LOCUS Exported 4474 bp ds-DNA circular SYN 12-MAY-2021 DEFINITION gateway entry vector for making C-terminal TurboID-mVenus fusion with non-nuclear proteins. ACCESSION . VERSION . KEYWORDS pDONR_P2R-P3_R2-Turbo-NES-mVenus-STOP-L3 SOURCE synthetic DNA construct ORGANISM synthetic DNA construct REFERENCE 1 (bases 1 to 4474) AUTHORS Mair A, Xu SL, Branon TC, Ting AY, Bergmann DC TITLE Proximity labeling of protein complexes and cell type-specific organellar proteomes in Arabidopsis enabled by TurboID. JOURNAL Elife. 2019 Sep 19;8. pii: 47864. doi: 10.7554/eLife.47864. PUBMED 31535972 REFERENCE 2 (bases 1 to 4474) AUTHORS . TITLE Direct Submission JOURNAL Exported May 12, 2021 from SnapGene Server 1.1.58 http://www.snapgene.com FEATURES Location/Qualifiers source 1..4474 /organism="synthetic DNA construct" /mol_type="other DNA" CDS 38..79 /codon_start=1 /product="epitope tag from simian virus 5" /label=V5 tag /translation="GKPIPNPLLGLDST" CDS 86..1042 /codon_start=1 /product="mutant of the E. coli biotin ligase BioID engineered for improved catalytic efficiency (Branon et al., 2018)" /label=TurboID /translation="KDNTVPLKLIALLANGEFHSGEQLGETLGMSRAAINKHIQTLRDW GVDVFTVPGKGYSLPEPIPLLNAKQILGQLDGGSVAVLPVVDSTNQYLLDRIGELKSGD ACIAEYQQAGRGSRGRKWFSPFGANLYLSMFWRLKRGPAAIGLGPVIGIVMAEALRKLG ADKVRVKWPNDLYLQDRKLAGILVELAGITGDAAQIVIGAGINVAMRRVEESVVNQGWI TLQEAGINLDRNTLAATLIRELRAALELFEQEGLAPYLPRWEKLDNFINRPVKLIIGDK EIFGISRGIDKQGALLLEQDGVIKPWMGGEISLRSAEK" CDS 1049..1075 /codon_start=1 /product="nuclear export signal from the HIV Rev protein (Fischer et al., 1995)" /label=NES /translation="LPPLERLTL" CDS 1115..1834 /codon_start=1 /product="yellow fluorescent protein (YFP) with fast and efficient maturation (Nagai et al., 2002)" /label=Venus /note="mammalian codon-optimized" /translation="MVSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTL KLICTTGKLPVPWPTLVTTLGYGLQCFARYPDHMKQHDFFKSAMPEGYVQERTIFFKDD GNYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYITADKQKNGIK ANFKIRHNIEDGGVQLADHYQQNTPIGDGPVLLPDNHYLSYQSALSKDPNEKRDHMVLL EFVTAAGITLGMDELYK" primer_bind complement(1160..1181) /label=EGFP-N /note="EGFP, reverse primer" primer_bind complement(1421..1440) /label=EXFP-R /note="For distinguishing EGFP variants, reverse primer" primer_bind 1768..1789 /label=EGFP-C /note="EGFP, forward primer" protein_bind complement(1835..1930) /gene="mutant version of attL" /label=attL3 /bound_moiety="LR Clonase(TM)" /note="recombination site for the Gateway(R) LR reaction" primer_bind complement(1967..1986) /label=T7 /note="T7 promoter, forward primer" promoter complement(1968..1986) /label=T7 promoter /note="promoter for bacteriophage T7 RNA polymerase" primer_bind complement(1991..2007) /label=M13 rev /note="common sequencing primer, one of multiple similar variants" primer_bind complement(1991..2007) /label=M13 Reverse /note="In lacZ gene. Also called M13-rev" primer_bind complement(2109..2128) /label=pENTR-R /note="pENTR vectors, reverse primer" CDS 2120..2929 /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(2187..2206) /label=Kan-R /note="Kanamycin resistance gene, reverse primer" rep_origin 3076..3664 /direction=RIGHT /label=ori /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of replication" primer_bind 3565..3584 /label=pBR322ori-F /note="pBR322 origin, forward primer" primer_bind 3818..3835 /label=L4440 /note="L4440 vector, forward primer" terminator 3994..4021 /label=rrnB T2 terminator /note="transcription terminator T2 from the E. coli rrnB gene" terminator 4113..4199 /gene="Escherichia coli rrnB" /label=rrnB T1 terminator /note="transcription terminator T1 from the E. coli rrnB gene" primer_bind 4248..4270 /label=M13/pUC Forward /note="In lacZ gene" primer_bind 4262..4279 /label=M13 Forward /note="In lacZ gene. Also called M13-F20 or M13 (-21) Forward" primer_bind 4263..4279 /label=M13 fwd /note="common sequencing primer, one of multiple similar variants" protein_bind complement(4350..4474) /gene="mutant version of attR" /label=attR2 /bound_moiety="LR Clonase(TM)" /note="recombination site for the Gateway(R) LR reaction" ORIGIN 1 tgggcgcgcc ggtggaggcg gttcaggagg tggcatgggc aagcccatcc ccaaccccct 61 gctgggcctg gacagcaccg ctagcaaaga caatactgtg cctctgaagc tgatcgctct 121 cctggctaat ggcgagttcc atagtggcga acagctggga gaaaccctgg gcatgtccag 181 ggccgctatc aacaagcaca ttcagactct gcgcgactgg ggcgtggacg tgttcaccgt 241 gcccggaaag ggctactctc tgcccgagcc tatcccgctg ctgaacgcta aacagattct 301 gggacagctg gacggcggga gcgtggcagt cctgcctgtg gtcgactcca ccaatcagta 361 cctgctggat cgaatcggcg agctgaagag tggggatgct tgcattgcag aatatcagca 421 ggcagggaga ggaagcagag ggaggaaatg gttctctcct tttggagcta acctgtacct 481 gagtatgttt tggcgcctga agcggggacc agcagcaatc ggcctgggcc cggtcatcgg 541 aattgtcatg gcagaagcgc tgcgaaagct gggagcagac aaggtgcgag tcaaatggcc 601 caatgacctg tatctgcagg atagaaagct ggcaggcatc ctggtggagc tggccggaat 661 aacaggcgat gctgcacaga tcgtcattgg cgccgggatt aacgtggcta tgaggcgcgt 721 ggaggaaagc gtggtcaatc agggctggat cacactgcag gaagcaggga ttaacctgga 781 caggaatact ctggccgcta cgctgatccg agagctgcgg gcagccctgg aactgttcga 841 gcaggaaggc ctggctccat atctgccacg gtgggagaag ctggataact tcatcaatag 901 acccgtgaag ctgatcattg gggacaaaga gattttcggg attagccggg ggattgataa 961 acagggagcc ctgctgctgg aacaggacgg agttatcaaa ccctggatgg gcggagaaat 1021 cagtctgcgg tctgccgaaa agctgcagct gcctcccctg gagcgcctga ccctggacgg 1081 aggcggtgga agcggcggag gtgggcgcgc ctccatggtg agcaagggcg aggagctgtt 1141 caccggggtg gtgcccatcc tggtcgagct ggacggcgac gtaaacggcc acaagttcag 1201 cgtgtccggc gagggcgagg gcgatgccac ctacggcaag ctgaccctga agctgatctg 1261 caccaccggc aagctgcccg tgccctggcc caccctcgtg accaccctgg gctacggcct 1321 gcagtgcttc gcccgctacc ccgaccacat gaagcagcac gacttcttca agtccgccat 1381 gcccgaaggc tacgtccagg agcgcaccat cttcttcaag gacgacggca actacaagac 1441 ccgcgccgag gtgaagttcg agggcgacac cctggtgaac cgcatcgagc tgaagggcat 1501 cgacttcaag gaggacggca acatcctggg gcacaagctg gagtacaact acaacagcca 1561 caacgtctat atcaccgccg acaagcagaa gaacggcatc aaggccaact tcaagatccg 1621 ccacaacatc gaggacggcg gcgtgcagct cgccgaccac taccagcaga acacccccat 1681 cggcgacggc cccgtgctgc tgcccgacaa ccactacctg agctaccagt ccgccctgag 1741 caaagacccc aacgagaagc gcgatcacat ggtcctgctg gagttcgtga ccgccgccgg 1801 gatcactctc ggcatggacg agctgtacaa gtgacaactt tattatacaa agttggcatt 1861 ataaaaaagc attgcttatc aatttgttgc aacgaacagg tcactatcag tcaaaataaa 1921 atcattattt ggagctccat ggtagcgtta acgcggccgc gatatcccct atagtgagtc 1981 gtattacatg gtcatagctg tttcctggca gctctggccc gtgtctcaaa atctctgatg 2041 ttacattgca caagataaaa atatatcatc atgaacaata aaactgtctg cttacataaa 2101 cagtaataca aggggtgtta tgagccatat tcaacgggaa acgtcgaggc cgcgattaaa 2161 ttccaacatg gatgctgatt tatatgggta taaatgggct cgcgataatg tcgggcaatc 2221 aggtgcgaca atctatcgct tgtatgggaa gcccgatgcg ccagagttgt ttctgaaaca 2281 tggcaaaggt agcgttgcca atgatgttac agatgagatg gtcagactaa actggctgac 2341 ggaatttatg cctcttccga ccatcaagca ttttatccgt actcctgatg atgcatggtt 2401 actcaccact gcgatccccg gaaaaacagc attccaggta ttagaagaat atcctgattc 2461 aggtgaaaat attgttgatg cgctggcagt gttcctgcgc cggttgcatt cgattcctgt 2521 ttgtaattgt ccttttaaca gcgatcgcgt atttcgtctc gctcaggcgc aatcacgaat 2581 gaataacggt ttggttgatg cgagtgattt tgatgacgag cgtaatggct ggcctgttga 2641 acaagtctgg aaagaaatgc ataaactttt gccattctca ccggattcag tcgtcactca 2701 tggtgatttc tcacttgata accttatttt tgacgagggg aaattaatag gttgtattga 2761 tgttggacga gtcggaatcg cagaccgata ccaggatctt gccatcctat ggaactgcct 2821 cggtgagttt tctccttcat tacagaaacg gctttttcaa aaatatggta ttgataatcc 2881 tgatatgaat aaattgcagt ttcatttgat gctcgatgag tttttctaat cagaattggt 2941 taattggttg taacactggc agagcattac gctgacttga cgggacggcg caagctcatg 3001 accaaaatcc cttaacgtga gttacgcgtc gttccactga gcgtcagacc ccgtagaaaa 3061 gatcaaagga tcttcttgag atcctttttt tctgcgcgta atctgctgct tgcaaacaaa 3121 aaaaccaccg ctaccagcgg tggtttgttt gccggatcaa gagctaccaa ctctttttcc 3181 gaaggtaact ggcttcagca gagcgcagat accaaatact gttcttctag tgtagccgta 3241 gttaggccac cacttcaaga actctgtagc accgcctaca tacctcgctc tgctaatcct 3301 gttaccagtg gctgctgcca gtggcgataa gtcgtgtctt accgggttgg actcaagacg 3361 atagttaccg gataaggcgc agcggtcggg ctgaacgggg ggttcgtgca cacagcccag 3421 cttggagcga acgacctaca ccgaactgag atacctacag cgtgagctat gagaaagcgc 3481 cacgcttccc gaagggagaa aggcggacag gtatccggta agcggcaggg tcggaacagg 3541 agagcgcacg agggagcttc cagggggaaa cgcctggtat ctttatagtc ctgtcgggtt 3601 tcgccacctc tgacttgagc gtcgattttt gtgatgctcg tcaggggggc ggagcctatg 3661 gaaaaacgcc agcaacgcgg cctttttacg gttcctggcc ttttgctggc cttttgctca 3721 catgttcttt cctgcgttat cccctgattc tgtggataac cgtattaccg cctttgagtg 3781 agctgatacc gctcgccgca gccgaacgac cgagcgcagc gagtcagtga gcgaggaagc 3841 ggaagagcgc ccaatacgca aaccgcctct ccccgcgcgt tggccgattc attaatgcag 3901 ctggcacgac aggtttcccg actggaaagc gggcagtgag cgcaacgcaa ttaatacgcg 3961 taccgctagc caggaagagt ttgtagaaac gcaaaaaggc catccgtcag gatggccttc 4021 tgcttagttt gatgcctggc agtttatggc gggcgtcctg cccgccaccc tccgggccgt 4081 tgcttcacaa cgttcaaatc cgctcccggc ggatttgtcc tactcaggag agcgttcacc 4141 gacaaacaac agataaaacg aaaggcccag tcttccgact gagcctttcg ttttatttga 4201 tgcctggcag ttccctactc tcgcgttaac gctagcatgg atgttttccc agtcacgacg 4261 ttgtaaaacg acggccagtc ttaagctcgg gccctgcagc tctagagctc gaattctaca 4321 ggtcactaat accatctaag tagttggttc atagtgactg catatgttgt gttttacagt 4381 attatgtagt ctgtttttta tgcaaaatct aatttaatat attgatattt atatcatttt 4441 acgtttctcg ttcaactttc ttgtacaaag tggc //