signal of Moloney murine leukemia virus 
                     (MMLV)"
     CDS             1192..1608
                     /label=gag (truncated)
                     /note="truncated Moloney murine leukemia virus (MMLV) gag 
                     gene lacking the start codon"
     primer_bind     1538..1560
                     /label=pLXSN 5'
                     /note="Murine stem cell virus, forward primer. Also called 
                     MSCV"
     primer_bind     1576..1592
                     /label=pBABE 5'
                     /note="Psi packaging signal, forward primer for pBABE 
                     vectors"
     misc_feature    1618..1992
                     /label=pol region
                     /note="Moloney murine leukemia virus (MMLV) pol region 
                     containing the splice acceptor site"
     primer_bind     1930..1946
                     /label=pBMN 5'
                     /note="MMLV sequence, for inserts in pBMN retroviral 
                     vector"
     regulatory      2019..2028
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     CDS             2025..2912
                     /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             2943..3365
                     /codon_start=1
                     /gene="Map1lc3b"
                     /product="rat LC3 protein"
                     /label=LC3
                     /note="Atg8 family member found on autophagosomes"
                     /translation="PSEKTFKQRRSFEQRVEDVRLIREQHPTKIPVIIERYKGEKQLPV
                     LDKTKFLVPDHVNMSELIKIIRRRLQLNANQAFFLLVNGHSMVSVSTPISEVYESERDE
                     DGFLYMVYASQETFGTALAVTYMSALKATATGREPCL"
     misc_feature    3689..4255
                     /label=IRES
                     /note="internal ribosome entry site (IRES) of the 
                     encephalomyocarditis virus (EMCV)"
     misc_feature    3705..4257
                     /label=IRES
                     /note="internal ribosome entry site (IRES) of the 
                     encephalomyocarditis virus (EMCV)"
     primer_bind     complement(3849..3866)
                     /label=IRES reverse
                     /note="IRES internal ribosome entry site, reverse primer. 
                     Also called pCDH-rev"
     primer_bind     4076..4095
                     /label=IRES-F
                     /note="IRES internal ribosome entry site, forward primer"
     LTR             4498..4967
                     /note="long terminal repeat from Moloney murine leukemia 
                     virus"
     primer_bind     complement(5097..5114)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(5268..5856)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(5348..5367)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(6020..6811,6812..6880))
                     /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     6643..6662
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(6881..6985)
                     /gene="bla"
                     /label=AmpR promoter
     primer_bind     7053..7071
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
ORIGIN
        1 attggctcat gtccaacatt accgccatgt tgacattgat tattgactag ttattaatag
       61 taatcaatta cggggtcatt agttcatagc ccatatatgg agttccgcgt tacataactt
      121 acggtaaatg gcccgcctgg ctgaccgccc aacgaccccc gcccattgac gtcaataatg
      181 acgtatgttc ccatagtaac gccaataggg actttccatt gacgtcaatg ggtggagtat
      241 ttacggtaaa ctgcccactt ggcagtacat caagtgtatc atatgccaag tacgccccct
      301 attgacgtca atgacggtaa atggcccgcc tggcattatg cccagtacat gaccttatgg
      361 gactttccta cttggcagta catctacgta ttagtcatcg ctattaccat gctagagaac
      421 catcagatgt ttccagggtg ccccaaggac ctgaaatgac cctgtgcctt atttgaacta
      481 accaatcagt tcgcttctcg cttctgttcg cgcgcttctg ctccccgagc tcaataaaag
      541 agcccacaac ccctcactcg gcgcgccagt cctccgattg actgagtcgc ccgggtaccc
      601 gtgtatccaa taaaccctct tgcagttgca tccgacttgt ggtctcgctg ttccttggga
      661 gggtctcctc tgagtgattg actacccgtc agcgggggtc tttcatttgg gggctcgtcc
      721 gggatcggga gacccctgcc cagggaccac cgacccacca ccgggaggta agctggccag
      781 caacttatct gtgtctgtcc gattgtctag tgtctatgac tgattttatg cgcctgcgtc
      841 ggtactagtt agctaactag ctctgtatct ggcggacccg tggtggaact gacgagttcg
      901 gaacacccgg ccgcaaccct gggagacgtc ccagggactt cgggggccgt ttttgtggcc
      961 cgacctgagt ccaaaaatcc cgatcgtttt ggactctttg gtgcaccccc cttagaggag
     1021 ggatatgtgg ttctggtagg agacgagaac ctaaaacagt tcccgcctcc gtctgaattt
     1081 ttgctttcgg tttgggaccg aagccgcgcc gcgcgtcttg tctgctgcag catcgttctg
     1141 tgttgtctct gtctgactgt gtttctgtat ttgtctgaaa atatgggccc gggccagact
     1201 gttaccactc ccttaagttt gaccttaggt cactggaaag atgtcgagcg gatcgctcac
     1261 aaccagtcgg tagatgtcaa gaagagacgt tgggttacct tctgctctgc agaatggcca
     1321 acctttaacg tcggatggcc gcgagacggc acctttaacc gagacctcat cacccaggtt
     1381 aagatcaagg tcttttcacc tggcccgcat ggacacccag accaggtccc ctacatcgtg
     1441 acctgggaag ccttggcttt tgacccccct ccctgggtca agccctttgt acaccctaag
     1501 cctccgcctc ctcttcctcc atccgccccg tctctccccc ttgaacctcc tcgttcgacc
     1561 ccgcctcgat cctcccttta tccagccctc actccttctc taggcgcccc catatggcca
     1621 tatgagatct tatatggggc acccccgccc cttgtaaact tccctgaccc tgacatgaca
     1681 agagttacta acagcccctc tctccaagct cacttacagg ctctctactt agtccagcac
     1741 gaagtctgga gacctctggc ggcagcctac caagaacaac tggaccgacc ggtggtacct
     1801 cacccttacc gagtcggcga cacagtgtgg gtccgccgac accagactaa gaacctagaa
     1861 cctcgctgga aaggacctta cacagtcctg ctgaccaccc ccaccgccct caaagtagac
     1921 ggcatcgcag cttggataca cgccgcccac gtgaaggctg ccgaccccgg gggtggacca
     1981 tcctctagac tgccggatct gccatcgata taaagcttgc caccatggaa atcggtactg
     2041 gctttccatt cgacccccat tatgtggaag tcctgggcga gcgcatgcac tacgtcgatg
     2101 ttggtccgcg cgatggcacc cctgtgctgt tcctgcacgg taacccgacc tcctcctacg
     2161 tgtggcgcaa catcatcccg catgttgcac cgacccatcg ctgcattgct ccagacctga
     2221 tcggtatggg caaatccgac aaaccagacc tgggttattt cttcgacgac cacgtccgct
     2281 tcatggatgc cttcatcgaa gccctgggtc tggaagaggt cgtcctggtc attcacgact
     2341 ggggctccgc tctgggtttc cactgggcca agcgcaatcc agagcgcgtc aaaggtattg
     2401 catttatgga gttcatccgc cctatcccga cctgggacga atggccagaa tttgcccgcg
     2461 agaccttcca ggccttccgc accaccgacg tcggccgcaa gctgatcatc gatcagaacg
     2521 tttttatcga gggtacgctg ccgatgggtg tcgtccgccc gctgactgaa gtcgagatgg
     2581 accattaccg cgagccgttc ctgaatcctg ttgaccgcga gccactgtgg cgcttcccaa
     2641 acgagctgcc aatcgccggt gagccagcga acatcgtcgc gctggtcgaa gaatacatgg
     2701 actggctgca ccagtcccct gtcccgaagc tgctgttctg gggcacccca ggcgttctga
     2761 tcccaccggc cgaagccgct cgcctggcca aaagcctgcc taactgcaag gctgtggaca
     2821 tcggcccagg tctgaatctg ctgcaagaag acaacccgga cctgatcggc agcgagatcg
     2881 cgcgctggct gtctactctg gagatttccg gtggttcagg ctcgggatcc gggaattcgg
     2941 gtccgtccga gaagaccttc aaacagcgcc ggagcttcga acaaagagtg gaagatgtcc
     3001 ggctcatccg ggagcagcac cccaccaaga tcccagtgat tatagagcga tacaagggtg
     3061 agaagcagct gcccgtcctg gacaagacca agttccttgt acctgatcac gtgaatatga
     3121 gcgaactcat caagataatt agaaggcgcc tgcagctcaa tgctaaccaa gccttcttcc
     3181 tcctggtgaa tgggcacagc atggtgagtg tgtccacacc catctctgaa gtgtacgaga
     3241 gcgagagaga tgaagacggc ttcctgtaca tggtctatgc ctcccaggag acgttcggga
     3301 cagcactggc tgttacatac atgtcagctc tgaaggcaac agcaacagga agagagccat
     3361 gcttgtgaca gacatacagc cacttccaac taaagcaagc ctctgctccc tgctacctgc
     3421 atggagccca ctgtgacact cagaccatcc ccggtcactc actcactcgt gtctgagaat
     3481 ctcagtgaga gctgcctctg tcacggacgg aagccaacac agccacctct cgacctggct
     3541 ccccacagca cccaccctcc ctgcatgcag ctgtccctgc taacccccaa tgttatgtta
     3601 cactgtgtaa gtccccactg ctgccgtgtg tgggttgtgt acgtcgtcat gtccctgggg
     3661 tctcgagtaa agcggccgct acgtaaattc cgcccccccc cctaacgtta ctggccgaag
     3721 ccgcttggaa taaggccggt gtgcgtttgt ctatatgtta ttttccacca tattgccgtc
     3781 ttttggcaat gtgagggccc ggaaacctgg ccctgtcttc ttgacgagca ttcctagggg
     3841 tctttcccct ctcgccaaag gaatgcaagg tctgttgaat gtcgtgaagg aagcagttcc
     3901 tctggaagtt tcttgaagac aaacaacgtc tgtagcgacc ctttgcaggc agcggaaccc
     3961 cccacctggc gacaggtgcc tctgcggcca aaagccacgt gtataagata cacctgcaaa
     4021 ggcggcacaa ccccagtgcc acgttgtgag ttggatagtt gtggaaagag tcaaatggct
     4081 ctcctcaagc gtattcaaca aggggctgaa ggatgcccag aaggtacccc attgtatggg
     4141 atctgatctg gggcctcggt gcacatgctt tacatgtgtt tagtcgaggt taaaaaaacg
     4201 tctaggcccc ccgaaccacg gggacgtggt tttcctttga aaaacacgat gataatatgg
     4261 ccacaaccgt cgaccgcccg ccccacgacc cgcagcgccc gaccgaaagg agcgcacgac
     4321 cccatggctc gacgataaaa taaaagattt tatttagtct ccagaaaaag gggggaatga
     4381 aagaccccac ctgtaggttt ggcaagctag cttaagtaac gccattttgc aaggcatgga
     4441 aaaatacata actgagaata gagaagttca gatcaaggtc aggaacagat ggaacagctg
     4501 aatatgggcc aaacaggata tctgtggtaa gcagttcctg ccccggctca gggccaagaa
     4561 cagatggaac agctgaatat gggccaaaca ggatatctgt ggtaagcagt tcctgccccg
     4621 gctcagggcc aagaacagat ggtccccaga tgcggtccag ccctcagcag tttctagaga
     4681 accatcagat gtttccaggg tgccccaagg acctgaaatg accctgtgcc ttatttgaac
     4741 taaccaatca gttcgcttct cgcttctgtt cgcgcgcttc tgctccccga gctcaataaa
     4801 agagcccaca acccctcact cggggcgcca gtcctccgat tgactgagtc gcccgggtac
     4861 ccgtgtatcc aataaaccct cttgcagttg catccgactt gtggtctcgc tgttccttgg
     4921 gagggtctcc tctgagtgat tgactacccg tcagcggggg tctttcacat gcagcatgta
     4981 tcaaaattaa tttggttttt tttcttaagt atttacatta aatggccata gttgcattaa
     5041 tgaatcggcc aacgcgcggg gagaggcggt ttgcgtattg ggcgctcttc cgcttcctcg
     5101 ctcactgact cgctgcgctc ggtcgttcgg ctgcggcgag cggtatcagc tcactcaaag
     5161 gcggtaatac ggttatccac agaatcaggg gataacgcag gaaagaacat gtgagcaaaa
     5221 ggccagcaaa aggccaggaa ccgtaaaaag gccgcgttgc tggcgttttt ccataggctc
     5281 cgcccccctg acgagcatca caaaaatcga cgctcaagtc agaggtggcg aaacccgaca
     5341 ggactataaa gataccaggc gtttccccct ggaagctccc tcgtgcgctc tcctgttccg
     5401 accctgccgc ttaccggata cctgtccgcc tttctccctt cgggaagcgt ggcgctttct
     5461 catagctcac gctgtaggta tctcagttcg gtgtaggtcg ttcgctccaa gctgggctgt
     5521 gtgcacgaac cccccgttca gcccgaccgc tgcgccttat ccggtaacta tcgtcttgag
     5581 tccaacccgg taagacacga cttatcgcca ctggcagcag ccactggtaa caggattagc
     5641 agagcgaggt atgtaggcgg tgctacagag ttcttgaagt ggtggcctaa ctacggctac
     5701 actagaagaa cagtatttgg tatctgcgct ctgctgaagc cagttacctt cggaaaaaga
     5761 gttggtagct cttgatccgg caaacaaacc accgctggta gcggtggttt ttttgtttgc
     5821 aagcagcaga ttacgcgcag aaaaaaagga tctcaagaag atcctttgat cttttctacg
     5881 gggtctgacg ctcagtggaa cgaaaactca cgttaaggga ttttggtcat gagattatca
     5941 aaaaggatct tcacctagat ccttttgcgg ccggccgcaa atcaatctaa agtatatatg
     6001 agtaaacttg gtctgacagt taccaatgct taatcagtga ggcacctatc tcagcgatct
     6061 gtctatttcg ttcatccata gttgcctgac tccccgtcgt gtagataact acgatacggg
     6121 agggcttacc atctggcccc agtgctgcaa tgataccgcg agacccacgc tcaccggctc
     6181 cagatttatc agcaataaac cagccagccg gaagggccga gcgcagaagt ggtcctgcaa
     6241 ctttatccgc ctccatccag tctattaatt gttgccggga agctagagta agtagttcgc
     6301 cagttaatag tttgcgcaac gttgttgcca ttgctacagg catcgtggtg tcacgctcgt
     6361 cgtttggtat ggcttcattc agctccggtt cccaacgatc aaggcgagtt acatgatccc
     6421 ccatgttgtg caaaaaagcg gttagctcct tcggtcctcc gatcgttgtc agaagtaagt
     6481 tggccgcagt gttatcactc atggttatgg cagcactgca taattctctt actgtcatgc
     6541 catccgtaag atgcttttct gtgactggtg agtactcaac caagtcattc tgagaatagt
     6601 gtatgcggcg accgagttgc tcttgcccgg cgtcaacacg ggataatacc gcgccacata
     6661 gcagaacttt aaaagtgctc atcattggaa aacgttcttc ggggcgaaaa ctctcaagga
     6721 tcttaccgct gttgagatcc agttcgatgt aacccactcg tgcacccaac tgatcttcag
     6781 catcttttac tttcaccagc gtttctgggt gagcaaaaac aggaaggcaa aatgccgcaa
     6841 aaaagggaat aagggcgaca cggaaatgtt gaatactcat actcttcctt tttcaatatt
     6901 attgaagcat ttatcagggt tattgtctca tgagcggata catatttgaa tgtatttaga
     6961 aaaataaaca aataggggtt ccgcgcacat ttccccgaaa agtgccacct gacgtctaag
     7021 aaaccattat tatcatgaca ttaacctata aaaataggcg tatcacgagg ccctttcgtc
     7081 ttcactatag gagcttggcc cattgcatac gttgtatcca tatcataata tgtacattta
     7141 t
//
