LOCUS Exported 7895 bp ds-DNA circular SYN 12-MAY-2021 DEFINITION targeting osTIR1-GFP into mouse Actb locus for auxin inducible degradation. ACCESSION . VERSION . KEYWORDS PBS Actb-2A-TIR1-GFP SOURCE synthetic DNA construct ORGANISM synthetic DNA construct REFERENCE 1 (bases 1 to 7895) AUTHORS Gu B, Posfai E, Rossant J TITLE Efficient generation of targeted large insertions by microinjection into two-cell-stage mouse embryos. JOURNAL Nat Biotechnol. 2018 Jun 11. pii: nbt.4166. doi: 10.1038/nbt.4166. PUBMED 29889212 REFERENCE 2 (bases 1 to 7895) AUTHORS . TITLE Direct Submission JOURNAL Exported May 12, 2021 from SnapGene Server 1.1.58 http://www.snapgene.com FEATURES Location/Qualifiers source 1..7895 /organism="synthetic DNA construct" /mol_type="other DNA" primer_bind complement(16..35) /label=F1ori-R /note="F1 origin, reverse primer" primer_bind 226..247 /label=F1ori-F /note="F1 origin, forward primer" promoter 410..514 /gene="bla" /label=AmpR promoter CDS 515..1375 /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(733..752) /label=Amp-R /note="Ampicillin resistance gene, reverse primer" rep_origin 1546..2134 /direction=RIGHT /label=ori /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of replication" primer_bind 2035..2054 /label=pBR322ori-F /note="pBR322 origin, forward primer" primer_bind 2288..2305 /label=L4440 /note="L4440 vector, forward primer" protein_bind 2422..2443 /label=CAP binding site /bound_moiety="E. coli catabolite activator protein" /note="CAP binding activates transcription in the presence of cAMP." promoter 2458..2488 /label=lac promoter /note="promoter for the E. coli lac operon" protein_bind 2496..2512 /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 2501..2523 /label=M13/pUC Reverse /note="In lacZ gene" primer_bind 2520..2536 /label=M13 rev /note="common sequencing primer, one of multiple similar variants" primer_bind 2520..2536 /label=M13 Reverse /note="In lacZ gene. Also called M13-rev" primer_bind 2555..2575 /label=T3 /note="T3 promoter, forward primer" promoter 2557..2575 /label=T3 promoter /note="promoter for bacteriophage T3 RNA polymerase" primer_bind 2604..2620 /label=pBluescriptKS /note="For pBluescript vector" primer_bind 2605..2621 /label=KS primer /note="common sequencing primer, one of multiple similar variants" CDS 3700..3753 /codon_start=1 /product="2A peptide from Thosea asigna virus capsid protein" /label=T2A /note="Eukaryotic ribosomes fail to insert a peptide bond between the Gly and Pro residues, yielding separate polypeptides." /translation="EGRGSLLTCGDVEENPGP" CDS 3760..5484 /codon_start=1 /product="rice transport inhibitor response 1 protein" /label=OsTIR1 /note="F-box protein that mediates auxin-dependent interaction of a ubiquitin ligase with an auxin-inducible degron (Tan et al., 2007)" /translation="MTYFPEEVVEHIFSFLPAQRDRNTVSLVCKVWYEIERLSRRGVFV GNCYAVRAGRVAARFPNVRALTVKGKPHFADFNLVPPDWGGYAGPWIEAAARGCHGLEE LRMKRMVVSDESLELLARSFPRFRALVLISCEGFSTDGLAAVASHCKLLRELDLQENEV EDRGPRWLSCFPDSCTSLVSLNFACIKGEVNAGSLERLVSRSPNLRSLRLNRSVSVDTL AKILLRTPNLEDLGTGNLTDDFQTESYFKLTSALEKCKMLRSLSGFWDASPVCLSFIYP LCAQLTGLNLSYAPTLDASDLTKMISRCVKLQRLWVLDCISDKGLQVVASSCKDLQELR VFPSDFYVAGYSAVTEEGLVAVSLGCPKLNSLLYFCHQMTNAALVTVAKNCPNFTRFRL CILEPGKPDVVTSQPLDEGFGAIVRECKGLQRLSISGLLTDKVFMYIGKYAKQLEMLSI AFAGDSDKGMMHVMNGCKNLRKLEIRDSPFGDAALLGNFARYETMRSLWMSSCNVTLKG CQVLASKMPMLNVEVINERDGSNEMEENHGDLPKVEKLYVYRTTAGARDDAPNFVKIL" CDS 5512..5541 /codon_start=1 /product="Myc (human c-Myc proto-oncogene) epitope tag" /label=Myc /translation="EQKLISEEDL" CDS 5548..5577 /codon_start=1 /product="Myc (human c-Myc proto-oncogene) epitope tag" /label=Myc /translation="EQKLISEEDL" CDS 5605..6318 /codon_start=1 /product="enhanced GFP" /label=EGFP /translation="VSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTLK FICTTGKLPVPWPTLVTTLTYGVQCFSRYPDHMKQHDFFKSAMPEGYVQERTIFFKDDG NYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIKV NFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSALSKDPNEKRDHMVLLE FVTAAGITLGMDELYK" primer_bind complement(5647..5668) /label=EGFP-N /note="EGFP, reverse primer" primer_bind complement(5908..5927) /label=EXFP-R /note="For distinguishing EGFP variants, reverse primer" primer_bind 6255..6276 /label=EGFP-C /note="EGFP, forward primer" primer_bind complement(7636..7655) /label=T7 /note="T7 promoter, forward primer" promoter complement(7637..7655) /label=T7 promoter /note="promoter for bacteriophage T7 RNA polymerase" primer_bind complement(7665..7682) /label=M13 Forward /note="In lacZ gene. Also called M13-F20 or M13 (-21) Forward" primer_bind complement(7665..7681) /label=M13 fwd /note="common sequencing primer, one of multiple similar variants" primer_bind complement(7674..7696) /label=M13/pUC Forward /note="In lacZ gene" rep_origin 7823..384 /direction=RIGHT /label=f1 ori /note="f1 bacteriophage origin of replication; arrow indicates direction of (+) strand synthesis" ORIGIN 1 gcgccctagc gcccgctcct ttcgctttct tcccttcctt tctcgccacg ttcgccggct 61 ttccccgtca agctctaaat cgggggctcc ctttagggtt ccgatttagt gctttacggc 121 acctcgaccc caaaaaactt gattagggtg atggttcacg tagtgggcca tcgccctgat 181 agacggtttt tcgccctttg acgttggagt ccacgttctt taatagtgga ctcttgttcc 241 aaactggaac aacactcaac cctatctcgg tctattcttt tgatttataa gggattttgc 301 cgatttcggc ctattggtta aaaaatgagc tgatttaaca aaaatttaac gcgaatttta 361 acaaaatatt aacgcttaca atttaggtgg cacttttcgg ggaaatgtgc gcggaacccc 421 tatttgttta tttttctaaa tacattcaaa tatgtatccg ctcatgagac aataaccctg 481 ataaatgctt caataatatt gaaaaaggaa gagtatgagt attcaacatt tccgtgtcgc 541 ccttattccc ttttttgcgg cattttgcct tcctgttttt gctcacccag aaacgctggt 601 gaaagtaaaa gatgctgaag atcagttggg tgcacgagtg ggttacatcg aactggatct 661 caacagcggt aagatccttg agagttttcg ccccgaagaa cgttttccaa tgatgagcac 721 ttttaaagtt ctgctatgtg gcgcggtatt atcccgtatt gacgccgggc aagagcaact 781 cggtcgccgc atacactatt ctcagaatga cttggttgag tactcaccag tcacagaaaa 841 gcatcttacg gatggcatga cagtaagaga attatgcagt gctgccataa ccatgagtga 901 taacactgcg gccaacttac ttctgacaac gatcggagga ccgaaggagc taaccgcttt 961 tttgcacaac atgggggatc atgtaactcg ccttgatcgt tgggaaccgg agctgaatga 1021 agccatacca aacgacgagc gtgacaccac gatgcctgta gcaatggcaa caacgttgcg 1081 caaactatta actggcgaac tacttactct agcttcccgg caacaattaa tagactggat 1141 ggaggcggat aaagttgcag gaccacttct gcgctcggcc cttccggctg gctggtttat 1201 tgctgataaa tctggagccg gtgagcgtgg gtctcgcggt atcattgcag cactggggcc 1261 agatggtaag ccctcccgta tcgtagttat ctacacgacg gggagtcagg caactatgga 1321 tgaacgaaat agacagatcg ctgagatagg tgcctcactg attaagcatt ggtaactgtc 1381 agaccaagtt tactcatata tactttagat tgatttaaaa cttcattttt aatttaaaag 1441 gatctaggtg aagatccttt ttgataatct catgaccaaa atcccttaac gtgagttttc 1501 gttccactga gcgtcagacc ccgtagaaaa gatcaaagga tcttcttgag atcctttttt 1561 tctgcgcgta atctgctgct tgcaaacaaa aaaaccaccg ctaccagcgg tggtttgttt 1621 gccggatcaa gagctaccaa ctctttttcc gaaggtaact ggcttcagca gagcgcagat 1681 accaaatact gtccttctag tgtagccgta gttaggccac cacttcaaga actctgtagc 1741 accgcctaca tacctcgctc tgctaatcct gttaccagtg gctgctgcca gtggcgataa 1801 gtcgtgtctt accgggttgg actcaagacg atagttaccg gataaggcgc agcggtcggg 1861 ctgaacgggg ggttcgtgca cacagcccag cttggagcga acgacctaca ccgaactgag 1921 atacctacag cgtgagctat gagaaagcgc cacgcttccc gaagggagaa aggcggacag 1981 gtatccggta agcggcaggg tcggaacagg agagcgcacg agggagcttc cagggggaaa 2041 cgcctggtat ctttatagtc ctgtcgggtt tcgccacctc tgacttgagc gtcgattttt 2101 gtgatgctcg tcaggggggc ggagcctatg gaaaaacgcc agcaacgcgg cctttttacg 2161 gttcctggcc ttttgctggc cttttgctca catgttcttt cctgcgttat cccctgattc 2221 tgtggataac cgtattaccg cctttgagtg agctgatacc gctcgccgca gccgaacgac 2281 cgagcgcagc gagtcagtga gcgaggaagc ggaagagcgc ccaatacgca aaccgcctct 2341 ccccgcgcgt tggccgattc attaatgcag ctggcacgac aggtttcccg actggaaagc 2401 gggcagtgag cgcaacgcaa ttaatgtgag ttagctcact cattaggcac cccaggcttt 2461 acactttatg cttccggctc gtatgttgtg tggaattgtg agcggataac aatttcacac 2521 aggaaacagc tatgaccatg attacgccaa gcgcgcaatt aaccctcact aaagggaaca 2581 aaagctgggt accgggcccc ccctcgaggt cgacggtatc gataagcttc agtttcacct 2641 gccctgagtg tttcttgtgg ctttctgaac ttgacaacat tatttatttt tctctacaga 2701 tcatgtttga gaccttcaac accccagcca tgtacgtagc catccaggct gtgctgtccc 2761 tgtatgcctc tggtcgtacc acaggcattg tgatggactc cggagacggg gtcacccaca 2821 ctgtgcccat ctacgagggc tatgctctcc ctcacgccat cctgcgtctg gacctggctg 2881 gccgggacct gacagactac ctcatgaaga tcctgaccga gcgtggctac agcttcacca 2941 ccacagctga gagggaaatc gtgcgtgaca tcaaagagaa gctgtgctat gttgctctag 3001 acttcgagca ggagatggcc actgccgcat cctcttcctc cctggagaag agctatgagc 3061 tgcctgacgg ccaggtcatc actattggca acgagcggtt ccgatgccct gaggctcttt 3121 tccagccttc cttcttgggt aagttgtagc ctagtccttt ctccatctaa aggtgacaaa 3181 actcctgagg ccatagtaca agttaagtct gatttctgtc actcttctct taggtatgga 3241 atcctgtggc atccatgaaa ctacattcaa ttccatcatg aagtgtgacg ttgacatccg 3301 taaagacctc tatgccaaca cagtgctgtc tggtggtacc accatgtacc caggcattgc 3361 tgacaggatg cagaaggaga ttactgctct ggctcctagc accatgaaga tcaaggtaag 3421 ctaagcatcc ttagcttggt gagggtgggc cctgtggttg tcagagcaac cttctaggtt 3481 taaggggaat cccagcaccc agagagctca ccattcacca tcttgtcttg ctttcttcag 3541 atcattgctc ctcctgagcg caagtactct gtgtggatcg gtggctccat cctggcctca 3601 ctgtccacct tccagcagat gtggatcagc aagcaggagt acgatgagtc cggcccctcc 3661 atcgtgcacc gcaagtgctt caagcttgat atcgaattcg agggcagagg aagtcttcta 3721 acatgcggtg acgtggagga gaatcccggc cctaccggta tgacgtactt cccggaggag 3781 gtggtggagc acatcttcag cttcctgccg gcgcagcgcg accgcaacac ggtctcgctc 3841 gtctgcaagg tgtggtacga gatcgagagg ctgagccgcc gcggcgtctt cgtgggcaac 3901 tgctacgccg tgcgcgccgg ccgcgtcgcc gcgcggttcc ccaacgtgcg ggcgctcacg 3961 gtgaagggga agccccactt cgccgacttc aacctcgtgc cccccgactg gggcggctac 4021 gcggggccgt ggatcgaggc ggccgcgagg ggatgccacg gcctggagga gctcaggatg 4081 aagcggatgg tggtgtccga cgagagcctc gagctgctgg ctcgctcgtt cccgcggttc 4141 agggctcttg ttcttatcag ctgcgagggg ttcagcactg acgggctagc cgccgtcgcg 4201 agccattgca agcttctgag ggagttggat ttgcaggaaa atgaagtgga ggatcgaggg 4261 cctaggtggc tttcctgctt ccctgattcc tgcacatcac ttgtctcatt gaattttgcc 4321 tgcatcaaag gggaggttaa tgctggttca ctggagagac ttgttagcag gtccccaaac 4381 ctgcggagtt tgaggctgaa tcgatctgta tcggtagata cacttgcaaa gatactactg 4441 cgtaccccta acttggagga tttggggaca gggaatttga cagatgactt ccaaactgag 4501 tcctacttta agcttaccag tgctctggag aaatgcaaga tgttgaggag tttgtctgga 4561 ttctgggatg cttctcctgt ttgcctgtca tttatctacc ccctgtgtgc tcaactgaca 4621 ggattgaact tgagctatgc ccccacactt gatgcttctg accttacaaa aatgattagc 4681 cgctgtgtga agctccaacg cctttgggta ctggattgta tctcggacaa aggcttgcaa 4741 gtggtggcct ccagttgcaa agacttgcaa gaactcaggg tatttccatc agatttctac 4801 gtagctggtt attctgcagt gacagaggag ggacttgttg cagtatcctt gggctgtcca 4861 aaactgaact cactactgta cttctgtcac caaatgacta atgctgcact agttactgtc 4921 gccaagaact gtccaaattt cacacgattc agactttgta ttcttgagcc agggaagcct 4981 gatgttgtga caagccaacc attagatgaa ggctttggag ctattgttcg tgagtgcaag 5041 ggattacaac gtttgtcaat atctggtctt ctcacagaca aagttttcat gtatattggg 5101 aaatatgcaa aacaacttga gatgctttct atagcatttg ctggtgacag tgataagggt 5161 atgatgcatg ttatgaatgg atgcaagaat ttaaggaaac tggagataag agatagcccg 5221 tttggtgatg ctgcactctt ggggaatttt gctaggtacg agacaatgcg atccctttgg 5281 atgtcatctt gcaatgtcac gttaaagggg tgccaagtcc ttgcgtcaaa gatgccgatg 5341 ctcaatgttg aggtcataaa tgagcgggat ggtagcaatg aaatggagga aaaccatgga 5401 gatctgccta aagtggagaa attatatgtg taccgcacaa ctgctggggc gagggatgat 5461 gcaccaaatt ttgttaaaat cctagtcgac tccggttctg ctgctagtgg tgaacaaaag 5521 ttgatttctg aagaagattt gaacggtgaa caaaagctaa tctccgagga agacttgaac 5581 ggtgaaggta gcggcagcgg tagcgtgagc aagggcgagg agctgttcac cggggtggtg 5641 cccatcctgg tcgagctgga cggcgacgta aacggccaca agttcagcgt gtccggcgag 5701 ggcgagggcg atgccaccta cggcaagctg accctgaagt tcatctgcac caccggcaag 5761 ctgcccgtgc cctggcccac cctcgtgacc accctgacct acggcgtgca gtgcttcagc 5821 cgctaccccg accacatgaa gcagcacgac ttcttcaagt ccgccatgcc cgaaggctac 5881 gtccaggagc gcaccatctt cttcaaggac gacggcaact acaagacccg cgccgaggtg 5941 aagttcgagg gcgacaccct ggtgaaccgc atcgagctga agggcatcga cttcaaggag 6001 gacggcaaca tcctggggca caagctggag tacaactaca acagccacaa cgtctatatc 6061 atggccgaca agcagaagaa cggcatcaag gtgaacttca agatccgcca caacatcgag 6121 gacggcagcg tgcagctcgc cgaccactac cagcagaaca cccccatcgg cgacggcccc 6181 gtgctgctgc ccgacaacca ctacctgagc acccagtccg ccctgagcaa agaccccaac 6241 gagaagcgcg atcacatggt cctgctggag ttcgtgaccg ccgccgggat cactctcggc 6301 atggacgagc tgtacaagtg agcggactgt tactgagctg cgttttacac cctttctttg 6361 acaaaaccta acttgcgcag aaaaaaaaaa aataagagac aacattggca tggctttgtt 6421 tttttaaatt ttttttaaag tttttttttt tttttttttt ttttttttta agtttttttg 6481 ttttgttttg gcgcttttga ctcaggattt aaaaactgga acggtgaagg cgacagcagt 6541 tggttggagc aaacatcccc caaagttcta caaatgtggc tgaggacttt gtacattgtt 6601 ttgttttttt ttttttttgg ttttgtcttt ttttaatagt cattccaagt atccatgaaa 6661 taagtggtta caggaagtcc ctcaccctcc caaaagccac ccccactcct aagaggagga 6721 tggtcgcgtc catgccctga gtccaccccg gggaaggtga cagcattgct tctgtgtaaa 6781 ttatgtactg caaaaatttt tttaaatctt ccgccttaat acttcatttt tgtttttaat 6841 ttctgaatgg cccaggtctg aggcctccct tttttttgtc cccccaactt gatgtatgaa 6901 ggctttggtc tccctgggag ggggttgagg tgttgaggca gccagggctg gcctgtacac 6961 tgacttgaga ccaataaaag tgcacacctt accttacaca aacagcttgt ggctctgtgg 7021 ctttgctggg tgtggggagc aggttgggtg ggtgtggagc tctattgggg ggggcatcta 7081 gggtgggcta ggccttgctg atggtatcta gtgggagggc tacaggccct ttaatgaagt 7141 ctcacaaata accacacctt cagactgtct tgaagagttg aaaaggtact cctccagaaa 7201 cctaggccaa gttggcctgc actaacacta ccttcctcaa ccgaggggtg tccacaccct 7261 ttgcctccat ctgcataact gggtttcaag cccctctgct agtctgaagc tgccctttcc 7321 agagctgtga ctgacagcag gaaggtgtga agcctatggc ttccttccca gggatgggca 7381 tcttttccct ggttttgttt tgagagccta ggactgagga acacctcaga gggaggaagg 7441 gggtcatcct ggaaatcagc cccttccaca tggcacgcag ctccagcctt tgtcagtcgt 7501 gatccttgga cttgggtgcc gcaagttagc tttgactggg atcatttgtt cacacatgca 7561 gagaacactg gttggttgag tcctagtcat ctccatccag ctatgcggcc gccaccgcgg 7621 tggagctcca attcgcccta tagtgagtcg tattacgcgc gctcactggc cgtcgtttta 7681 caacgtcgtg actgggaaaa ccctggcgtt acccaactta atcgccttgc agcacatccc 7741 cctttcgcca gctggcgtaa tagcgaagag gcccgcaccg atcgcccttc ccaacagttg 7801 cgcagcctga atggcgaatg ggacgcgccc tgtagcggcg cattaagcgc ggcgggtgtg 7861 gtggttacgc gcagcgtgac cgctacactt gccag //