LOCUS       sequence-221593-        6468 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 6468)
  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 6468)
  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..6468
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     LTR             22..615
                     /note="long terminal repeat from Moloney murine leukemia 
                     virus"
     misc_feature    678..1035
                     /label=MMLV Psi
                     /note="packaging signal of Moloney murine leukemia virus 
                     (MMLV)"
     CDS             1094..1510
                     /label=gag (truncated)
                     /note="truncated Moloney murine leukemia virus (MMLV) gag 
                     gene lacking the start codon"
     primer_bind     1440..1462
                     /label=pLXSN 5'
                     /note="Murine stem cell virus, forward primer. Also called 
                     MSCV"
     primer_bind     1478..1494
                     /label=pBABE 5'
                     /note="Psi packaging signal, forward primer for pBABE 
                     vectors"
     misc_feature    1520..1894
                     /label=pol region
                     /note="Moloney murine leukemia virus (MMLV) pol region 
                     containing the splice acceptor site"
     primer_bind     1832..1848
                     /label=pBMN 5'
                     /note="MMLV sequence, for inserts in pBMN retroviral 
                     vector"
     CDS             join(1923..1925,1926..1928,1929..2639)
                     /codon_start=1
                     /product="enhanced GFP"
                     /label=EGFP
                     /translation="MVSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTL
                     KFICTTGKLPVPWPTLVTTLTYGVQCFSRYPDHMKQHDFFKSAMPEGYVQERTIFFKDD
                     GNYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIK
                     VNFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSALSKDPNEKRDHMVLL
                     EFVTAAGITLGMDELYK"
     CDS             join(1923..1925,1926..1928,1929..2639)
                     /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(1968..1989)
                     /label=EGFP-N
                     /note="EGFP, reverse primer"
     primer_bind     complement(2229..2248)
                     /label=EXFP-R
                     /note="For distinguishing EGFP variants, reverse primer"
     primer_bind     2576..2597
                     /label=EGFP-C
                     /note="EGFP, forward primer"
     CDS             3063..3740
                     /codon_start=1
                     /product="monomeric derivative of DsRed (Campbell et al., 
                     2002)"
                     /label=mRFP1
                     /translation="MASSEDVIKEFMRFKVRMEGSVNGHEFEIEGEGEGRPYEGTQTAK
                     LKVTKGGPLPFAWDILSPQFQYGSKAYVKHPADIPDYLKLSFPEGFKWERVMNFEDGGV
                     VTVTQDSSLQDGEFIYKVKLRGTNFPSDGPVMQKKTMGWEASTERMYPEDGALKGEIKM
                     RLKLKDGGHYDAEVKTTYMAKKPVQLPGAYKTDIKLDITSHNEDYTIVEQYERAEGRHS
                     TGA"
     CDS             3063..3740
                     /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(3193..3211)
                     /label=mCherry-R
                     /note="mCherry, reverse primer"
     primer_bind     3461..3480
                     /label=mCherry-F
                     /note="mCherry, forward primer"
     primer_bind     3655..3678
                     /label=DsRed1-C
                     /note="DsRed1, forward primer"
     LTR             3973..4442
                     /note="long terminal repeat from Moloney murine leukemia 
                     virus"
     primer_bind     complement(4572..4589)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(4743..5331)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(4823..4842)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(5495..6286,6287..6355))
                     /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     6118..6137
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(6356..6460)
                     /gene="bla"
                     /label=AmpR promoter
ORIGIN
        1 cccgaaaagt gccacctgca taatgaaaga ccccacctgt aggtttggca agctagctta
       61 agtaacgcca ttttgcaagg catggaaaaa tacataactg agaatagaaa agttcagatc
      121 aaggtcagga acagatggaa cagctgaata tgggccaaac aggatatctg tggtaagcag
      181 ttcctgcccc ggctcagggc caagaacaga tggaacagct gaatatgggc caaacaggat
      241 atctgtggta agcagttcct gccccggctc agggccaaga acagatggtc cccagatgcg
      301 gtccagccct cagcagtttc tagagaacca tcagatgttt ccagggtgcc ccaaggacct
      361 gaaatgaccc tgtgccttat ttgaactaac caatcagttc gcttctcgct tctgttcgcg
      421 cgcttctgct ccccgagctc aataaaagag cccacaaccc ctcactcggc gcgccagtcc
      481 tccgattgac tgagtcgccc gggtacccgt gtatccaata aaccctcttg cagttgcatc
      541 cgacttgtgg tctcgctgtt ccttgggagg gtctcctctg agtgattgac tacccgtcag
      601 cgggggtctt tcatttgggg gctcgtccgg gatcgggaga cccctgccca gggaccaccg
      661 acccaccacc gggaggtaag ctggccagca acttatctgt gtctgtccga ttgtctagtg
      721 tctatgactg attttatgcg cctgcgtcgg tactagttag ctaactagct ctgtatctgg
      781 cggacccgtg gtggaactga cgagttcgga acacccggcc gcaaccctgg gagacgtccc
      841 agggacttcg ggggccgttt ttgtggcccg acctgagtcc aaaaatcccg atcgttttgg
      901 actctttggt gcacccccct aataggaggg atatgtggtt ctggtaggag acgagaacct
      961 aaaacagttc ccgcctccgt ctgaattttt gctttcggtt tgggaccgaa gccgcgccgc
     1021 gcgtcttgtc tgctgcagca tcgttctgtg ttgtctctgt ctgactgtgt ttctgtattt
     1081 gtctgaaaat tagggccaga ctgttaccac tcccttaagt ttgaccttag gtcactggaa
     1141 agatgtcgag cggatcgctc acaaccagtc ggtagatgtc aagaagagac gttgggttac
     1201 cttctgctct gcagaatggc caacctttaa cgtcggatgg ccgcgagacg gcacctttaa
     1261 ccgagacctc atcacccagg ttaagatcaa ggtcttttca cctggcccgc atggacaccc
     1321 agaccaggtc ccctacatcg tgacctggga agccttggct tttgaccccc ctccctgggt
     1381 caagcccttt gtacacccta agcctccgcc tcctcttcct ccatccgccc cgtctctccc
     1441 ccttgaacct cctcgttcga ccccgcctcg atcctccctt tatccagccc tcactccttc
     1501 tctaggcgcc cccatatggc catatgagat cttatatggg gcacccccgc cccttgtaaa
     1561 cttccctgac cctgacatga caagagttac taacagcccc tctctccaag ctcacttaca
     1621 ggctctctac ttagtccagc acgaagtctg gagacctctg gcggcagcct accaagaaca
     1681 actggaccga ccggtggtac ctcaccctta ccgagtcggc gacacagtgt gggtccgccg
     1741 acaccagact aagaacctag aacctcgctg gaaaggacct tacacagtcc tgctgaccac
     1801 ccccaccgcc ctcaaagtag acggcatcgc agcttggata cacgccgccc acgtgaaggc
     1861 tgccgacccc gggggtggac catcctctag actgccggat ctagctagtt aattaaggat
     1921 ctatggtgag caagggcgag gagctgttca ccggggtggt gcccatcctg gtcgagctgg
     1981 acggcgacgt aaacggccac aagttcagcg tgtccggcga gggcgagggc gatgccacct
     2041 acggcaagct gaccctgaag ttcatctgca ccaccggcaa gctgcccgtg ccctggccca
     2101 ccctcgtgac caccctgacc tacggcgtgc agtgcttcag ccgctacccc gaccacatga
     2161 agcagcacga cttcttcaag tccgccatgc ccgaaggcta cgtccaggag cgcaccatct
     2221 tcttcaagga cgacggcaac tacaagaccc gcgccgaggt gaagttcgag ggcgacaccc
     2281 tggtgaaccg catcgagctg aagggcatcg acttcaagga ggacggcaac atcctggggc
     2341 acaagctgga gtacaactac aacagccaca acgtctatat catggccgac aagcagaaga
     2401 acggcatcaa ggtgaacttc aagatccgcc acaacatcga ggacggcagc gtgcagctcg
     2461 ccgaccacta ccagcagaac acccccatcg gcgacggccc cgtgctgctg cccgacaacc
     2521 actacctgag cacccagtcc gccctgagca aagaccccaa cgagaagcgc gatcacatgg
     2581 tcctgctgga gttcgtgacc gccgccggga tcactctcgg catggacgag ctgtacaagg
     2641 gtggcggtgg ctcgggcggt ggtgggtcgg gtggcggcgg atctatcgat gaattgaatt
     2701 ccatgccgtc cgagaagacc ttcaaacagc gccggagctt cgaacaaaga gtggaagatg
     2761 tccggctcat ccgggagcag caccccacca agatcccagt gattatagag cgatacaagg
     2821 gtgagaagca gctgcccgtc ctggacaaga ccaagttcct tgtacctgat cacgtgaata
     2881 tgagcgaact catcaagata attagaaggc gcctgcagct caatgctaac caagccttct
     2941 tcctcctggt gaatgggcac agcatggtga gtgtgtccac acccatctct gaagtgtacg
     3001 agagcgagag agatgaagac ggcttcctgt acatggtcta tgcctcccag gagacgttcg
     3061 ggatggcctc ctccgaggac gtcatcaagg agttcatgcg cttcaaggtg cgcatggagg
     3121 gctccgtgaa cggccacgag ttcgagatcg agggcgaggg cgagggccgc ccctacgagg
     3181 gcacccagac cgccaagctg aaggtgacca agggcggccc cctgcccttc gcctgggaca
     3241 tcctgtcccc tcagttccag tacggctcca aggcctacgt gaagcacccc gccgacatcc
     3301 ccgactactt gaagctgtcc ttccccgagg gcttcaagtg ggagcgcgtg atgaacttcg
     3361 aggacggcgg cgtggtgacc gtgacccagg actcctccct gcaggacggc gagttcatct
     3421 acaaggtgaa gctgcgcggc accaacttcc cctccgacgg ccccgtaatg cagaagaaga
     3481 ccatgggctg ggaggcctcc accgagcgga tgtaccccga ggacggcgcc ctgaagggcg
     3541 agatcaagat gaggctgaag ctgaaggacg gcggccacta cgacgccgag gtcaagacca
     3601 cctacatggc caagaagccc gtgcagctgc ccggcgccta caagaccgac atcaagctgg
     3661 acatcacctc ccacaacgag gactacacca tcgtggaaca gtacgagcgc gccgagggcc
     3721 gccactccac cggcgcctaa agatcccagt gtggtggtac gggaattcct gcaggcctcg
     3781 agggccggcg cgccgcggcc gctacgacga taaaataaaa gattttattt agtctccaga
     3841 aaaagggggg aatgaaagac cccacctgta ggtttggcaa gctagcttaa gtaacgccat
     3901 tttgcaaggc atggaaaaat acataactga gaatagagaa gttcagatca aggtcaggaa
     3961 cagatggaac agctgaatat gggccaaaca ggatatctgt ggtaagcagt tcctgccccg
     4021 gctcagggcc aagaacagat ggaacagctg aatatgggcc aaacaggata tctgtggtaa
     4081 gcagttcctg ccccggctca gggccaagaa cagatggtcc ccagatgcgg tccagccctc
     4141 agcagtttct agagaaccat cagatgtttc cagggtgccc caaggacctg aaatgaccct
     4201 gtgccttatt tgaactaacc aatcagttcg cttctcgctt ctgttcgcgc gcttctgctc
     4261 cccgagctca ataaaagagc ccacaacccc tcactcgggg cgccagtcct ccgattgact
     4321 gagtcgcccg ggtacccgtg tatccaataa accctcttgc agttgcatcc gacttgtggt
     4381 ctcgctgttc cttgggaggg tctcctctga gtgattgact acccgtcagc gggggtcttt
     4441 cacatgcagc atgtatcaaa attaatttgg ttttttttct taagtattta cattaaatgg
     4501 ccatagttgc attaatgaat cggccaacgc gcggggagag gcggtttgcg tattgggcgc
     4561 tcttccgctt cctcgctcac tgactcgctg cgctcggtcg ttcggctgcg gcgagcggta
     4621 tcagctcact caaaggcggt aatacggtta tccacagaat caggggataa cgcaggaaag
     4681 aacatgtgag caaaaggcca gcaaaaggcc aggaaccgta aaaaggccgc gttgctggcg
     4741 tttttccata ggctccgccc ccctgacgag catcacaaaa atcgacgctc aagtcagagg
     4801 tggcgaaacc cgacaggact ataaagatac caggcgtttc cccctggaag ctccctcgtg
     4861 cgctctcctg ttccgaccct gccgcttacc ggatacctgt ccgcctttct cccttcggga
     4921 agcgtggcgc tttctcatag ctcacgctgt aggtatctca gttcggtgta ggtcgttcgc
     4981 tccaagctgg gctgtgtgca cgaacccccc gttcagcccg accgctgcgc cttatccggt
     5041 aactatcgtc ttgagtccaa cccggtaaga cacgacttat cgccactggc agcagccact
     5101 ggtaacagga ttagcagagc gaggtatgta ggcggtgcta cagagttctt gaagtggtgg
     5161 cctaactacg gctacactag aagaacagta tttggtatct gcgctctgct gaagccagtt
     5221 accttcggaa aaagagttgg tagctcttga tccggcaaac aaaccaccgc tggtagcggt
     5281 ggtttttttg tttgcaagca gcagattacg cgcagaaaaa aaggatctca agaagatcct
     5341 ttgatctttt ctacggggtc tgacgctcag tggaacgaaa actcacgtta agggattttg
     5401 gtcatgagat tatcaaaaag gatcttcacc tagatccttt tgcggccggc cgcaaatcaa
     5461 tctaaagtat atatgagtaa acttggtctg acagttacca atgcttaatc agtgaggcac
     5521 ctatctcagc gatctgtcta tttcgttcat ccatagttgc ctgactcccc gtcgtgtaga
     5581 taactacgat acgggagggc ttaccatctg gccccagtgc tgcaatgata ccgcgagacc
     5641 cacgctcacc ggctccagat ttatcagcaa taaaccagcc agccggaagg gccgagcgca
     5701 gaagtggtcc tgcaacttta tccgcctcca tccagtctat taattgttgc cgggaagcta
     5761 gagtaagtag ttcgccagtt aatagtttgc gcaacgttgt tgccattgct acaggcatcg
     5821 tggtgtcacg ctcgtcgttt ggtatggctt cattcagctc cggttcccaa cgatcaaggc
     5881 gagttacatg atcccccatg ttgtgcaaaa aagcggttag ctccttcggt cctccgatcg
     5941 ttgtcagaag taagttggcc gcagtgttat cactcatggt tatggcagca ctgcataatt
     6001 ctcttactgt catgccatcc gtaagatgct tttctgtgac tggtgagtac tcaaccaagt
     6061 cattctgaga atagtgtatg cggcgaccga gttgctcttg cccggcgtca atacgggata
     6121 ataccgcgcc acatagcaga actttaaaag tgctcatcat tggaaaacgt tcttcggggc
     6181 gaaaactctc aaggatctta ccgctgttga gatccagttc gatgtaaccc actcgtgcac
     6241 ccaactgatc ttcagcatct tttactttca ccagcgtttc tgggtgagca aaaacaggaa
     6301 ggcaaaatgc cgcaaaaaag ggaataaggg cgacacggaa atgttgaata ctcatactct
     6361 tcctttttca atattattga agcatttatc agggttattg tctcatgagc ggatacatat
     6421 ttgaatgtat ttagaaaaat aaacaaatag gggttccgcg cacatttc
//
