LOCUS Exported 6820 bp ds-DNA circular SYN 13-MAY-2021 DEFINITION Expresses GFP-LC3-RFP-LC3-Delta-G in zebrafish to measure autophagic flux. ACCESSION . VERSION . KEYWORDS pT2AL200R150G-EGFP-LC3B-RFP-LC3B-Delta-G SOURCE synthetic DNA construct ORGANISM synthetic DNA construct REFERENCE 1 (bases 1 to 6820) 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 6820) AUTHORS . TITLE Direct Submission JOURNAL Exported May 13, 2021 from SnapGene Server 1.1.58 http://www.snapgene.com FEATURES Location/Qualifiers source 1..6820 /organism="synthetic DNA construct" /mol_type="other DNA" primer_bind 19..39 /label=T3 /note="T3 promoter, forward primer" promoter 21..39 /label=T3 promoter /note="promoter for bacteriophage T3 RNA polymerase" primer_bind 76..92 /label=SK primer /note="common sequencing primer, one of multiple similar variants" primer_bind 76..92 /label=pBluescriptSK /note="For pBluescript vector" intron 856..1428 /label=-beta-globin intron /note="intron from rabbit beta-globin gene" CDS 1498..2214 /codon_start=1 /product="enhanced GFP" /label=EGFP /translation="MVSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTL KFICTTGKLPVPWPTLVTTLTYGVQCFSRYPDHMKQHDFFKSAMPEGYVQERTIFFKDD GNYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIK VNFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSALSKDPNEKRDHMVLL EFVTAAGITLGMDELYK" CDS 1498..2214 /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(1543..1564) /label=EGFP-N /note="EGFP, reverse primer" primer_bind complement(1804..1823) /label=EXFP-R /note="For distinguishing EGFP variants, reverse primer" primer_bind 2151..2172 /label=EGFP-C /note="EGFP, forward primer" CDS 2638..3312 /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(2768..2786) /label=mCherry-R /note="mCherry, reverse primer" primer_bind 3036..3055 /label=mCherry-F /note="mCherry, forward primer" primer_bind 3230..3253 /label=DsRed1-C /note="DsRed1, forward primer" polyA_signal 3718..3852 /label=SV40 poly(A) signal /note="SV40 polyadenylation signal" primer_bind complement(3742..3761) /label=EBV-rev /note="SV40 polyA terminator, reverse primer" primer_bind 3796..3815 /label=SV40pA-R /note="SV40 polyA, reverse primer" primer_bind complement(4153..4172) /label=T7 /note="T7 promoter, forward primer" promoter complement(4154..4172) /label=T7 promoter /note="promoter for bacteriophage T7 RNA polymerase" primer_bind complement(4182..4199) /label=M13 Forward /note="In lacZ gene. Also called M13-F20 or M13 (-21) Forward" primer_bind complement(4182..4198) /label=M13 fwd /note="common sequencing primer, one of multiple similar variants" primer_bind complement(4191..4213) /label=M13/pUC Forward /note="In lacZ gene" primer_bind 4510..4531 /label=F1ori-F /note="F1 origin, forward primer" promoter 4694..4798 /gene="bla" /label=AmpR promoter CDS 4799..5659 /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 complement(5017..5036) /label=Amp-R /note="Ampicillin resistance gene, reverse primer" rep_origin 5830..6418 /direction=RIGHT /label=ori /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of replication" primer_bind 6319..6338 /label=pBR322ori-F /note="pBR322 origin, forward primer" primer_bind 6572..6589 /label=L4440 /note="L4440 vector, forward primer" protein_bind 6706..6727 /label=CAP binding site /bound_moiety="E. coli catabolite activator protein" /note="CAP binding activates transcription in the presence of cAMP." promoter 6742..6772 /label=lac promoter /note="promoter for the E. coli lac operon" protein_bind 6780..6796 /label=lac operator /bound_moiety="lac repressor encoded by lacI" /note="The lac repressor binds to the lac operator to inhibit transcription in E. coli. This inhibition can be relieved by adding lactose or isopropyl-beta-D-thiogalactopyranoside (IPTG)." primer_bind 6785..6807 /label=M13/pUC Reverse /note="In lacZ gene" primer_bind 6804..6820 /label=M13 rev /note="common sequencing primer, one of multiple similar variants" primer_bind 6804..6820 /label=M13 Reverse /note="In lacZ gene. Also called M13-rev" ORIGIN 1 catgattacg ccaagcgcgc aattaaccct cactaaaggg aacaaaagct ggagctccac 61 cgcggtggcg gccgctctag aactagtgga tctgctgggc ttgctgaagg tagggggtca 121 agaaccagag gtgtaaagta cttgagtaat tttacttgat tactgtactt aagtattatt 181 tttggggatt tttactttac ttgagtacaa ttaaaaatca atacttttac ttttacttaa 241 ttacattttt ttagaaaaaa aagtactttt tactccttac aattttattt acagtcaaaa 301 agtacttatt ttttggagat cacttgggcc cggctcgagc agggggatca tctaatcaag 361 cacaaataag gggcgtgtaa cacaaaagcc agcgaccctt tccaatgcaa atcaaacttg 421 caattctttg ccgtttttat catttaagtg tcggcttaag gtccactatc agatgtaaac 481 agccttatct aacaaaggta tcattacatt ctgaaattct caggcatgca agctagctta 541 tgacgcacta gggagtgcca cccttccttt cgccctaact tcgtgataac tcgcgcgttt 601 cactcaacag ctgcatccgc cctagtgcta ctgggagttg tagtatacaa gacgcttaca 661 ggctgaatgt tctgtcaaga ccccgcctct agcactttgg gaattctgga cttgatgatg 721 tcatggttaa tccccgccca gtagaggcgg ctatataaag ggtggttaag gcccggttcg 781 ctctcttcct caccgggtct gcggcgagtt ctagctgaag cttcctgcag gtcgaccgat 841 cctgagaact tcagggtgag tttggggacc cttgattgtt ctttcttttt cgctattgta 901 aaattcatgt tatatggagg gggcaaagtt ttcagggtgt tgtttagaat gggaagatgt 961 cccttgtatc accatggacc ctcatgataa ttttgtttct ttcactttct actctgttga 1021 caaccattgt ctcctcttat tttcttttca ttttctgtaa ctttttcgtt aaactttagc 1081 ttgcatttgt aacgaatttt taaattcact tttgtttatt tgtcagattg taagtacttt 1141 ctctaatcac ttttttttca aggcaatcag ggtatattat attgtacttc agcacagttt 1201 tagagaacaa ttgttataat taaatgataa ggtagaatat ttctgcatat aaattctggc 1261 tggcgtggaa atattcttat tggtagaaac aactacaccc tggtcatcat cctgcctttc 1321 tctttatggt tacaatgata tacactgttt gagatgagga taaaatactc tgagtccaaa 1381 ccgggcccct ctgctaacca tgttcatgcc ttcttctctt tcctacagct cctgggcaac 1441 gtgctggttg ttgtgctgtc tcatcatttt ggcaaagaat tcctcgacgg atccaccatg 1501 gtgagcaagg gcgaggagct gttcaccggg gtggtgccca tcctggtcga gctggacggc 1561 gacgtaaacg gccacaagtt cagcgtgtcc ggcgagggcg agggcgatgc cacctacggc 1621 aagctgaccc tgaagttcat ctgcaccacc ggcaagctgc ccgtgccctg gcccaccctc 1681 gtgaccaccc tgacctacgg cgtgcagtgc ttcagccgct accccgacca catgaagcag 1741 cacgacttct tcaagtccgc catgcccgaa ggctacgtcc aggagcgcac catcttcttc 1801 aaggacgacg gcaactacaa gacccgcgcc gaggtgaagt tcgagggcga caccctggtg 1861 aaccgcatcg agctgaaggg catcgacttc aaggaggacg gcaacatcct ggggcacaag 1921 ctggagtaca actacaacag ccacaacgtc tatatcatgg ccgacaagca gaagaacggc 1981 atcaaggtga acttcaagat ccgccacaac atcgaggacg gcagcgtgca gctcgccgac 2041 cactaccagc agaacacccc catcggcgac ggccccgtgc tgctgcccga caaccactac 2101 ctgagcaccc agtccgccct gagcaaagac cccaacgaga agcgcgatca catggtcctg 2161 ctggagttcg tgaccgccgc cgggatcact ctcggcatgg acgagctgta caagggtggc 2221 ggtggctcgg gcggtggtgg gtcgggtggc ggcggatcta tcgatgaatt gaattccatg 2281 ccgtccgaga agaccttcaa acagcgccgg agcttcgaac aaagagtgga agatgtccgg 2341 ctcatccggg agcagcaccc caccaagatc ccagtgatta tagagcgata caagggtgag 2401 aagcagctgc ccgtcctgga caagaccaag ttccttgtac ctgatcacgt gaatatgagc 2461 gaactcatca agataattag aaggcgcctg cagctcaatg ctaaccaagc cttcttcctc 2521 ctggtgaatg ggcacagcat ggtgagtgtg tccacaccca tctctgaagt gtacgagagc 2581 gagagagatg aagacggctt cctgtacatg gtctatgcct cccaggagac gttcgggatg 2641 gcctcctccg aggacgtcat caaggagttc atgcgcttca aggtgcgcat ggagggctcc 2701 gtgaacggcc acgagttcga gatcgagggc gagggcgagg gccgccccta cgagggcacc 2761 cagaccgcca agctgaaggt gaccaagggc ggccccctgc ccttcgcctg ggacatcctg 2821 tcccctcagt tccagtacgg ctccaaggcc tacgtgaagc accccgccga catccccgac 2881 tacttgaagc tgtccttccc cgagggcttc aagtgggagc gcgtgatgaa cttcgaggac 2941 ggcggcgtgg tgaccgtgac ccaggactcc tccctgcagg acggcgagtt catctacaag 3001 gtgaagctgc gcggcaccaa cttcccctcc gacggccccg taatgcagaa gaagaccatg 3061 ggctgggagg cctccaccga gcggatgtac cccgaggacg gcgccctgaa gggcgagatc 3121 aagatgaggc tgaagctgaa ggacggcggc cactacgacg ccgaggtcaa gaccacctac 3181 atggccaaga agcccgtgca gctgcccggc gcctacaaga ccgacatcaa gctggacatc 3241 acctcccaca acgaggacta caccatcgtg gaacagtacg agcgcgccga gggccgccac 3301 tccaccggcg ccatcgatga attgaattcc atgccgtccg agaagacctt caaacagcgc 3361 cggagcttcg aacaaagagt ggaagatgtc cggctcatcc gggagcagca ccccaccaag 3421 atcccagtga ttatagagcg atacaagggt gagaagcagc tgcccgtcct ggacaagacc 3481 aagttccttg tacctgatca cgtgaatatg agcgaactca tcaagataat tagaaggcgc 3541 ctgcagctca atgctaacca agccttcttc ctcctggtga atgggcacag catggtgagt 3601 gtgtccacac ccatctctga agtgtacgag agcgagagag atgaagacgg cttcctgtac 3661 atggtctatg cctcccagga gacgttctaa ggatcccccg ggtcgcgaat cgatgatgat 3721 ccagacatga taagatacat tgatgagttt ggacaaacca caactagaat gcagtgaaaa 3781 aaatgcttta tttgtgaaat ttgtgatgct attgctttat ttgtaaccat tataagctgc 3841 aataaacaag ttaacaacaa caattgcatt cattttatgt ttcaggttca gggggaggtg 3901 tgggaggttt tttaaagcaa gtaaaacctc tacaaatgtg gtatggctga ttatgatcct 3961 ctagatcaga tctaatactc aagtacaatt ttaatggagt acttttttac ttttactcaa 4021 gtaagattct agccagatac ttttactttt aattgagtaa aattttccct aagtacttgt 4081 actttcactt gagtaaaatt tttgagtact ttttacacct ctgtcaagaa ccatatgccg 4141 gtacccaatt cgccctatag tgagtcgtat tacgcgcgct cactggccgt cgttttacaa 4201 cgtcgtgact gggaaaaccc tggcgttacc caacttaatc gccttgcagc acatccccct 4261 ttcgccagct ggcgtaatag cgaagaggcc cgcaccgatc gcccttccca acagttgcgc 4321 agcctgaatg gcgaatggga cgcgccctcc gtcaagctct aaatcggggg ctccctttag 4381 ggttccgatt tagtgcttta cggcacctcg accccaaaaa acttgattag ggtgatggtt 4441 cacgtagtgg gccatcgccc tgatagacgg tttttcgccc tttgacgttg gagtccacgt 4501 tctttaatag tggactcttg ttccaaactg gaacaacact caaccctatc tcggtctatt 4561 cttttgattt ataagggatt ttgccgattt cggcctattg gttaaaaaat gagctgattt 4621 aacaaaaatt taacgcgaat tttaacaaaa tattaacgct tacaatttag gtggcacttt 4681 tcggggaaat gtgcgcggaa cccctatttg tttatttttc taaatacatt caaatatgta 4741 tccgctcatg agacaataac cctgataaat gcttcaataa tattgaaaaa ggaagagtat 4801 gagtattcaa catttccgtg tcgcccttat tccctttttt gcggcatttt gccttcctgt 4861 ttttgctcac ccagaaacgc tggtgaaagt aaaagatgct gaagatcagt tgggtgcacg 4921 agtgggttac atcgaactgg atctcaacag cggtaagatc cttgagagtt ttcgccccga 4981 agaacgtttt ccaatgatga gcacttttaa agttctgcta tgtggcgcgg tattatcccg 5041 tattgacgcc gggcaagagc aactcggtcg ccgcatacac tattctcaga atgacttggt 5101 tgagtactca ccagtcacag aaaagcatct tacggatggc atgacagtaa gagaattatg 5161 cagtgctgcc ataaccatga gtgataacac tgcggccaac ttacttctga caacgatcgg 5221 aggaccgaag gagctaaccg cttttttgca caacatgggg gatcatgtaa ctcgccttga 5281 tcgttgggaa ccggagctga atgaagccat accaaacgac gagcgtgaca ccacgatgcc 5341 tgtagcaatg gcaacaacgt tgcgcaaact attaactggc gaactactta ctctagcttc 5401 ccggcaacaa ttaatagact ggatggaggc ggataaagtt gcaggaccac ttctgcgctc 5461 ggcccttccg gctggctggt ttattgctga taaatctgga gccggtgagc gtgggtctcg 5521 cggtatcatt gcagcactgg ggccagatgg taagccctcc cgtatcgtag ttatctacac 5581 gacggggagt caggcaacta tggatgaacg aaatagacag atcgctgaga taggtgcctc 5641 actgattaag cattggtaac tgtcagacca agtttactca tatatacttt agattgattt 5701 aaaacttcat ttttaattta aaaggatcta ggtgaagatc ctttttgata atctcatgac 5761 caaaatccct taacgtgagt tttcgttcca ctgagcgtca gaccccgtag aaaagatcaa 5821 aggatcttct tgagatcctt tttttctgcg cgtaatctgc tgcttgcaaa caaaaaaacc 5881 accgctacca gcggtggttt gtttgccgga tcaagagcta ccaactcttt ttccgaaggt 5941 aactggcttc agcagagcgc agataccaaa tactgttctt ctagtgtagc cgtagttagg 6001 ccaccacttc aagaactctg tagcaccgcc tacatacctc gctctgctaa tcctgttacc 6061 agtggctgct gccagtggcg ataagtcgtg tcttaccggg ttggactcaa gacgatagtt 6121 accggataag gcgcagcggt cgggctgaac ggggggttcg tgcacacagc ccagcttgga 6181 gcgaacgacc tacaccgaac tgagatacct acagcgtgag ctatgagaaa gcgccacgct 6241 tcccgaaggg agaaaggcgg acaggtatcc ggtaagcggc agggtcggaa caggagagcg 6301 cacgagggag cttccagggg gaaacgcctg gtatctttat agtcctgtcg ggtttcgcca 6361 cctctgactt gagcgtcgat ttttgtgatg ctcgtcaggg gggcggagcc tatggaaaaa 6421 cgccagcaac gcggcctttt tacggttcct ggccttttgc tggccttttg ctcacatgtt 6481 ctttcctgcg ttatcccctg attctgtgga taaccgtatt accgcctttg agtgagctga 6541 taccgctcgc cgcagccgaa cgaccgagcg cagcgagtca gtgagcgagg aagcggaaga 6601 gcgcccaata cgcaaaccgc ctctccccgc gcgttggccg attcattaat gcagctggca 6661 cgacaggttt cccgactgga aagcgggcag tgagcgcaac gcaattaatg tgagttagct 6721 cactcattag gcaccccagg ctttacactt tatgcttccg gctcgtatgt tgtgtggaat 6781 tgtgagcgga taacaatttc acacaggaaa cagctatgac //