LOCUS Exported 5005 bp ds-DNA circular SYN 12-MAY-2021 DEFINITION pMVP L5-L4 entry plasmid, contains Tir1-P2A-AID for 4-component MultiSite Gateway Pro assembly. Allows expression of N-term Tir1 linked by P2A to AID domain fused to gene of interest.. ACCESSION . VERSION . KEYWORDS JE301: pMVP (L5-L4) Tir1-P2A-AID SOURCE synthetic DNA construct ORGANISM synthetic DNA construct REFERENCE 1 (bases 1 to 5005) AUTHORS Haldeman JM, Conway AE, Arlotto ME, Slentz DH, Muoio DM, Becker TC, Newgard CB TITLE Creation of versatile cloning platforms for transgene expression and dCas9-based epigenome editing. JOURNAL Nucleic Acids Res. 2018 Dec 27. pii: 5264291. doi: 10.1093/nar/gky1286. PUBMED 30590691 REFERENCE 2 (bases 1 to 5005) AUTHORS . TITLE Direct Submission JOURNAL Exported May 12, 2021 from SnapGene Server 1.1.58 http://www.snapgene.com FEATURES Location/Qualifiers source 1..5005 /organism="synthetic DNA construct" /mol_type="other DNA" primer_bind 92..109 /label=L4440 /note="L4440 vector, forward primer" terminator 268..295 /label=rrnB T2 terminator /note="transcription terminator T2 from the E. coli rrnB gene" terminator 387..473 /gene="Escherichia coli rrnB" /label=rrnB T1 terminator /note="transcription terminator T1 from the E. coli rrnB gene" primer_bind 522..544 /label=M13/pUC Forward /note="In lacZ gene" primer_bind 536..553 /label=M13 Forward /note="In lacZ gene. Also called M13-F20 or M13 (-21) Forward" primer_bind 537..553 /label=M13 fwd /note="common sequencing primer, one of multiple similar variants" protein_bind 570..665 /gene="mutant version of attL" /label=attL5 /bound_moiety="LR Clonase(TM)" /note="recombination site for the Gateway(R) LR reaction" regulatory 668..677 /regulatory_class="other" /note="vertebrate consensus sequence for strong initiation of translation (Kozak, 1987)" CDS 722..2443 /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="TYFPEEVVEHIFSFLPAQRDRNTVSLVCKVWYEIERLSRRGVFVG NCYAVRAGRVAARFPNVRALTVKGKPHFADFNLVPPDWGGYAGPWIEAAARGCHGLEEL RMKRMVVSDESLELLARSFPRFRALVLISCEGFSTDGLAAVASHCKLLRELDLQENEVE DRGPRWLSCFPDSCTSLVSLNFACIKGEVNAGSLERLVSRSPNLRSLRLNRSVSVDTLA KILLRTPNLEDLGTGNLTDDFQTESYFKLTSALEKCKMLRSLSGFWDASPVCLSFIYPL CAQLTGLNLSYAPTLDASDLTKMISRCVKLQRLWVLDCISDKGLQVVASSCKDLQELRV FPSDFYVAGYSAVTEEGLVAVSLGCPKLNSLLYFCHQMTNAALVTVAKNCPNFTRFRLC ILEPGKPDVVTSQPLDEGFGAIVRECKGLQRLSISGLLTDKVFMYIGKYAKQLEMLSIA FAGDSDKGMMHVMNGCKNLRKLEIRDSPFGDAALLGNFARYETMRSLWMSSCNVTLKGC QVLASKMPMLNVEVINERDGSNEMEENHGDLPKVEKLYVYRTTAGARDDAPNFVKIL" CDS 2471..2500 /codon_start=1 /product="Myc (human c-Myc proto-oncogene) epitope tag" /label=Myc /translation="EQKLISEEDL" CDS 2507..2536 /codon_start=1 /product="Myc (human c-Myc proto-oncogene) epitope tag" /label=Myc /translation="EQKLISEEDL" CDS 2543..2572 /codon_start=1 /product="Myc (human c-Myc proto-oncogene) epitope tag" /label=Myc /translation="EQKLISEEDL" CDS 2591..2620 /codon_start=1 /product="Myc (human c-Myc proto-oncogene) epitope tag" /label=Myc /translation="EQKLISEEDL" CDS 2627..2656 /codon_start=1 /product="Myc (human c-Myc proto-oncogene) epitope tag" /label=Myc /translation="EQKLISEEDL" CDS 2663..2692 /codon_start=1 /product="Myc (human c-Myc proto-oncogene) epitope tag" /label=Myc /translation="EQKLISEEDL" CDS 2711..2740 /codon_start=1 /product="Myc (human c-Myc proto-oncogene) epitope tag" /label=Myc /translation="EQKLISEEDL" CDS 2747..2776 /codon_start=1 /product="Myc (human c-Myc proto-oncogene) epitope tag" /label=Myc /translation="EQKLISEEDL" CDS 2783..2812 /codon_start=1 /product="Myc (human c-Myc proto-oncogene) epitope tag" /label=Myc /translation="EQKLISEEDL" CDS 2846..2902 /codon_start=1 /product="2A peptide from porcine teschovirus-1 polyprotein" /label=P2A /note="Eukaryotic ribosomes fail to insert a peptide bond between the Gly and Pro residues, yielding separate polypeptides." /translation="ATNFSLLKQAGDVEENPGP" CDS 2903..3106 /codon_start=1 /product="minimal degron from the auxin-responsive IAA17 protein of Arabidopsis thaliana (Kubota et al., 2013)" /label=mini-AID /note="acts as an auxin-inducible degron (AID) in the presence of TIR1" /translation="KEKSACPKDPAKPPAKAQVVGWPPVRSYRKNVMVSCQKSSGGPEA AAFVKVSMDGAPYLRKIDLRMYK" protein_bind complement(3120..3215) /gene="mutant version of attL" /label=attL4 /bound_moiety="LR Clonase(TM)" /note="recombination site for the Gateway(R) LR reaction" primer_bind complement(3246..3265) /label=T7 /note="T7 promoter, forward primer" promoter complement(3247..3265) /label=T7 promoter /note="promoter for bacteriophage T7 RNA polymerase" primer_bind complement(3270..3286) /label=M13 rev /note="common sequencing primer, one of multiple similar variants" primer_bind complement(3270..3286) /label=M13 Reverse /note="In lacZ gene. Also called M13-rev" primer_bind complement(3388..3407) /label=pENTR-R /note="pENTR vectors, reverse primer" CDS 3399..4208 /codon_start=1 /gene="aph(3')-Ia" /product="aminoglycoside phosphotransferase" /label=KanR /note="confers resistance to kanamycin in bacteria or G418 (Geneticin(R)) in eukaryotes" /translation="MSHIQRETSRPRLNSNMDADLYGYKWARDNVGQSGATIYRLYGKP DAPELFLKHGKGSVANDVTDEMVRLNWLTEFMPLPTIKHFIRTPDDAWLLTTAIPGKTA FQVLEEYPDSGENIVDALAVFLRRLHSIPVCNCPFNSDRVFRLAQAQSRMNNGLVDASD FDDERNGWPVEQVWKEMHKLLPFSPDSVVTHGDFSLDNLIFDEGKLIGCIDVGRVGIAD RYQDLAILWNCLGEFSPSLQKRLFQKYGIDNPDMNKLQFHLMLDEFF" primer_bind complement(3466..3485) /label=Kan-R /note="Kanamycin resistance gene, reverse primer" rep_origin 4355..4943 /direction=RIGHT /label=ori /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of replication" primer_bind 4844..4863 /label=pBR322ori-F /note="pBR322 origin, forward primer" ORIGIN 1 ctttcctgcg ttatcccctg attctgtgga taaccgtatt accgcctttg agtgagctga 61 taccgctcgc cgcagccgaa cgaccgagcg cagcgagtca gtgagcgagg aagcggaaga 121 gcgcccaata cgcaaaccgc ctctccccgc gcgttggccg attcattaat gcagctggca 181 cgacaggttt cccgactgga aagcgggcag tgagcgcaac gcaattaata cgcgtaccgc 241 tagccaggaa gagtttgtag aaacgcaaaa aggccatccg tcaggatggc cttctgctta 301 gtttgatgcc tggcagttta tggcgggcgt cctgcccgcc accctccggg ccgttgcttc 361 acaacgttca aatccgctcc cggcggattt gtcctactca ggagagcgtt caccgacaaa 421 caacagataa aacgaaaggc ccagtcttcc gactgagcct ttcgttttat ttgatgcctg 481 gcagttccct actctcgcgt taacgctagc atggatgttt tcccagtcac gacgttgtaa 541 aacgacggcc agtcttaagc tcgggcccca aataatgatt ttattttgac tgatagtgac 601 ctgttcgttg caacaaattg atgagcaatg cttttttata atgccaactt tgtatacaaa 661 agttgtcgcc accatggcat tacaaaaaaa attagaagaa ttagaattag atgaaggcgg 721 cacgtacttc ccggaggagg tggtggagca catcttcagc ttcctgccgg cgcagcgcga 781 ccgcaacacg gtctcgctcg tctgcaaggt gtggtacgag atcgagaggc tgagccgccg 841 cggcgtcttc gtgggcaact gctacgccgt gcgcgccggc cgcgtcgccg cgcggttccc 901 caacgtgcgg gcgctcacgg tgaaggggaa gccccacttc gccgacttca acctcgtgcc 961 ccccgactgg ggcggctacg cggggccgtg gatcgaggcg gccgcgaggg gatgccacgg 1021 cctggaggag ctcaggatga agcggatggt ggtgtccgac gagagcctcg agctgctggc 1081 tcgctcgttc ccgcggttca gggctcttgt tcttatcagc tgcgaggggt tcagcactga 1141 cgggctagcc gccgtcgcga gccattgcaa gcttctgagg gagttggatt tgcaggaaaa 1201 tgaagtggag gatcgagggc ctaggtggct ttcctgcttc cctgattcct gcacatcact 1261 tgtctcattg aattttgcct gcatcaaagg ggaggttaat gctggttcac tggagagact 1321 tgttagcagg tccccaaacc tgcggagttt gaggctgaat cgatctgtat cggtagatac 1381 acttgcaaag atactactgc gtacccctaa cttggaggat ttggggacag ggaatttgac 1441 agatgacttc caaactgagt cctactttaa gcttaccagt gctctggaga aatgcaagat 1501 gttgaggagt ttgtctggat tctgggatgc ttctcctgtt tgcctgtcat ttatctaccc 1561 cctgtgtgct caactgacag gattgaactt gagctatgca cccacacttg atgcttctga 1621 ccttacaaaa atgattagcc gctgtgtgaa gctccaacgc ctttgggtac tggattgtat 1681 ctcggacaaa ggcttgcaag tggtggcctc cagttgcaaa gacttgcaag aactcagggt 1741 atttccatca gatttctacg tagctggtta ttctgcagtg acagaggagg gacttgttgc 1801 agtatccttg ggctgtccaa aactgaactc actactgtac ttctgtcacc aaatgactaa 1861 tgctgcacta gttactgtcg ccaagaactg tccaaatttc acacgattca gactttgtat 1921 tcttgagcca gggaagcctg atgttgtgac aagccaacca ttagatgaag gctttggagc 1981 tattgttcgt gagtgcaagg gattacaacg tttgtcaata tctggtcttc tcacagacaa 2041 agttttcatg tatatcggga aatatgcaaa acaacttgag atgctttcta tagcatttgc 2101 tggtgacagt gataagggta tgatgcatgt tatgaatgga tgcaagaatt taaggaaact 2161 ggagataaga gatagcccgt ttggtgatgc tgcactcttg gggaattttg ctaggtacga 2221 gacaatgcga tccctttgga tgtcatcttg caatgtcacg ttaaaggggt gccaagtcct 2281 tgcgtcaaag atgccgatgc tcaatgttga ggtcataaat gagcgggatg gtagcaatga 2341 aatggaggaa aaccatggag atctgcctaa agtggagaaa ttatatgtgt accgcacaac 2401 tgctggggcg agggatgatg caccaaattt tgttaaaatc ctagtcgact ccggttctgc 2461 tgctagtggt gaacaaaagt tgatttctga agaagatttg aacggtgaac aaaagctaat 2521 ctccgaggaa gacttgaacg gtgaacaaaa attaatctca gaagaagact tgaacggatc 2581 ctctagaggt gaacaaaagt tgatttctga agaagatttg aacggtgaac aaaagctaat 2641 ctccgaggaa gacttgaacg gtgaacaaaa attaatctca gaagaagact tgaacggatc 2701 ctctagaggt gaacaaaagt tgatttctga agaagatttg aacggtgaac aaaagctaat 2761 ctccgaggaa gacttgaacg gtgaacaaaa attaatctca gaagaagact tgaacggatc 2821 cactagcggt acagccggat caggagctac taatttctct ctgttaaagc aggcaggaga 2881 tgtggaggag aatccaggtc ctaaggagaa gagtgcttgt cctaaagatc cagccaaacc 2941 tccggccaag gcacaagttg tgggatggcc accggtgaga tcataccgga agaacgtgat 3001 ggtttcctgc caaaaatcaa gcggtggccc ggaggcggcg gcgttcgtga aggtatcaat 3061 ggacggagca ccgtacttga ggaaaatcga tttgaggatg tataaaggca gtaccaaccc 3121 aacttttcta tacaaagttg gcattataag aaagcattgc ttatcaattt gttgcaacga 3181 acaggtcact atcagtcaaa ataaaatcat tatttgccat ccagctgcag ggcggccgcg 3241 atatccccta tagtgagtcg tattacatgg tcatagctgt ttcctggcag ctctggcccg 3301 tgtctcaaaa tctctgatgt tacattgcac aagataaaaa tatatcatca tgaacaataa 3361 aactgtctgc ttacataaac agtaatacaa ggggtgttat gagccatatt caacgggaaa 3421 cgtcgaggcc gcgattaaat tccaacatgg atgctgattt atatgggtat aaatgggctc 3481 gcgataatgt cgggcaatca ggtgcgacaa tctatcgctt gtatgggaag cccgatgcgc 3541 cagagttgtt tctgaaacat ggcaaaggta gcgttgccaa tgatgttaca gatgagatgg 3601 tcagactaaa ctggctgacg gaatttatgc ctcttccgac catcaagcat tttatccgta 3661 ctcctgatga tgcatggtta ctcaccactg cgatccccgg aaaaacagca ttccaggtat 3721 tagaagaata tcctgattca ggtgaaaata ttgttgatgc gctggcagtg ttcctgcgcc 3781 ggttgcattc gattcctgtt tgtaattgtc cttttaacag cgatcgcgta tttcgtctcg 3841 ctcaggcgca atcacgaatg aataacggtt tggttgatgc gagtgatttt gatgacgagc 3901 gtaatggctg gcctgttgaa caagtctgga aagaaatgca taaacttttg ccattctcac 3961 cggattcagt cgtcactcat ggtgatttct cacttgataa ccttattttt gacgagggga 4021 aattaatagg ttgtattgat gttggacgag tcggaatcgc agaccgatac caggatcttg 4081 ccatcctatg gaactgcctc ggtgagtttt ctccttcatt acagaaacgg ctttttcaaa 4141 aatatggtat tgataatcct gatatgaata aattgcagtt tcatttgatg ctcgatgagt 4201 ttttctaatc agaattggtt aattggttgt aacactggca gagcattacg ctgacttgac 4261 gggacggcgc aagctcatga ccaaaatccc ttaacgtgag ttacgcgtcg ttccactgag 4321 cgtcagaccc cgtagaaaag atcaaaggat cttcttgaga tccttttttt ctgcgcgtaa 4381 tctgctgctt gcaaacaaaa aaaccaccgc taccagcggt ggtttgtttg ccggatcaag 4441 agctaccaac tctttttccg aaggtaactg gcttcagcag agcgcagata ccaaatactg 4501 ttcttctagt gtagccgtag ttaggccacc acttcaagaa ctctgtagca ccgcctacat 4561 acctcgctct gctaatcctg ttaccagtgg ctgctgccag tggcgataag tcgtgtctta 4621 ccgggttgga ctcaagacga tagttaccgg ataaggcgca gcggtcgggc tgaacggggg 4681 gttcgtgcac acagcccagc ttggagcgaa cgacctacac cgaactgaga tacctacagc 4741 gtgagctatg agaaagcgcc acgcttcccg aagggagaaa ggcggacagg tatccggtaa 4801 gcggcagggt cggaacagga gagcgcacga gggagcttcc agggggaaac gcctggtatc 4861 tttatagtcc tgtcgggttt cgccacctct gacttgagcg tcgatttttg tgatgctcgt 4921 caggggggcg gagcctatgg aaaaacgcca gcaacgcggc ctttttacgg ttcctggcct 4981 tttgctggcc ttttgctcac atgtt //