LOCUS       sequence-224908-        6465 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Expresses GFP-LC3-RFP in mammalian cells to measure autophagic flux 
            without any drug resistance..
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 6465)
  AUTHORS   Morita K, Hama Y, Izume T, Tamura N, Ueno T, Yamashita Y, Sakamaki 
            Y, Mimura K, Morishita H, Shihoya W, Nureki O, Mano H, Mizushima N
  TITLE     Genome-wide CRISPR screen identifies TMEM41B as a gene required for 
            autophagosome formation.
  JOURNAL   J Cell Biol. 2018 Aug 9. pii: jcb.201804132. doi: 
            10.1083/jcb.201804132.
  PUBMED    30093494
REFERENCE   2  (bases 1 to 6465)
  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 
            Biol. 2018 Aug 9. pii: jcb.201804132. doi: 10.1083/jcb.201804132."
COMMENT     Sequence Label: pMXs GFP-LC3-RFP
FEATURES             Location/Qualifiers
     source          1..6465
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     CDS             1..417
                     /label=gag (truncated)
                     /note="truncated Moloney murine leukemia virus (MMLV) gag 
                     gene lacking the start codon"
     primer_bind     347..369
                     /label=pLXSN 5'
                     /note="Murine stem cell virus, forward primer. Also called 
                     MSCV"
     primer_bind     385..401
                     /label=pBABE 5'
                     /note="Psi packaging signal, forward primer for pBABE 
                     vectors"
     misc_feature    427..801
                     /label=pol region
                     /note="Moloney murine leukemia virus (MMLV) pol region 
                     containing the splice acceptor site"
     primer_bind     739..755
                     /label=pBMN 5'
                     /note="MMLV sequence, for inserts in pBMN retroviral 
                     vector"
     regulatory      821..830
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     CDS             join(827..829,830..832,833..1543)
                     /codon_start=1
                     /product="enhanced GFP"
                     /label=EGFP
                     /translation="MVSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTL
                     KFICTTGKLPVPWPTLVTTLTYGVQCFSRYPDHMKQHDFFKSAMPEGYVQERTIFFKDD
                     GNYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIK
                     VNFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSALSKDPNEKRDHMVLL
                     EFVTAAGITLGMDELYK"
     CDS             join(827..829,830..832,833..1543)
                     /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(872..893)
                     /label=EGFP-N
                     /note="EGFP, reverse primer"
     primer_bind     complement(1133..1152)
                     /label=EXFP-R
                     /note="For distinguishing EGFP variants, reverse primer"
     primer_bind     1480..1501
                     /label=EGFP-C
                     /note="EGFP, forward primer"
     CDS             1967..2644
                     /codon_start=1
                     /product="monomeric derivative of DsRed (Campbell et al., 
                     2002)"
                     /label=mRFP1
                     /translation="MASSEDVIKEFMRFKVRMEGSVNGHEFEIEGEGEGRPYEGTQTAK
                     LKVTKGGPLPFAWDILSPQFQYGSKAYVKHPADIPDYLKLSFPEGFKWERVMNFEDGGV
                     VTVTQDSSLQDGEFIYKVKLRGTNFPSDGPVMQKKTMGWEASTERMYPEDGALKGEIKM
                     RLKLKDGGHYDAEVKTTYMAKKPVQLPGAYKTDIKLDITSHNEDYTIVEQYERAEGRHS
                     TGA"
     CDS             1967..2644
                     /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(2097..2115)
                     /label=mCherry-R
                     /note="mCherry, reverse primer"
     primer_bind     2365..2384
                     /label=mCherry-F
                     /note="mCherry, forward primer"
     primer_bind     2559..2582
                     /label=DsRed1-C
                     /note="DsRed1, forward primer"
     LTR             2877..3346
                     /note="long terminal repeat from Moloney murine leukemia 
                     virus"
     primer_bind     complement(3476..3493)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(3647..4235)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(3727..3746)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(4399..5190,5191..5259))
                     /codon_start=1
                     /gene="bla"
                     /product="beta-lactamase"
                     /label=AmpR
                     /note="confers resistance to ampicillin, carbenicillin, and
                     related antibiotics"
                     /translation="MSIQHFRVALIPFFAAFCLPVFAHPETLVKVKDAEDQLGARVGYI
                     ELDLNSGKILESFRPEERFPMMSTFKVLLCGAVLSRIDAGQEQLGRRIHYSQNDLVEYS
                     PVTEKHLTDGMTVRELCSAAITMSDNTAANLLLTTIGGPKELTAFLHNMGDHVTRLDRW
                     EPELNEAIPNDERDTTMPVAMATTLRKLLTGELLTLASRQQLIDWMEADKVAGPLLRSA
                     LPAGWFIADKSGAGERGSRGIIAALGPDGKPSRIVVIYTTGSQATMDERNRQIAEIGAS
                     LIKHW"
     primer_bind     5022..5041
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(5260..5364)
                     /gene="bla"
                     /label=AmpR promoter
     LTR             5394..5987
                     /note="long terminal repeat from Moloney murine leukemia 
                     virus"
     misc_feature    6050..6407
                     /label=MMLV Psi
                     /note="packaging signal of Moloney murine leukemia virus 
                     (MMLV)"
ORIGIN
        1 ggccagactg ttaccactcc cttaagtttg accttaggtc actggaaaga tgtcgagcgg
       61 atcgctcaca accagtcggt agatgtcaag aagagacgtt gggttacctt ctgctctgca
      121 gaatggccaa cctttaacgt cggatggccg cgagacggca cctttaaccg agacctcatc
      181 acccaggtta agatcaaggt cttttcacct ggcccgcatg gacacccaga ccaggtcccc
      241 tacatcgtga cctgggaagc cttggctttt gacccccctc cctgggtcaa gccctttgta
      301 caccctaagc ctccgcctcc tcttcctcca tccgccccgt ctctccccct tgaacctcct
      361 cgttcgaccc cgcctcgatc ctccctttat ccagccctca ctccttctct aggcgccccc
      421 atatggccat atgagatctt atatggggca cccccgcccc ttgtaaactt ccctgaccct
      481 gacatgacaa gagttactaa cagcccctct ctccaagctc acttacaggc tctctactta
      541 gtccagcacg aagtctggag acctctggcg gcagcctacc aagaacaact ggaccgaccg
      601 gtggtacctc acccttaccg agtcggcgac acagtgtggg tccgccgaca ccagactaag
      661 aacctagaac ctcgctggaa aggaccttac acagtcctgc tgaccacccc caccgccctc
      721 aaagtagacg gcatcgcagc ttggatacac gccgcccacg tgaaggctgc cgaccccggg
      781 ggtggaccat cctctagact gccggatcta gcctcccgcc gccaccatgg tgagcaaggg
      841 cgaggagctg ttcaccgggg tggtgcccat cctggtcgag ctggacggcg acgtaaacgg
      901 ccacaagttc agcgtgtccg gcgagggcga gggcgatgcc acctacggca agctgaccct
      961 gaagttcatc tgcaccaccg gcaagctgcc cgtgccctgg cccaccctcg tgaccaccct
     1021 gacctacggc gtgcagtgct tcagccgcta ccccgaccac atgaagcagc acgacttctt
     1081 caagtccgcc atgcccgaag gctacgtcca ggagcgcacc atcttcttca aggacgacgg
     1141 caactacaag acccgcgccg aggtgaagtt cgagggcgac accctggtga accgcatcga
     1201 gctgaagggc atcgacttca aggaggacgg caacatcctg gggcacaagc tggagtacaa
     1261 ctacaacagc cacaacgtct atatcatggc cgacaagcag aagaacggca tcaaggtgaa
     1321 cttcaagatc cgccacaaca tcgaggacgg cagcgtgcag ctcgccgacc actaccagca
     1381 gaacaccccc atcggcgacg gccccgtgct gctgcccgac aaccactacc tgagcaccca
     1441 gtccgccctg agcaaagacc ccaacgagaa gcgcgatcac atggtcctgc tggagttcgt
     1501 gaccgccgcc gggatcactc tcggcatgga cgagctgtac aagggtggcg gtggctcggg
     1561 cggtggtggg tcgggtggcg gcggatctat cgatgaattg aattccatgc cgtccgagaa
     1621 gaccttcaaa cagcgccgga gcttcgaaca aagagtggaa gatgtccggc tcatccggga
     1681 gcagcacccc accaagatcc cagtgattat agagcgatac aagggtgaga agcagctgcc
     1741 cgtcctggac aagaccaagt tccttgtacc tgatcacgtg aatatgagcg aactcatcaa
     1801 gataattaga aggcgcctgc agctcaatgc taaccaagcc ttcttcctcc tggtgaatgg
     1861 gcacagcatg gtgagtgtgt ccacacccat ctctgaagtg tacgagagcg agagagatga
     1921 agacggcttc ctgtacatgg tctatgcctc ccaggagacg ttcgggatgg cctcctccga
     1981 ggacgtcatc aaggagttca tgcgcttcaa ggtgcgcatg gagggctccg tgaacggcca
     2041 cgagttcgag atcgagggcg agggcgaggg ccgcccctac gagggcaccc agaccgccaa
     2101 gctgaaggtg accaagggcg gccccctgcc cttcgcctgg gacatcctgt cccctcagtt
     2161 ccagtacggc tccaaggcct acgtgaagca ccccgccgac atccccgact acttgaagct
     2221 gtccttcccc gagggcttca agtgggagcg cgtgatgaac ttcgaggacg gcggcgtggt
     2281 gaccgtgacc caggactcct ccctgcagga cggcgagttc atctacaagg tgaagctgcg
     2341 cggcaccaac ttcccctccg acggccccgt aatgcagaag aagaccatgg gctgggaggc
     2401 ctccaccgag cggatgtacc ccgaggacgg cgccctgaag ggcgagatca agatgaggct
     2461 gaagctgaag gacggcggcc actacgacgc cgaggtcaag accacctaca tggccaagaa
     2521 gcccgtgcag ctgcccggcg cctacaagac cgacatcaag ctggacatca cctcccacaa
     2581 cgaggactac accatcgtgg aacagtacga gcgcgccgag ggccgccact ccaccggcgc
     2641 ctaaagatcc cagtgtggtg gtacgggaat tcctgcaggc ctcgagggcc ggcgcgccgc
     2701 ggccgctacg acgataaaat aaaagatttt atttagtctc cagaaaaagg ggggaatgaa
     2761 agaccccacc tgtaggtttg gcaagctagc ttaagtaacg ccattttgca aggcatggaa
     2821 aaatacataa ctgagaatag agaagttcag atcaaggtca ggaacagatg gaacagctga
     2881 atatgggcca aacaggatat ctgtggtaag cagttcctgc cccggctcag ggccaagaac
     2941 agatggaaca gctgaatatg ggccaaacag gatatctgtg gtaagcagtt cctgccccgg
     3001 ctcagggcca agaacagatg gtccccagat gcggtccagc cctcagcagt ttctagagaa
     3061 ccatcagatg tttccagggt gccccaagga cctgaaatga ccctgtgcct tatttgaact
     3121 aaccaatcag ttcgcttctc gcttctgttc gcgcgcttct gctccccgag ctcaataaaa
     3181 gagcccacaa cccctcactc ggggcgccag tcctccgatt gactgagtcg cccgggtacc
     3241 cgtgtatcca ataaaccctc ttgcagttgc atccgacttg tggtctcgct gttccttggg
     3301 agggtctcct ctgagtgatt gactacccgt cagcgggggt ctttcacatg cagcatgtat
     3361 caaaattaat ttggtttttt ttcttaagta tttacattaa atggccatag ttgcattaat
     3421 gaatcggcca acgcgcgggg agaggcggtt tgcgtattgg gcgctcttcc gcttcctcgc
     3481 tcactgactc gctgcgctcg gtcgttcggc tgcggcgagc ggtatcagct cactcaaagg
     3541 cggtaatacg gttatccaca gaatcagggg ataacgcagg aaagaacatg tgagcaaaag
     3601 gccagcaaaa ggccaggaac cgtaaaaagg ccgcgttgct ggcgtttttc cataggctcc
     3661 gcccccctga cgagcatcac aaaaatcgac gctcaagtca gaggtggcga aacccgacag
     3721 gactataaag ataccaggcg tttccccctg gaagctccct cgtgcgctct cctgttccga
     3781 ccctgccgct taccggatac ctgtccgcct ttctcccttc gggaagcgtg gcgctttctc
     3841 atagctcacg ctgtaggtat ctcagttcgg tgtaggtcgt tcgctccaag ctgggctgtg
     3901 tgcacgaacc ccccgttcag cccgaccgct gcgccttatc cggtaactat cgtcttgagt
     3961 ccaacccggt aagacacgac ttatcgccac tggcagcagc cactggtaac aggattagca
     4021 gagcgaggta tgtaggcggt gctacagagt tcttgaagtg gtggcctaac tacggctaca
     4081 ctagaagaac agtatttggt atctgcgctc tgctgaagcc agttaccttc ggaaaaagag
     4141 ttggtagctc ttgatccggc aaacaaacca ccgctggtag cggtggtttt tttgtttgca
     4201 agcagcagat tacgcgcaga aaaaaaggat ctcaagaaga tcctttgatc ttttctacgg
     4261 ggtctgacgc tcagtggaac gaaaactcac gttaagggat tttggtcatg agattatcaa
     4321 aaaggatctt cacctagatc cttttgcggc cggccgcaaa tcaatctaaa gtatatatga
     4381 gtaaacttgg tctgacagtt accaatgctt aatcagtgag gcacctatct cagcgatctg
     4441 tctatttcgt tcatccatag ttgcctgact ccccgtcgtg tagataacta cgatacggga
     4501 gggcttacca tctggcccca gtgctgcaat gataccgcga gacccacgct caccggctcc
     4561 agatttatca gcaataaacc agccagccgg aagggccgag cgcagaagtg gtcctgcaac
     4621 tttatccgcc tccatccagt ctattaattg ttgccgggaa gctagagtaa gtagttcgcc
     4681 agttaatagt ttgcgcaacg ttgttgccat tgctacaggc atcgtggtgt cacgctcgtc
     4741 gtttggtatg gcttcattca gctccggttc ccaacgatca aggcgagtta catgatcccc
     4801 catgttgtgc aaaaaagcgg ttagctcctt cggtcctccg atcgttgtca gaagtaagtt
     4861 ggccgcagtg ttatcactca tggttatggc agcactgcat aattctctta ctgtcatgcc
     4921 atccgtaaga tgcttttctg tgactggtga gtactcaacc aagtcattct gagaatagtg
     4981 tatgcggcga ccgagttgct cttgcccggc gtcaatacgg gataataccg cgccacatag
     5041 cagaacttta aaagtgctca tcattggaaa acgttcttcg gggcgaaaac tctcaaggat
     5101 cttaccgctg ttgagatcca gttcgatgta acccactcgt gcacccaact gatcttcagc
     5161 atcttttact ttcaccagcg tttctgggtg agcaaaaaca ggaaggcaaa atgccgcaaa
     5221 aaagggaata agggcgacac ggaaatgttg aatactcata ctcttccttt ttcaatatta
     5281 ttgaagcatt tatcagggtt attgtctcat gagcggatac atatttgaat gtatttagaa
     5341 aaataaacaa ataggggttc cgcgcacatt tccccgaaaa gtgccacctg cataatgaaa
     5401 gaccccacct gtaggtttgg caagctagct taagtaacgc cattttgcaa ggcatggaaa
     5461 aatacataac tgagaataga aaagttcaga tcaaggtcag gaacagatgg aacagctgaa
     5521 tatgggccaa acaggatatc tgtggtaagc agttcctgcc ccggctcagg gccaagaaca
     5581 gatggaacag ctgaatatgg gccaaacagg atatctgtgg taagcagttc ctgccccggc
     5641 tcagggccaa gaacagatgg tccccagatg cggtccagcc ctcagcagtt tctagagaac
     5701 catcagatgt ttccagggtg ccccaaggac ctgaaatgac cctgtgcctt atttgaacta
     5761 accaatcagt tcgcttctcg cttctgttcg cgcgcttctg ctccccgagc tcaataaaag
     5821 agcccacaac ccctcactcg gcgcgccagt cctccgattg actgagtcgc ccgggtaccc
     5881 gtgtatccaa taaaccctct tgcagttgca tccgacttgt ggtctcgctg ttccttggga
     5941 gggtctcctc tgagtgattg actacccgtc agcgggggtc tttcatttgg gggctcgtcc
     6001 gggatcggga gacccctgcc cagggaccac cgacccacca ccgggaggta agctggccag
     6061 caacttatct gtgtctgtcc gattgtctag tgtctatgac tgattttatg cgcctgcgtc
     6121 ggtactagtt agctaactag ctctgtatct ggcggacccg tggtggaact gacgagttcg
     6181 gaacacccgg ccgcaaccct gggagacgtc ccagggactt cgggggccgt ttttgtggcc
     6241 cgacctgagt ccaaaaatcc cgatcgtttt ggactctttg gtgcaccccc ctaataggag
     6301 ggatatgtgg ttctggtagg agacgagaac ctaaaacagt tcccgcctcc gtctgaattt
     6361 ttgctttcgg tttgggaccg aagccgcgcc gcgcgtcttg tctgctgcag catcgttctg
     6421 tgttgtctct gtctgactgt gtttctgtat ttgtctgaaa attag
//
