LOCUS       sequence-340539-        7968 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Expresses GFP-LC3-RFP in mammalian cells to measure autophagic flux.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 7968)
  AUTHORS   Kaizuka T, Morishita H, Hama Y, Tsukamoto S, Matsui T, Toyota Y, 
            Kodama A, Ishihara T, Mizushima T, Mizushima N
  TITLE     An Autophagic Flux Probe that Releases an Internal Control.
  JOURNAL   Mol Cell. 2016 Nov 17;64(4):835-849. doi: 
            10.1016/j.molcel.2016.09.037. Epub 2016 Nov 3.
  PUBMED    27818143
REFERENCE   2  (bases 1 to 7968)
  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"; doi: 
            "10.1016/j.molcel.2016.09.037"; journalName: "Mol Cell"; date: 
            "2016-11-17- 17"; volume: "64"; issue: "4"; pages: "835-849"
COMMENT     Sequence Label: pMRX-IP-GFP-LC3-RFP
FEATURES             Location/Qualifiers
     source          1..7968
                     /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      1976..1985
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     CDS             join(1982..1984,1985..1987,1988..2698)
                     /codon_start=1
                     /product="enhanced GFP"
                     /label=EGFP
                     /translation="MVSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTL
                     KFICTTGKLPVPWPTLVTTLTYGVQCFSRYPDHMKQHDFFKSAMPEGYVQERTIFFKDD
                     GNYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIK
                     VNFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSALSKDPNEKRDHMVLL
                     EFVTAAGITLGMDELYK"
     CDS             join(1982..1984,1985..1987,1988..2698)
                     /codon_start=1
                     /product="the original enhanced GFP (Yang et al., 1996)"
                     /label=EGFP
                     /note="mammalian codon-optimized"
                     /translation="MVSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTL
                     KFICTTGKLPVPWPTLVTTLTYGVQCFSRYPDHMKQHDFFKSAMPEGYVQERTIFFKDD
                     GNYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIK
                     VNFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSALSKDPNEKRDHMVLL
                     EFVTAAGITLGMDELYK"
     primer_bind     complement(2027..2048)
                     /label=EGFP-N
                     /note="EGFP, reverse primer"
     primer_bind     complement(2288..2307)
                     /label=EXFP-R
                     /note="For distinguishing EGFP variants, reverse primer"
     primer_bind     2635..2656
                     /label=EGFP-C
                     /note="EGFP, forward primer"
     CDS             3122..3799
                     /codon_start=1
                     /product="monomeric derivative of DsRed (Campbell et al., 
                     2002)"
                     /label=mRFP1
                     /translation="MASSEDVIKEFMRFKVRMEGSVNGHEFEIEGEGEGRPYEGTQTAK
                     LKVTKGGPLPFAWDILSPQFQYGSKAYVKHPADIPDYLKLSFPEGFKWERVMNFEDGGV
                     VTVTQDSSLQDGEFIYKVKLRGTNFPSDGPVMQKKTMGWEASTERMYPEDGALKGEIKM
                     RLKLKDGGHYDAEVKTTYMAKKPVQLPGAYKTDIKLDITSHNEDYTIVEQYERAEGRHS
                     TGA"
     CDS             3122..3799
                     /codon_start=1
                     /product="monomeric derivative of DsRed (Campbell et al., 
                     2002)"
                     /label=mRFP1
                     /note="mammalian codon-optimized"
                     /translation="MASSEDVIKEFMRFKVRMEGSVNGHEFEIEGEGEGRPYEGTQTAK
                     LKVTKGGPLPFAWDILSPQFQYGSKAYVKHPADIPDYLKLSFPEGFKWERVMNFEDGGV
                     VTVTQDSSLQDGEFIYKVKLRGTNFPSDGPVMQKKTMGWEASTERMYPEDGALKGEIKM
                     RLKLKDGGHYDAEVKTTYMAKKPVQLPGAYKTDIKLDITSHNEDYTIVEQYERAEGRHS
                     TGA"
     primer_bind     complement(3252..3270)
                     /label=mCherry-R
                     /note="mCherry, reverse primer"
     primer_bind     3520..3539
                     /label=mCherry-F
                     /note="mCherry, forward primer"
     primer_bind     3714..3737
                     /label=DsRed1-C
                     /note="DsRed1, forward primer"
     misc_feature    join(3884..4458,4459..4461,4462..4470)
                     /label=IRES2
                     /note="internal ribosome entry site (IRES) of the 
                     encephalomyocarditis virus (EMCV)"
     primer_bind     complement(4051..4068)
                     /label=IRES reverse
                     /note="IRES internal ribosome entry site, reverse primer. 
                     Also called pCDH-rev"
     primer_bind     4278..4297
                     /label=IRES-F
                     /note="IRES internal ribosome entry site, forward primer"
     CDS             4471..5070
                     /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(4471..4490)
                     /label=Puro-R
                     /note="Puromycin resistance gene, reverse primer. Also 
                     called puro-variant-R"
     primer_bind     4967..4987
                     /label=Puro-F
                     /note="Puromycin resistance gene, forward primer"
     LTR             5294..5763
                     /note="long terminal repeat from Moloney murine leukemia 
                     virus"
     primer_bind     complement(5893..5910)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(6064..6652)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(6144..6163)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(6816..7607,7608..7676))
                     /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     7439..7458
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(7677..7781)
                     /gene="bla"
                     /label=AmpR promoter
     primer_bind     7849..7867
                     /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 gccggatctc ccgccgccac
     1981 catggtgagc aagggcgagg agctgttcac cggggtggtg cccatcctgg tcgagctgga
     2041 cggcgacgta aacggccaca agttcagcgt gtccggcgag ggcgagggcg atgccaccta
     2101 cggcaagctg accctgaagt tcatctgcac caccggcaag ctgcccgtgc cctggcccac
     2161 cctcgtgacc accctgacct acggcgtgca gtgcttcagc cgctaccccg accacatgaa
     2221 gcagcacgac ttcttcaagt ccgccatgcc cgaaggctac gtccaggagc gcaccatctt
     2281 cttcaaggac gacggcaact acaagacccg cgccgaggtg aagttcgagg gcgacaccct
     2341 ggtgaaccgc atcgagctga agggcatcga cttcaaggag gacggcaaca tcctggggca
     2401 caagctggag tacaactaca acagccacaa cgtctatatc atggccgaca agcagaagaa
     2461 cggcatcaag gtgaacttca agatccgcca caacatcgag gacggcagcg tgcagctcgc
     2521 cgaccactac cagcagaaca cccccatcgg cgacggcccc gtgctgctgc ccgacaacca
     2581 ctacctgagc acccagtccg ccctgagcaa agaccccaac gagaagcgcg atcacatggt
     2641 cctgctggag ttcgtgaccg ccgccgggat cactctcggc atggacgagc tgtacaaggg
     2701 tggcggtggc tcgggcggtg gtgggtcggg tggcggcgga tctatcgatg aattgaattc
     2761 catgccgtcc gagaagacct tcaaacagcg ccggagcttc gaacaaagag tggaagatgt
     2821 ccggctcatc cgggagcagc accccaccaa gatcccagtg attatagagc gatacaaggg
     2881 tgagaagcag ctgcccgtcc tggacaagac caagttcctt gtacctgatc acgtgaatat
     2941 gagcgaactc atcaagataa ttagaaggcg cctgcagctc aatgctaacc aagccttctt
     3001 cctcctggtg aatgggcaca gcatggtgag tgtgtccaca cccatctctg aagtgtacga
     3061 gagcgagaga gatgaagacg gcttcctgta catggtctat gcctcccagg agacgttcgg
     3121 gatggcctcc tccgaggacg tcatcaagga gttcatgcgc ttcaaggtgc gcatggaggg
     3181 ctccgtgaac ggccacgagt tcgagatcga gggcgagggc gagggccgcc cctacgaggg
     3241 cacccagacc gccaagctga aggtgaccaa gggcggcccc ctgcccttcg cctgggacat
     3301 cctgtcccct cagttccagt acggctccaa ggcctacgtg aagcaccccg ccgacatccc
     3361 cgactacttg aagctgtcct tccccgaggg cttcaagtgg gagcgcgtga tgaacttcga
     3421 ggacggcggc gtggtgaccg tgacccagga ctcctccctg caggacggcg agttcatcta
     3481 caaggtgaag ctgcgcggca ccaacttccc ctccgacggc cccgtaatgc agaagaagac
     3541 catgggctgg gaggcctcca ccgagcggat gtaccccgag gacggcgccc tgaagggcga
     3601 gatcaagatg aggctgaagc tgaaggacgg cggccactac gacgccgagg tcaagaccac
     3661 ctacatggcc aagaagcccg tgcagctgcc cggcgcctac aagaccgaca tcaagctgga
     3721 catcacctcc cacaacgagg actacaccat cgtggaacag tacgagcgcg ccgagggccg
     3781 ccactccacc ggcgcctaaa gatcccagtg tggtggtacg ggaattcctg caggcctcga
     3841 gggccggcgc gccgcggccg ctacgtaaat tccgcccccc ccccccctct ccctcccccc
     3901 cccctaacgt tactggccga agccgcttgg aataaggccg gtgtgcgttt gtctatatgt
     3961 tattttccac catattgccg tcttttggca atgtgagggc ccggaaacct ggccctgtct
     4021 tcttgacgag cattcctagg ggtctttccc ctctcgccaa aggaatgcaa ggtctgttga
     4081 atgtcgtgaa ggaagcagtt cctctggaag cttcttgaag acaaacaacg tctgtagcga
     4141 ccctttgcag gcagcggaac cccccacctg gcgacaggtg cctctgcggc caaaagccac
     4201 gtgtataaga tacacctgca aaggcggcac aaccccagtg ccacgttgtg agttggatag
     4261 ttgtggaaag agtcaaatgg ctctcctcaa gcgtattcaa caaggggctg aaggatgccc
     4321 agaaggtacc ccattgtatg ggatctgatc tggggcctcg gtgcacatgc tttacatgtg
     4381 tttagtcgag gttaaaaaaa cgtctaggcc ccccgaacca cggggacgtg gttttccttt
     4441 gaaaaacacg atgataatat ggccacaacc atgaccgagt acaagcccac ggtgcgcctc
     4501 gccacccgcg acgacgtccc ccgggccgta cgcaccctcg ccgccgcgtt cgccgactac
     4561 cccgccacgc gccacaccgt cgacccggac cgccacatcg agcgggtcac cgagctgcaa
     4621 gaactcttcc tcacgcgcgt cgggctcgac atcggcaagg tgtgggtcgc ggacgacggc
     4681 gccgcggtgg cggtctggac cacgccggag agcgtcgaag cgggggcggt gttcgccgag
     4741 atcggcccgc gcatggccga gttgagcggt tcccggctgg ccgcgcagca acagatggaa
     4801 ggcctcctgg cgccgcaccg gcccaaggag cccgcgtggt tcctggccac cgtcggcgtc
     4861 tcgcccgacc accagggcaa gggtctgggc agcgccgtcg tgctccccgg agtggaggcg
     4921 gccgagcgcg ccggggtgcc cgccttcctg gagacctccg cgccccgcaa cctccccttc
     4981 tacgagcggc tcggcttcac cgtcaccgcc gacgtcgagg tgcccgaagg accgcgcacc
     5041 tggtgcatga cccgcaagcc cggtgcctga cgcccgcccc acgacccgca gcgcccgacc
     5101 gaaaggagcg cacgacccca tggctcgacg ataaaataaa agattttatt tagtctccag
     5161 aaaaaggggg gaatgaaaga ccccacctgt aggtttggca agctagctta agtaacgcca
     5221 ttttgcaagg catggaaaaa tacataactg agaatagaga agttcagatc aaggtcagga
     5281 acagatggaa cagctgaata tgggccaaac aggatatctg tggtaagcag ttcctgcccc
     5341 ggctcagggc caagaacaga tggaacagct gaatatgggc caaacaggat atctgtggta
     5401 agcagttcct gccccggctc agggccaaga acagatggtc cccagatgcg gtccagccct
     5461 cagcagtttc tagagaacca tcagatgttt ccagggtgcc ccaaggacct gaaatgaccc
     5521 tgtgccttat ttgaactaac caatcagttc gcttctcgct tctgttcgcg cgcttctgct
     5581 ccccgagctc aataaaagag cccacaaccc ctcactcggg gcgccagtcc tccgattgac
     5641 tgagtcgccc gggtacccgt gtatccaata aaccctcttg cagttgcatc cgacttgtgg
     5701 tctcgctgtt ccttgggagg gtctcctctg agtgattgac tacccgtcag cgggggtctt
     5761 tcacatgcag catgtatcaa aattaatttg gttttttttc ttaagtattt acattaaatg
     5821 gccatagttg cattaatgaa tcggccaacg cgcggggaga ggcggtttgc gtattgggcg
     5881 ctcttccgct tcctcgctca ctgactcgct gcgctcggtc gttcggctgc ggcgagcggt
     5941 atcagctcac tcaaaggcgg taatacggtt atccacagaa tcaggggata acgcaggaaa
     6001 gaacatgtga gcaaaaggcc agcaaaaggc caggaaccgt aaaaaggccg cgttgctggc
     6061 gtttttccat aggctccgcc cccctgacga gcatcacaaa aatcgacgct caagtcagag
     6121 gtggcgaaac ccgacaggac tataaagata ccaggcgttt ccccctggaa gctccctcgt
     6181 gcgctctcct gttccgaccc tgccgcttac cggatacctg tccgcctttc tcccttcggg
     6241 aagcgtggcg ctttctcata gctcacgctg taggtatctc agttcggtgt aggtcgttcg
     6301 ctccaagctg ggctgtgtgc acgaaccccc cgttcagccc gaccgctgcg ccttatccgg
     6361 taactatcgt cttgagtcca acccggtaag acacgactta tcgccactgg cagcagccac
     6421 tggtaacagg attagcagag cgaggtatgt aggcggtgct acagagttct tgaagtggtg
     6481 gcctaactac ggctacacta gaagaacagt atttggtatc tgcgctctgc tgaagccagt
     6541 taccttcgga aaaagagttg gtagctcttg atccggcaaa caaaccaccg ctggtagcgg
     6601 tggttttttt gtttgcaagc agcagattac gcgcagaaaa aaaggatctc aagaagatcc
     6661 tttgatcttt tctacggggt ctgacgctca gtggaacgaa aactcacgtt aagggatttt
     6721 ggtcatgaga ttatcaaaaa ggatcttcac ctagatcctt ttgcggccgg ccgcaaatca
     6781 atctaaagta tatatgagta aacttggtct gacagttacc aatgcttaat cagtgaggca
     6841 cctatctcag cgatctgtct atttcgttca tccatagttg cctgactccc cgtcgtgtag
     6901 ataactacga tacgggaggg cttaccatct ggccccagtg ctgcaatgat accgcgagac
     6961 ccacgctcac cggctccaga tttatcagca ataaaccagc cagccggaag ggccgagcgc
     7021 agaagtggtc ctgcaacttt atccgcctcc atccagtcta ttaattgttg ccgggaagct
     7081 agagtaagta gttcgccagt taatagtttg cgcaacgttg ttgccattgc tacaggcatc
     7141 gtggtgtcac gctcgtcgtt tggtatggct tcattcagct ccggttccca acgatcaagg
     7201 cgagttacat gatcccccat gttgtgcaaa aaagcggtta gctccttcgg tcctccgatc
     7261 gttgtcagaa gtaagttggc cgcagtgtta tcactcatgg ttatggcagc actgcataat
     7321 tctcttactg tcatgccatc cgtaagatgc ttttctgtga ctggtgagta ctcaaccaag
     7381 tcattctgag aatagtgtat gcggcgaccg agttgctctt gcccggcgtc aacacgggat
     7441 aataccgcgc cacatagcag aactttaaaa gtgctcatca ttggaaaacg ttcttcgggg
     7501 cgaaaactct caaggatctt accgctgttg agatccagtt cgatgtaacc cactcgtgca
     7561 cccaactgat cttcagcatc ttttactttc accagcgttt ctgggtgagc aaaaacagga
     7621 aggcaaaatg ccgcaaaaaa gggaataagg gcgacacgga aatgttgaat actcatactc
     7681 ttcctttttc aatattattg aagcatttat cagggttatt gtctcatgag cggatacata
     7741 tttgaatgta tttagaaaaa taaacaaata ggggttccgc gcacatttcc ccgaaaagtg
     7801 ccacctgacg tctaagaaac cattattatc atgacattaa cctataaaaa taggcgtatc
     7861 acgaggccct ttcgtcttca ctcgaggagc ttggcccatt gcatacgttg tatccatatc
     7921 ataatatgta catttatatt ggctcatgtc caacattacc gccatgtt
//
