LOCUS       sequence-364105-        8243 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Stable expression of GFP-LC3-mRuby3 in mammalian cells to measure 
            autophagic flux.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 8243)
  AUTHORS   Okawa F, Hama Y, Zhang S, Morishita H, Yamamoto H, Levine TP, 
            Mizushima N
  TITLE     Evolution and insights into the structure and function of the DedA 
            superfamily containing TMEM41B and VMP1.
  JOURNAL   J Cell Sci. 2021 Apr 15;134(8). pii: 237813. doi: 
            10.1242/jcs.255877. Epub 2021 Apr 28.
  PUBMED    33771928
REFERENCE   2  (bases 1 to 8243)
  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: "J Cell 
            Sci."; date: "2021-04-15"; volume: "134(8). pii: 237813. doi"; 
            pages: " 10.1242/jcs.255877. Epub 2021 Apr 28"
COMMENT     Sequence Label: pMRX-IP-GFP-LC3-mRuby3
FEATURES             Location/Qualifiers
     source          1..8243
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     enhancer        25..404
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
     misc_feature    763..1120
                     /label=MMLV Psi
                     /note="packaging signal of Moloney murine leukemia virus 
                     (MMLV)"
     CDS             1185..1601
                     /label=gag (truncated)
                     /note="truncated Moloney murine leukemia virus (MMLV) gag 
                     gene lacking the start codon"
     primer_bind     1531..1553
                     /label=pLXSN 5'
                     /note="Murine stem cell virus, forward primer. Also called 
                     MSCV"
     primer_bind     1569..1585
                     /label=pBABE 5'
                     /note="Psi packaging signal, forward primer for pBABE 
                     vectors"
     misc_feature    1611..1985
                     /label=pol region
                     /note="Moloney murine leukemia virus (MMLV) pol region 
                     containing the splice acceptor site"
     primer_bind     1923..1939
                     /label=pBMN 5'
                     /note="MMLV sequence, for inserts in pBMN retroviral 
                     vector"
     regulatory      2012..2021
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     CDS             join(2018..2020,2021..2023,2024..2734)
                     /codon_start=1
                     /product="enhanced GFP"
                     /label=EGFP
                     /translation="MVSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTL
                     KFICTTGKLPVPWPTLVTTLTYGVQCFSRYPDHMKQHDFFKSAMPEGYVQERTIFFKDD
                     GNYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIK
                     VNFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSALSKDPNEKRDHMVLL
                     EFVTAAGITLGMDELYK"
     CDS             join(2018..2020,2021..2023,2024..2734)
                     /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(2063..2084)
                     /label=EGFP-N
                     /note="EGFP, reverse primer"
     primer_bind     complement(2324..2343)
                     /label=EXFP-R
                     /note="For distinguishing EGFP variants, reverse primer"
     primer_bind     2671..2692
                     /label=EGFP-C
                     /note="EGFP, forward primer"
     misc_feature    4178..4744
                     /label=IRES
                     /note="internal ribosome entry site (IRES) of the 
                     encephalomyocarditis virus (EMCV)"
     misc_feature    4194..4746
                     /label=IRES
                     /note="internal ribosome entry site (IRES) of the 
                     encephalomyocarditis virus (EMCV)"
     primer_bind     complement(4338..4355)
                     /label=IRES reverse
                     /note="IRES internal ribosome entry site, reverse primer. 
                     Also called pCDH-rev"
     primer_bind     4565..4584
                     /label=IRES-F
                     /note="IRES internal ribosome entry site, forward primer"
     CDS             4764..5363
                     /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(4764..4783)
                     /label=Puro-R
                     /note="Puromycin resistance gene, reverse primer. Also 
                     called puro-variant-R"
     primer_bind     5260..5280
                     /label=Puro-F
                     /note="Puromycin resistance gene, forward primer"
     LTR             5593..6062
                     /note="long terminal repeat from Moloney murine leukemia 
                     virus"
     primer_bind     complement(6192..6209)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(6363..6951)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(6443..6462)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(7115..7906,7907..7975))
                     /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     7738..7757
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(7976..8080)
                     /gene="bla"
                     /label=AmpR promoter
     primer_bind     8148..8166
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
ORIGIN
        1 catgtccaac attaccgcca tgttgacatt gattattgac tagttattaa tagtaatcaa
       61 ttacggggtc attagttcat agcccatata tggagttccg cgttacataa cttacggtaa
      121 atggcccgcc tggctgaccg cccaacgacc cccgcccatt gacgtcaata atgacgtatg
      181 ttcccatagt aacgccaata gggactttcc attgacgtca atgggtggag tatttacggt
      241 aaactgccca cttggcagta catcaagtgt atcatatgcc aagtacgccc cctattgacg
      301 tcaatgacgg taaatggccc gcctggcatt atgcccagta catgacctta tgggactttc
      361 ctacttggca gtacatctac gtattagtca tcgctattac catgctagag aaccatcaga
      421 tgtttccagg gtgccccaag gacctgaaat gaccctgtgc cttatttgaa ctaaccaatc
      481 agttcgcttc tcgcttctgt tcgcgcgctt ctgctccccg agctcaataa aagagcccac
      541 aacccctcac tcggcgcgcc agtcctccga ttgactgagt cgcccgggta cccgtgtatc
      601 caataaaccc tcttgcagtt gcatccgact tgtggtctcg ctgttccttg ggagggtctc
      661 ctctgagtga ttgactaccc gtcagcgggg gtctttcatt tgggggctcg tccgggatcg
      721 ggagacccct gcccagggac caccgaccca ccaccgggag gtaagctggc cagcaactta
      781 tctgtgtctg tccgattgtc tagtgtctat gactgatttt atgcgcctgc gtcggtacta
      841 gttagctaac tagctctgta tctggcggac ccgtggtgga actgacgagt tcggaacacc
      901 cggccgcaac cctgggagac gtcccaggga cttcgggggc cgtttttgtg gcccgacctg
      961 agtccaaaaa tcccgatcgt tttggactct ttggtgcacc ccccttagag gagggatatg
     1021 tggttctggt aggagacgag aacctaaaac agttcccgcc tccgtctgaa tttttgcttt
     1081 cggtttggga ccgaagccgc gccgcgcgtc ttgtctgctg cagcatcgtt ctgtgttgtc
     1141 tctgtctgac tgtgtttctg tatttgtctg aaaatatggg cccgggccag actgttacca
     1201 ctcccttaag tttgacctta ggtcactgga aagatgtcga gcggatcgct cacaaccagt
     1261 cggtagatgt caagaagaga cgttgggtta ccttctgctc tgcagaatgg ccaaccttta
     1321 acgtcggatg gccgcgagac ggcaccttta accgagacct catcacccag gttaagatca
     1381 aggtcttttc acctggcccg catggacacc cagaccaggt cccctacatc gtgacctggg
     1441 aagccttggc ttttgacccc cctccctggg tcaagccctt tgtacaccct aagcctccgc
     1501 ctcctcttcc tccatccgcc ccgtctctcc cccttgaacc tcctcgttcg accccgcctc
     1561 gatcctccct ttatccagcc ctcactcctt ctctaggcgc ccccatatgg ccatatgaga
     1621 tcttatatgg ggcacccccg ccccttgtaa acttccctga ccctgacatg acaagagtta
     1681 ctaacagccc ctctctccaa gctcacttac aggctctcta cttagtccag cacgaagtct
     1741 ggagacctct ggcggcagcc taccaagaac aactggaccg accggtggta cctcaccctt
     1801 accgagtcgg cgacacagtg tgggtccgcc gacaccagac taagaaccta gaacctcgct
     1861 ggaaaggacc ttacacagtc ctgctgacca cccccaccgc cctcaaagta gacggcatcg
     1921 cagcttggat acacgccgcc cacgtgaagg ctgccgaccc cgggggtgga ccatcctcta
     1981 gactgccgga tctgccatcg atataaagct tgccaccatg gtgagcaagg gcgaggagct
     2041 gttcaccggg gtggtgccca tcctggtcga gctggacggc gacgtaaacg gccacaagtt
     2101 cagcgtgtcc ggcgagggcg agggcgatgc cacctacggc aagctgaccc tgaagttcat
     2161 ctgcaccacc ggcaagctgc ccgtgccctg gcccaccctc gtgaccaccc tgacctacgg
     2221 cgtgcagtgc ttcagccgct accccgacca catgaagcag cacgacttct tcaagtccgc
     2281 catgcccgaa ggctacgtcc aggagcgcac catcttcttc aaggacgacg gcaactacaa
     2341 gacccgcgcc gaggtgaagt tcgagggcga caccctggtg aaccgcatcg agctgaaggg
     2401 catcgacttc aaggaggacg gcaacatcct ggggcacaag ctggagtaca actacaacag
     2461 ccacaacgtc tatatcatgg ccgacaagca gaagaacggc atcaaggtga acttcaagat
     2521 ccgccacaac atcgaggacg gcagcgtgca gctcgccgac cactaccagc agaacacccc
     2581 catcggcgac ggccccgtgc tgctgcccga caaccactac ctgagcaccc agtccgccct
     2641 gagcaaagac cccaacgaga agcgcgatca catggtcctg ctggagttcg tgaccgccgc
     2701 cgggatcact ctcggcatgg acgagctgta caagggttca ggctcgggat ccgggaactc
     2761 gggtccgtcc 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 gatggtgaat tcaaagggcg aggagctcat caaggagaac atgcggatga aagtcgtcat
     3181 ggaaggctct gtcaatgggc accagttcaa gtgtaccgga gaaggggaag ggcgtcccta
     3241 tgaaggcgta caaaccatgc gcatcaaagt gatagagggc gggccactcc cttttgcctt
     3301 cgacattctg gctacctcct tcatgtatgg cagtcggaca ttcatcaagt atcccgcaga
     3361 cattccggac ttcttcaaac agtcctttcc agaaggcttt acatgggaga gggtgactag
     3421 gtacgaagat ggcggagtgg ttactgtgac tcaggacacg tcattggagg acggagagct
     3481 ggtgtacaat gtgaaagtac gcggagtgaa ctttcccagc aatggtcctg ttatgcagaa
     3541 gaaaaccaag ggttgggaac ccaatacgga gatgatgtat ccagctgatg gtggcctgag
     3601 gggatacaca gacatagccc tgaaagtcga tggaggtggc catctgcact gcaactttgt
     3661 gaccacctac agaagcaaga aaactgtggg gaacattaag atgcctggag tgcatgcagt
     3721 cgatcacaga cttgagagaa tcgaggagtc tgataacgag acatacgtgg tacaacgaga
     3781 agttgccgtt gcgaagtaca gcaatctggg cgggggcatg gacgagctgt acaagtaatg
     3841 agaattcgac agcactgtga cagacataca gccacttcca actaaagcaa gcctctgctc
     3901 cctgctacct gcatggagcc cactgtgaca ctcagaccat ccccggtcac tcactcactc
     3961 gtgtctgaga atctcagtga gagctgcctc tgtcacggac ggaagccaac acagccacct
     4021 ctcgacctgg ctccccacag cacccaccct ccctgcatgc agctgtccct gctaaccccc
     4081 aatgttatgt tacactgtgt aagtccccac tgctgccgtg tgtgggttgt gtacgtcgtc
     4141 atgtccctgg ggtctcgaga gcggccgcta cgtaaattcc gccccccccc ctaacgttac
     4201 tggccgaagc cgcttggaat aaggccggtg tgcgtttgtc tatatgttat tttccaccat
     4261 attgccgtct tttggcaatg tgagggcccg gaaacctggc cctgtcttct tgacgagcat
     4321 tcctaggggt ctttcccctc tcgccaaagg aatgcaaggt ctgttgaatg tcgtgaagga
     4381 agcagttcct ctggaagttt cttgaagaca aacaacgtct gtagcgaccc tttgcaggca
     4441 gcggaacccc ccacctggcg acaggtgcct ctgcggccaa aagccacgtg tataagatac
     4501 acctgcaaag gcggcacaac cccagtgcca cgttgtgagt tggatagttg tggaaagagt
     4561 caaatggctc tcctcaagcg tattcaacaa ggggctgaag gatgcccaga aggtacccca
     4621 ttgtatggga tctgatctgg ggcctcggtg cacatgcttt acatgtgttt agtcgaggtt
     4681 aaaaaaacgt ctaggccccc cgaaccacgg ggacgtggtt ttcctttgaa aaacacgatg
     4741 ataatatggc cacaaccgtc gacatgaccg agtacaagcc cacggtgcgc ctcgccaccc
     4801 gcgacgacgt cccccgggcc gtacgcaccc tcgccgccgc gttcgccgac taccccgcca
     4861 cgcgccacac cgtcgacccg gaccgccaca tcgagcgggt caccgagctg caagaactct
     4921 tcctcacgcg cgtcgggctc gacatcggca aggtgtgggt cgcggacgac ggcgccgcgg
     4981 tggcggtctg gaccacgccg gagagcgtcg aagcgggggc ggtgttcgcc gagatcggcc
     5041 cgcgcatggc cgagttgagc ggttcccggc tggccgcgca gcaacagatg gaaggcctcc
     5101 tggcgccgca ccggcccaag gagcccgcgt ggttcctggc caccgtcggc gtctcgcccg
     5161 accaccaggg caagggtctg ggcagcgccg tcgtgctccc cggagtggag gcggccgagc
     5221 gcgccggggt gcccgccttc ctggagacct ccgcgccccg caacctcccc ttctacgagc
     5281 ggctcggctt caccgtcacc gccgacgtcg aggtgcccga aggaccgcgc acctggtgca
     5341 tgacccgcaa gcccggtgcc tgagtcgacc gcccgcccca cgacccgcag cgcccgaccg
     5401 aaaggagcgc acgaccccat ggctcgacga taaaataaaa gattttattt agtctccaga
     5461 aaaagggggg aatgaaagac cccacctgta ggtttggcaa gctagcttaa gtaacgccat
     5521 tttgcaaggc atggaaaaat acataactga gaatagagaa gttcagatca aggtcaggaa
     5581 cagatggaac agctgaatat gggccaaaca ggatatctgt ggtaagcagt tcctgccccg
     5641 gctcagggcc aagaacagat ggaacagctg aatatgggcc aaacaggata tctgtggtaa
     5701 gcagttcctg ccccggctca gggccaagaa cagatggtcc ccagatgcgg tccagccctc
     5761 agcagtttct agagaaccat cagatgtttc cagggtgccc caaggacctg aaatgaccct
     5821 gtgccttatt tgaactaacc aatcagttcg cttctcgctt ctgttcgcgc gcttctgctc
     5881 cccgagctca ataaaagagc ccacaacccc tcactcgggg cgccagtcct ccgattgact
     5941 gagtcgcccg ggtacccgtg tatccaataa accctcttgc agttgcatcc gacttgtggt
     6001 ctcgctgttc cttgggaggg tctcctctga gtgattgact acccgtcagc gggggtcttt
     6061 cacatgcagc atgtatcaaa attaatttgg ttttttttct taagtattta cattaaatgg
     6121 ccatagttgc attaatgaat cggccaacgc gcggggagag gcggtttgcg tattgggcgc
     6181 tcttccgctt cctcgctcac tgactcgctg cgctcggtcg ttcggctgcg gcgagcggta
     6241 tcagctcact caaaggcggt aatacggtta tccacagaat caggggataa cgcaggaaag
     6301 aacatgtgag caaaaggcca gcaaaaggcc aggaaccgta aaaaggccgc gttgctggcg
     6361 tttttccata ggctccgccc ccctgacgag catcacaaaa atcgacgctc aagtcagagg
     6421 tggcgaaacc cgacaggact ataaagatac caggcgtttc cccctggaag ctccctcgtg
     6481 cgctctcctg ttccgaccct gccgcttacc ggatacctgt ccgcctttct cccttcggga
     6541 agcgtggcgc tttctcatag ctcacgctgt aggtatctca gttcggtgta ggtcgttcgc
     6601 tccaagctgg gctgtgtgca cgaacccccc gttcagcccg accgctgcgc cttatccggt
     6661 aactatcgtc ttgagtccaa cccggtaaga cacgacttat cgccactggc agcagccact
     6721 ggtaacagga ttagcagagc gaggtatgta ggcggtgcta cagagttctt gaagtggtgg
     6781 cctaactacg gctacactag aagaacagta tttggtatct gcgctctgct gaagccagtt
     6841 accttcggaa aaagagttgg tagctcttga tccggcaaac aaaccaccgc tggtagcggt
     6901 ggtttttttg tttgcaagca gcagattacg cgcagaaaaa aaggatctca agaagatcct
     6961 ttgatctttt ctacggggtc tgacgctcag tggaacgaaa actcacgtta agggattttg
     7021 gtcatgagat tatcaaaaag gatcttcacc tagatccttt tgcggccggc cgcaaatcaa
     7081 tctaaagtat atatgagtaa acttggtctg acagttacca atgcttaatc agtgaggcac
     7141 ctatctcagc gatctgtcta tttcgttcat ccatagttgc ctgactcccc gtcgtgtaga
     7201 taactacgat acgggagggc ttaccatctg gccccagtgc tgcaatgata ccgcgagacc
     7261 cacgctcacc ggctccagat ttatcagcaa taaaccagcc agccggaagg gccgagcgca
     7321 gaagtggtcc tgcaacttta tccgcctcca tccagtctat taattgttgc cgggaagcta
     7381 gagtaagtag ttcgccagtt aatagtttgc gcaacgttgt tgccattgct acaggcatcg
     7441 tggtgtcacg ctcgtcgttt ggtatggctt cattcagctc cggttcccaa cgatcaaggc
     7501 gagttacatg atcccccatg ttgtgcaaaa aagcggttag ctccttcggt cctccgatcg
     7561 ttgtcagaag taagttggcc gcagtgttat cactcatggt tatggcagca ctgcataatt
     7621 ctcttactgt catgccatcc gtaagatgct tttctgtgac tggtgagtac tcaaccaagt
     7681 cattctgaga atagtgtatg cggcgaccga gttgctcttg cccggcgtca acacgggata
     7741 ataccgcgcc acatagcaga actttaaaag tgctcatcat tggaaaacgt tcttcggggc
     7801 gaaaactctc aaggatctta ccgctgttga gatccagttc gatgtaaccc actcgtgcac
     7861 ccaactgatc ttcagcatct tttactttca ccagcgtttc tgggtgagca aaaacaggaa
     7921 ggcaaaatgc cgcaaaaaag ggaataaggg cgacacggaa atgttgaata ctcatactct
     7981 tcctttttca atattattga agcatttatc agggttattg tctcatgagc ggatacatat
     8041 ttgaatgtat ttagaaaaat aaacaaatag gggttccgcg cacatttccc cgaaaagtgc
     8101 cacctgacgt ctaagaaacc attattatca tgacattaac ctataaaaat aggcgtatca
     8161 cgaggccctt tcgtcttcac tataggagct tggcccattg catacgttgt atccatatca
     8221 taatatgtac atttatattg gct
//
