LOCUS       sequence-364036-        8188 bp    DNA     circular SYN 14-APR-2025
DEFINITION  Stable expression of HaloTag7-mGFP-LC3-mRFP in mammalian cells to 
            measure autophagic flux.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 8188)
  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 8188)
  AUTHORS   .
  TITLE     Direct Submission
  JOURNAL   Exported Apr 14, 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-mRFP
FEATURES             Location/Qualifiers
     source          1..8188
                     /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(2919..2921,2922..3632)
                     /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(2961..2982)
                     /label=EGFP-N
                     /note="EGFP, reverse primer"
     primer_bind     complement(3222..3241)
                     /label=EXFP-R
                     /note="For distinguishing EGFP variants, reverse primer"
     primer_bind     3569..3590
                     /label=EGFP-C
                     /note="EGFP, forward primer"
     CDS             4020..4700
                     /codon_start=1
                     /product="monomeric derivative of DsRed (Campbell et al., 
                     2002)"
                     /label=mRFP1
                     /translation="MVASSEDVIKEFMRFKVRMEGSVNGHEFEIEGEGEGRPYEGTQTA
                     KLKVTKGGPLPFAWDILSPQFQYGSKAYVKHPADIPDYLKLSFPEGFKWERVMNFEDGG
                     VVTVTQDSSLQDGEFIYKVKLRGTNFPSDGPVMQKKTMGWEASTERMYPEDGALKGEIK
                     MRLKLKDGGHYDAEVKTTYMAKKPVQLPGAYKTDIKLDITSHNEDYTIVEQYERAEGRH
                     STGA"
     CDS             4020..4700
                     /codon_start=1
                     /product="monomeric derivative of DsRed (Campbell et al., 
                     2002)"
                     /label=mRFP1
                     /note="mammalian codon-optimized"
                     /translation="MVASSEDVIKEFMRFKVRMEGSVNGHEFEIEGEGEGRPYEGTQTA
                     KLKVTKGGPLPFAWDILSPQFQYGSKAYVKHPADIPDYLKLSFPEGFKWERVMNFEDGG
                     VVTVTQDSSLQDGEFIYKVKLRGTNFPSDGPVMQKKTMGWEASTERMYPEDGALKGEIK
                     MRLKLKDGGHYDAEVKTTYMAKKPVQLPGAYKTDIKLDITSHNEDYTIVEQYERAEGRH
                     STGA"
     primer_bind     complement(4153..4171)
                     /label=mCherry-R
                     /note="mCherry, reverse primer"
     primer_bind     4421..4440
                     /label=mCherry-F
                     /note="mCherry, forward primer"
     primer_bind     4615..4638
                     /label=DsRed1-C
                     /note="DsRed1, forward primer"
     misc_feature    4721..5287
                     /label=IRES
                     /note="internal ribosome entry site (IRES) of the 
                     encephalomyocarditis virus (EMCV)"
     misc_feature    4737..5289
                     /label=IRES
                     /note="internal ribosome entry site (IRES) of the 
                     encephalomyocarditis virus (EMCV)"
     primer_bind     complement(4881..4898)
                     /label=IRES reverse
                     /note="IRES internal ribosome entry site, reverse primer. 
                     Also called pCDH-rev"
     primer_bind     5108..5127
                     /label=IRES-F
                     /note="IRES internal ribosome entry site, forward primer"
     LTR             5530..5999
                     /note="long terminal repeat from Moloney murine leukemia 
                     virus"
     primer_bind     complement(6129..6146)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(6300..6888)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(6380..6399)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(7052..7843,7844..7912))
                     /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     7675..7694
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(7913..8017)
                     /gene="bla"
                     /label=AmpR promoter
     primer_bind     8085..8103
                     /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 ttccggtggt tcaggctcgg gatccggggt gagcaagggc gaggagctgt
     2941 tcaccggggt ggtgcccatc ctggtcgagc tggacggcga cgtaaacggc cacaagttca
     3001 gcgtgtccgg cgagggcgag ggcgatgcca cctacggcaa gctgaccctg aagttcatct
     3061 gcaccaccgg caagctgccc gtgccctggc ccaccctcgt gaccaccctg acctacggcg
     3121 tgcagtgctt cagccgctac cccgaccaca tgaagcagca cgacttcttc aagtccgcca
     3181 tgcccgaagg ctacgtccag gagcgcacca tcttcttcaa ggacgacggc aactacaaga
     3241 cccgcgccga ggtgaagttc gagggcgaca ccctggtgaa ccgcatcgag ctgaagggca
     3301 tcgacttcaa ggaggacggc aacatcctgg ggcacaagct ggagtacaac tacaacagcc
     3361 acaacgtcta tatcatggcc gacaagcaga agaacggcat caaggtgaac ttcaagatcc
     3421 gccacaacat cgaggacggc agcgtgcagc tcgccgacca ctaccagcag aacaccccca
     3481 tcggcgacgg ccccgtgctg ctgcccgaca accactacct gagcacccag tccaaactga
     3541 gcaaagaccc caacgagaag cgcgatcaca tggtcctgct ggagttcgtg accgccgccg
     3601 ggatcactct cggcatggac gagctgtaca agggttcagg ctcgggatct gggaattcgg
     3661 gtccgtccga gaagaccttc aaacagcgcc ggagcttcga acaaagagtg gaagatgtcc
     3721 ggctcatccg ggagcagcac cccaccaaga tcccagtgat tatagagcga tacaagggtg
     3781 agaagcagct gcccgtcctg gacaagacca agttccttgt acctgatcac gtgaatatga
     3841 gcgaactcat caagataatt agaaggcgcc tgcagctcaa tgctaaccaa gccttcttcc
     3901 tcctggtgaa tgggcacagc atggtgagtg tgtccacacc catctctgaa gtgtacgaga
     3961 gcgagagaga tgaagacggc ttcctgtaca tggtctatgc ctcccaggag acgttcggga
     4021 tggtggcctc ctccgaggac gtcatcaagg agttcatgcg cttcaaggtg cgcatggagg
     4081 gctccgtgaa cggccacgag ttcgagatcg agggcgaggg cgagggccgc ccctacgagg
     4141 gcacccagac cgccaagctg aaggtgacca agggcggccc cctgcccttc gcctgggaca
     4201 tcctgtcccc tcagttccag tacggctcca aggcctacgt gaagcacccc gccgacatcc
     4261 ccgactactt gaagctgtcc ttccccgagg gcttcaagtg ggagcgcgtg atgaacttcg
     4321 aggacggcgg cgtggtgacc gtgacccagg actcctccct gcaggacggc gagttcatct
     4381 acaaggtgaa gctgcgcggc accaacttcc cctccgacgg ccccgtaatg cagaagaaga
     4441 ccatgggctg ggaggcctcc accgagcgga tgtaccccga ggacggcgcc ctgaagggcg
     4501 agatcaagat gaggctgaag ctgaaggacg gcggccacta cgacgccgag gtcaagacca
     4561 cctacatggc caagaagccc gtgcagctgc ccggcgccta caagaccgac atcaagctgg
     4621 acatcacctc ccacaacgag gactacacca tcgtggaaca gtacgagcgc gccgagggcc
     4681 gccactccac cggcgcctaa tgagcggccg ctacgtaaat tccgcccccc cccctaacgt
     4741 tactggccga agccgcttgg aataaggccg gtgtgcgttt gtctatatgt tattttccac
     4801 catattgccg tcttttggca atgtgagggc ccggaaacct ggccctgtct tcttgacgag
     4861 cattcctagg ggtctttccc ctctcgccaa aggaatgcaa ggtctgttga atgtcgtgaa
     4921 ggaagcagtt cctctggaag tttcttgaag acaaacaacg tctgtagcga ccctttgcag
     4981 gcagcggaac cccccacctg gcgacaggtg cctctgcggc caaaagccac gtgtataaga
     5041 tacacctgca aaggcggcac aaccccagtg ccacgttgtg agttggatag ttgtggaaag
     5101 agtcaaatgg ctctcctcaa gcgtattcaa caaggggctg aaggatgccc agaaggtacc
     5161 ccattgtatg ggatctgatc tggggcctcg gtgcacatgc tttacatgtg tttagtcgag
     5221 gttaaaaaaa cgtctaggcc ccccgaacca cggggacgtg gttttccttt gaaaaacacg
     5281 atgataatat ggccacaacc gtcgaccgcc cgccccacga cccgcagcgc ccgaccgaaa
     5341 ggagcgcacg accccatggc tcgacgataa aataaaagat tttatttagt ctccagaaaa
     5401 aggggggaat gaaagacccc acctgtaggt ttggcaagct agcttaagta acgccatttt
     5461 gcaaggcatg gaaaaataca taactgagaa tagagaagtt cagatcaagg tcaggaacag
     5521 atggaacagc tgaatatggg ccaaacagga tatctgtggt aagcagttcc tgccccggct
     5581 cagggccaag aacagatgga acagctgaat atgggccaaa caggatatct gtggtaagca
     5641 gttcctgccc cggctcaggg ccaagaacag atggtcccca gatgcggtcc agccctcagc
     5701 agtttctaga gaaccatcag atgtttccag ggtgccccaa ggacctgaaa tgaccctgtg
     5761 ccttatttga actaaccaat cagttcgctt ctcgcttctg ttcgcgcgct tctgctcccc
     5821 gagctcaata aaagagccca caacccctca ctcggggcgc cagtcctccg attgactgag
     5881 tcgcccgggt acccgtgtat ccaataaacc ctcttgcagt tgcatccgac ttgtggtctc
     5941 gctgttcctt gggagggtct cctctgagtg attgactacc cgtcagcggg ggtctttcac
     6001 atgcagcatg tatcaaaatt aatttggttt tttttcttaa gtatttacat taaatggcca
     6061 tagttgcatt aatgaatcgg ccaacgcgcg gggagaggcg gtttgcgtat tgggcgctct
     6121 tccgcttcct cgctcactga ctcgctgcgc tcggtcgttc ggctgcggcg agcggtatca
     6181 gctcactcaa aggcggtaat acggttatcc acagaatcag gggataacgc aggaaagaac
     6241 atgtgagcaa aaggccagca aaaggccagg aaccgtaaaa aggccgcgtt gctggcgttt
     6301 ttccataggc tccgcccccc tgacgagcat cacaaaaatc gacgctcaag tcagaggtgg
     6361 cgaaacccga caggactata aagataccag gcgtttcccc ctggaagctc cctcgtgcgc
     6421 tctcctgttc cgaccctgcc gcttaccgga tacctgtccg cctttctccc ttcgggaagc
     6481 gtggcgcttt ctcatagctc acgctgtagg tatctcagtt cggtgtaggt cgttcgctcc
     6541 aagctgggct gtgtgcacga accccccgtt cagcccgacc gctgcgcctt atccggtaac
     6601 tatcgtcttg agtccaaccc ggtaagacac gacttatcgc cactggcagc agccactggt
     6661 aacaggatta gcagagcgag gtatgtaggc ggtgctacag agttcttgaa gtggtggcct
     6721 aactacggct acactagaag aacagtattt ggtatctgcg ctctgctgaa gccagttacc
     6781 ttcggaaaaa gagttggtag ctcttgatcc ggcaaacaaa ccaccgctgg tagcggtggt
     6841 ttttttgttt gcaagcagca gattacgcgc agaaaaaaag gatctcaaga agatcctttg
     6901 atcttttcta cggggtctga cgctcagtgg aacgaaaact cacgttaagg gattttggtc
     6961 atgagattat caaaaaggat cttcacctag atccttttgc ggccggccgc aaatcaatct
     7021 aaagtatata tgagtaaact tggtctgaca gttaccaatg cttaatcagt gaggcaccta
     7081 tctcagcgat ctgtctattt cgttcatcca tagttgcctg actccccgtc gtgtagataa
     7141 ctacgatacg ggagggctta ccatctggcc ccagtgctgc aatgataccg cgagacccac
     7201 gctcaccggc tccagattta tcagcaataa accagccagc cggaagggcc gagcgcagaa
     7261 gtggtcctgc aactttatcc gcctccatcc agtctattaa ttgttgccgg gaagctagag
     7321 taagtagttc gccagttaat agtttgcgca acgttgttgc cattgctaca ggcatcgtgg
     7381 tgtcacgctc gtcgtttggt atggcttcat tcagctccgg ttcccaacga tcaaggcgag
     7441 ttacatgatc ccccatgttg tgcaaaaaag cggttagctc cttcggtcct ccgatcgttg
     7501 tcagaagtaa gttggccgca gtgttatcac tcatggttat ggcagcactg cataattctc
     7561 ttactgtcat gccatccgta agatgctttt ctgtgactgg tgagtactca accaagtcat
     7621 tctgagaata gtgtatgcgg cgaccgagtt gctcttgccc ggcgtcaaca cgggataata
     7681 ccgcgccaca tagcagaact ttaaaagtgc tcatcattgg aaaacgttct tcggggcgaa
     7741 aactctcaag gatcttaccg ctgttgagat ccagttcgat gtaacccact cgtgcaccca
     7801 actgatcttc agcatctttt actttcacca gcgtttctgg gtgagcaaaa acaggaaggc
     7861 aaaatgccgc aaaaaaggga ataagggcga cacggaaatg ttgaatactc atactcttcc
     7921 tttttcaata ttattgaagc atttatcagg gttattgtct catgagcgga tacatatttg
     7981 aatgtattta gaaaaataaa caaatagggg ttccgcgcac atttccccga aaagtgccac
     8041 ctgacgtcta agaaaccatt attatcatga cattaaccta taaaaatagg cgtatcacga
     8101 ggccctttcg tcttcactat aggagcttgg cccattgcat acgttgtatc catatcataa
     8161 tatgtacatt tatattggct catgtcca
//
