LOCUS Exported 6465 bp ds-DNA circular SYN 12-MAY-2021 DEFINITION Expresses GFP-LC3-RFP in mammalian cells to measure autophagic flux without any drug resistance.. ACCESSION . VERSION . KEYWORDS pMXs GFP-LC3-RFP 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 May 12, 2021 from SnapGene Server 1.1.58 http://www.snapgene.com FEATURES Location/Qualifiers source 1..6465 /organism="synthetic DNA construct" /mol_type="other DNA" 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 /regulatory_class="other" /note="vertebrate consensus sequence for strong initiation of translation (Kozak, 1987)" CDS 827..1543 /codon_start=1 /product="enhanced GFP" /label=EGFP /translation="MVSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTL KFICTTGKLPVPWPTLVTTLTYGVQCFSRYPDHMKQHDFFKSAMPEGYVQERTIFFKDD GNYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIK VNFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSALSKDPNEKRDHMVLL EFVTAAGITLGMDELYK" CDS 827..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 /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(4399..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 //