LOCUS       sequence-364090-        7547 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Stable expression of HaloTag7-mGFP in mammalian cells to measure 
            autophagic flux.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 7547)
  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 7547)
  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-HaloTag7-mGFP
FEATURES             Location/Qualifiers
     source          1..7547
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     enhancer        17..396
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
     misc_feature    755..1112
                     /label=MMLV Psi
                     /note="packaging signal of Moloney murine leukemia virus 
                     (MMLV)"
     CDS             1177..1593
                     /label=gag (truncated)
                     /note="truncated Moloney murine leukemia virus (MMLV) gag 
                     gene lacking the start codon"
     primer_bind     1523..1545
                     /label=pLXSN 5'
                     /note="Murine stem cell virus, forward primer. Also called 
                     MSCV"
     primer_bind     1561..1577
                     /label=pBABE 5'
                     /note="Psi packaging signal, forward primer for pBABE 
                     vectors"
     misc_feature    1603..1977
                     /label=pol region
                     /note="Moloney murine leukemia virus (MMLV) pol region 
                     containing the splice acceptor site"
     primer_bind     1915..1931
                     /label=pBMN 5'
                     /note="MMLV sequence, for inserts in pBMN retroviral 
                     vector"
     regulatory      2004..2013
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     CDS             2010..2897
                     /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(2904..2906,2907..3620)
                     /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(2946..2967)
                     /label=EGFP-N
                     /note="EGFP, reverse primer"
     primer_bind     complement(3207..3226)
                     /label=EXFP-R
                     /note="For distinguishing EGFP variants, reverse primer"
     primer_bind     3554..3575
                     /label=EGFP-C
                     /note="EGFP, forward primer"
     misc_feature    3645..4211
                     /label=IRES
                     /note="internal ribosome entry site (IRES) of the 
                     encephalomyocarditis virus (EMCV)"
     misc_feature    3661..4213
                     /label=IRES
                     /note="internal ribosome entry site (IRES) of the 
                     encephalomyocarditis virus (EMCV)"
     primer_bind     complement(3805..3822)
                     /label=IRES reverse
                     /note="IRES internal ribosome entry site, reverse primer. 
                     Also called pCDH-rev"
     primer_bind     4032..4051
                     /label=IRES-F
                     /note="IRES internal ribosome entry site, forward primer"
     LTR             4889..5358
                     /note="long terminal repeat from Moloney murine leukemia 
                     virus"
     primer_bind     complement(5488..5505)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(5659..6247)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(5739..5758)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(6411..7202,7203..7271))
                     /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     7034..7053
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(7272..7376)
                     /gene="bla"
                     /label=AmpR promoter
     primer_bind     7444..7462
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
ORIGIN
        1 acattaccgc catgttgaca ttgattattg actagttatt aatagtaatc aattacgggg
       61 tcattagttc atagcccata tatggagttc cgcgttacat aacttacggt aaatggcccg
      121 cctggctgac cgcccaacga cccccgccca ttgacgtcaa taatgacgta tgttcccata
      181 gtaacgccaa tagggacttt ccattgacgt caatgggtgg agtatttacg gtaaactgcc
      241 cacttggcag tacatcaagt gtatcatatg ccaagtacgc cccctattga cgtcaatgac
      301 ggtaaatggc ccgcctggca ttatgcccag tacatgacct tatgggactt tcctacttgg
      361 cagtacatct acgtattagt catcgctatt accatgctag agaaccatca gatgtttcca
      421 gggtgcccca aggacctgaa atgaccctgt gccttatttg aactaaccaa tcagttcgct
      481 tctcgcttct gttcgcgcgc ttctgctccc cgagctcaat aaaagagccc acaacccctc
      541 actcggcgcg ccagtcctcc gattgactga gtcgcccggg tacccgtgta tccaataaac
      601 cctcttgcag ttgcatccga cttgtggtct cgctgttcct tgggagggtc tcctctgagt
      661 gattgactac ccgtcagcgg gggtctttca tttgggggct cgtccgggat cgggagaccc
      721 ctgcccaggg accaccgacc caccaccggg aggtaagctg gccagcaact tatctgtgtc
      781 tgtccgattg tctagtgtct atgactgatt ttatgcgcct gcgtcggtac tagttagcta
      841 actagctctg tatctggcgg acccgtggtg gaactgacga gttcggaaca cccggccgca
      901 accctgggag acgtcccagg gacttcgggg gccgtttttg tggcccgacc tgagtccaaa
      961 aatcccgatc gttttggact ctttggtgca ccccccttag aggagggata tgtggttctg
     1021 gtaggagacg agaacctaaa acagttcccg cctccgtctg aatttttgct ttcggtttgg
     1081 gaccgaagcc gcgccgcgcg tcttgtctgc tgcagcatcg ttctgtgttg tctctgtctg
     1141 actgtgtttc tgtatttgtc tgaaaatatg ggcccgggcc agactgttac cactccctta
     1201 agtttgacct taggtcactg gaaagatgtc gagcggatcg ctcacaacca gtcggtagat
     1261 gtcaagaaga gacgttgggt taccttctgc tctgcagaat ggccaacctt taacgtcgga
     1321 tggccgcgag acggcacctt taaccgagac ctcatcaccc aggttaagat caaggtcttt
     1381 tcacctggcc cgcatggaca cccagaccag gtcccctaca tcgtgacctg ggaagccttg
     1441 gcttttgacc cccctccctg ggtcaagccc tttgtacacc ctaagcctcc gcctcctctt
     1501 cctccatccg ccccgtctct cccccttgaa cctcctcgtt cgaccccgcc tcgatcctcc
     1561 ctttatccag ccctcactcc ttctctaggc gcccccatat ggccatatga gatcttatat
     1621 ggggcacccc cgccccttgt aaacttccct gaccctgaca tgacaagagt tactaacagc
     1681 ccctctctcc aagctcactt acaggctctc tacttagtcc agcacgaagt ctggagacct
     1741 ctggcggcag cctaccaaga acaactggac cgaccggtgg tacctcaccc ttaccgagtc
     1801 ggcgacacag tgtgggtccg ccgacaccag actaagaacc tagaacctcg ctggaaagga
     1861 ccttacacag tcctgctgac cacccccacc gccctcaaag tagacggcat cgcagcttgg
     1921 atacacgccg cccacgtgaa ggctgccgac cccgggggtg gaccatcctc tagactgccg
     1981 gatctgccat cgatataaag cttgccacca tggaaatcgg tactggcttt ccattcgacc
     2041 cccattatgt ggaagtcctg ggcgagcgca tgcactacgt cgatgttggt ccgcgcgatg
     2101 gcacccctgt gctgttcctg cacggtaacc cgacctcctc ctacgtgtgg cgcaacatca
     2161 tcccgcatgt tgcaccgacc catcgctgca ttgctccaga cctgatcggt atgggcaaat
     2221 ccgacaaacc agacctgggt tatttcttcg acgaccacgt ccgcttcatg gatgccttca
     2281 tcgaagccct gggtctggaa gaggtcgtcc tggtcattca cgactggggc tccgctctgg
     2341 gtttccactg ggccaagcgc aatccagagc gcgtcaaagg tattgcattt atggagttca
     2401 tccgccctat cccgacctgg gacgaatggc cagaatttgc ccgcgagacc ttccaggcct
     2461 tccgcaccac cgacgtcggc cgcaagctga tcatcgatca gaacgttttt atcgagggta
     2521 cgctgccgat gggtgtcgtc cgcccgctga ctgaagtcga gatggaccat taccgcgagc
     2581 cgttcctgaa tcctgttgac cgcgagccac tgtggcgctt cccaaacgag ctgccaatcg
     2641 ccggtgagcc agcgaacatc gtcgcgctgg tcgaagaata catggactgg ctgcaccagt
     2701 cccctgtccc gaagctgctg ttctggggca ccccaggcgt tctgatccca ccggccgaag
     2761 ccgctcgcct ggccaaaagc ctgcctaact gcaaggctgt ggacatcggc ccaggtctga
     2821 atctgctgca agaagacaac ccggacctga tcggcagcga gatcgcgcgc tggctgtcta
     2881 ctctggagat ttccggtctc gaggtgagca agggcgagga gctgttcacc ggggtggtgc
     2941 ccatcctggt cgagctggac ggcgacgtaa acggccacaa gttcagcgtg tccggcgagg
     3001 gcgagggcga tgccacctac ggcaagctga ccctgaagtt catctgcacc accggcaagc
     3061 tgcccgtgcc ctggcccacc ctcgtgacca ccctgaccta cggcgtgcag tgcttcagcc
     3121 gctaccccga ccacatgaag cagcacgact tcttcaagtc cgccatgccc gaaggctacg
     3181 tccaggagcg caccatcttc ttcaaggacg acggcaacta caagacccgc gccgaggtga
     3241 agttcgaggg cgacaccctg gtgaaccgca tcgagctgaa gggcatcgac ttcaaggagg
     3301 acggcaacat cctggggcac aagctggagt acaactacaa cagccacaac gtctatatca
     3361 tggccgacaa gcagaagaac ggcatcaagg tgaacttcaa gatccgccac aacatcgagg
     3421 acggcagcgt gcagctcgcc gaccactacc agcagaacac ccccatcggc gacggccccg
     3481 tgctgctgcc cgacaaccac tacctgagca cccagtccaa actgagcaaa gaccccaacg
     3541 agaagcgcga tcacatggtc ctgctggagt tcgtgaccgc cgccgggatc actctcggca
     3601 tggacgagct gtacaagtaa ctcgagagcg gccgctacgt aaattccgcc ccccccccta
     3661 acgttactgg ccgaagccgc ttggaataag gccggtgtgc gtttgtctat atgttatttt
     3721 ccaccatatt gccgtctttt ggcaatgtga gggcccggaa acctggccct gtcttcttga
     3781 cgagcattcc taggggtctt tcccctctcg ccaaaggaat gcaaggtctg ttgaatgtcg
     3841 tgaaggaagc agttcctctg gaagtttctt gaagacaaac aacgtctgta gcgacccttt
     3901 gcaggcagcg gaacccccca cctggcgaca ggtgcctctg cggccaaaag ccacgtgtat
     3961 aagatacacc tgcaaaggcg gcacaacccc agtgccacgt tgtgagttgg atagttgtgg
     4021 aaagagtcaa atggctctcc tcaagcgtat tcaacaaggg gctgaaggat gcccagaagg
     4081 taccccattg tatgggatct gatctggggc ctcggtgcac atgctttaca tgtgtttagt
     4141 cgaggttaaa aaaacgtcta ggccccccga accacgggga cgtggttttc ctttgaaaaa
     4201 cacgatgata atatggccac aaccgtcgac atggtcatga aaacatttaa catttctcaa
     4261 caagatctag aattagtaga agtagcgaca gagaagatta caatgcttta tgaggataat
     4321 aaacatcatg tgggagcggc aattcgtacg aaaacaggag aaatcatttc ggcagtacat
     4381 attgaagcgt atataggacg agtaactgtt tgtgcagaag ccattgcgat tggtagtgca
     4441 gtttcgaatg gacaaaagga ttttgacacg attgtagctg ttagacaccc ttattctgac
     4501 gaagtagata gaagtattcg agtggtaagt ccttgtggta tgtgtaggga gttgatttca
     4561 gactatgcac cagattgttt tgtgttaata gaaatgaatg gcaagttagt caaaactacg
     4621 attgaagaac tcattccact caaatatacc cgaaattaag tcgaccgccc gccccacgac
     4681 ccgcagcgcc cgaccgaaag gagcgcacga ccccatggct cgacgataaa ataaaagatt
     4741 ttatttagtc tccagaaaaa ggggggaatg aaagacccca cctgtaggtt tggcaagcta
     4801 gcttaagtaa cgccattttg caaggcatgg aaaaatacat aactgagaat agagaagttc
     4861 agatcaaggt caggaacaga tggaacagct gaatatgggc caaacaggat atctgtggta
     4921 agcagttcct gccccggctc agggccaaga acagatggaa cagctgaata tgggccaaac
     4981 aggatatctg tggtaagcag ttcctgcccc ggctcagggc caagaacaga tggtccccag
     5041 atgcggtcca gccctcagca gtttctagag aaccatcaga tgtttccagg gtgccccaag
     5101 gacctgaaat gaccctgtgc cttatttgaa ctaaccaatc agttcgcttc tcgcttctgt
     5161 tcgcgcgctt ctgctccccg agctcaataa aagagcccac aacccctcac tcggggcgcc
     5221 agtcctccga ttgactgagt cgcccgggta cccgtgtatc caataaaccc tcttgcagtt
     5281 gcatccgact tgtggtctcg ctgttccttg ggagggtctc ctctgagtga ttgactaccc
     5341 gtcagcgggg gtctttcaca tgcagcatgt atcaaaatta atttggtttt ttttcttaag
     5401 tatttacatt aaatggccat agttgcatta atgaatcggc caacgcgcgg ggagaggcgg
     5461 tttgcgtatt gggcgctctt ccgcttcctc gctcactgac tcgctgcgct cggtcgttcg
     5521 gctgcggcga gcggtatcag ctcactcaaa ggcggtaata cggttatcca cagaatcagg
     5581 ggataacgca ggaaagaaca tgtgagcaaa aggccagcaa aaggccagga accgtaaaaa
     5641 ggccgcgttg ctggcgtttt tccataggct ccgcccccct gacgagcatc acaaaaatcg
     5701 acgctcaagt cagaggtggc gaaacccgac aggactataa agataccagg cgtttccccc
     5761 tggaagctcc ctcgtgcgct ctcctgttcc gaccctgccg cttaccggat acctgtccgc
     5821 ctttctccct tcgggaagcg tggcgctttc tcatagctca cgctgtaggt atctcagttc
     5881 ggtgtaggtc gttcgctcca agctgggctg tgtgcacgaa ccccccgttc agcccgaccg
     5941 ctgcgcctta tccggtaact atcgtcttga gtccaacccg gtaagacacg acttatcgcc
     6001 actggcagca gccactggta acaggattag cagagcgagg tatgtaggcg gtgctacaga
     6061 gttcttgaag tggtggccta actacggcta cactagaaga acagtatttg gtatctgcgc
     6121 tctgctgaag ccagttacct tcggaaaaag agttggtagc tcttgatccg gcaaacaaac
     6181 caccgctggt agcggtggtt tttttgtttg caagcagcag attacgcgca gaaaaaaagg
     6241 atctcaagaa gatcctttga tcttttctac ggggtctgac gctcagtgga acgaaaactc
     6301 acgttaaggg attttggtca tgagattatc aaaaaggatc ttcacctaga tccttttgcg
     6361 gccggccgca aatcaatcta aagtatatat gagtaaactt ggtctgacag ttaccaatgc
     6421 ttaatcagtg aggcacctat ctcagcgatc tgtctatttc gttcatccat agttgcctga
     6481 ctccccgtcg tgtagataac tacgatacgg gagggcttac catctggccc cagtgctgca
     6541 atgataccgc gagacccacg ctcaccggct ccagatttat cagcaataaa ccagccagcc
     6601 ggaagggccg agcgcagaag tggtcctgca actttatccg cctccatcca gtctattaat
     6661 tgttgccggg aagctagagt aagtagttcg ccagttaata gtttgcgcaa cgttgttgcc
     6721 attgctacag gcatcgtggt gtcacgctcg tcgtttggta tggcttcatt cagctccggt
     6781 tcccaacgat caaggcgagt tacatgatcc cccatgttgt gcaaaaaagc ggttagctcc
     6841 ttcggtcctc cgatcgttgt cagaagtaag ttggccgcag tgttatcact catggttatg
     6901 gcagcactgc ataattctct tactgtcatg ccatccgtaa gatgcttttc tgtgactggt
     6961 gagtactcaa ccaagtcatt ctgagaatag tgtatgcggc gaccgagttg ctcttgcccg
     7021 gcgtcaacac gggataatac cgcgccacat agcagaactt taaaagtgct catcattgga
     7081 aaacgttctt cggggcgaaa actctcaagg atcttaccgc tgttgagatc cagttcgatg
     7141 taacccactc gtgcacccaa ctgatcttca gcatctttta ctttcaccag cgtttctggg
     7201 tgagcaaaaa caggaaggca aaatgccgca aaaaagggaa taagggcgac acggaaatgt
     7261 tgaatactca tactcttcct ttttcaatat tattgaagca tttatcaggg ttattgtctc
     7321 atgagcggat acatatttga atgtatttag aaaaataaac aaataggggt tccgcgcaca
     7381 tttccccgaa aagtgccacc tgacgtctaa gaaaccatta ttatcatgac attaacctat
     7441 aaaaataggc gtatcacgag gccctttcgt cttcactata ggagcttggc ccattgcata
     7501 cgttgtatcc atatcataat atgtacattt atattggctc atgtcca
//
