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" 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 complement(624..748) /gene="mutant version of attR" /label=attR2 /bound_moiety="LR Clonase(TM)" /note="recombination site for the Gateway(R) LR reaction" CDS 786..827 /codon_start=1 /product="epitope tag from simian virus 5" /label=V5 tag /translation="GKPIPNPLLGLDST" CDS 834..1790 /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 1797..1823 /codon_start=1 /product="nuclear export signal from the HIV Rev protein (Fischer et al., 1995)" /label=NES /translation="LPPLERLTL" CDS 1863..2582 /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(1908..1929) /label=EGFP-N /note="EGFP, reverse primer" primer_bind complement(2169..2188) /label=EXFP-R /note="For distinguishing EGFP variants, reverse primer" primer_bind 2516..2537 /label=EGFP-C /note="EGFP, forward primer" protein_bind complement(2583..2678) /gene="mutant version of attL" /label=attL3 /bound_moiety="LR Clonase(TM)" /note="recombination site for the Gateway(R) LR reaction" primer_bind complement(2715..2734) /label=T7 /note="T7 promoter, forward primer" promoter complement(2716..2734) /label=T7 promoter /note="promoter for bacteriophage T7 RNA polymerase" primer_bind complement(2739..2755) /label=M13 rev /note="common sequencing primer, one of multiple similar variants" primer_bind complement(2739..2755) /label=M13 Reverse /note="In lacZ gene. Also called M13-rev" primer_bind complement(2857..2876) /label=pENTR-R /note="pENTR vectors, reverse primer" CDS 2868..3677 /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(2935..2954) /label=Kan-R /note="Kanamycin resistance gene, reverse primer" rep_origin 3824..4412 /direction=RIGHT /label=ori /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of replication" primer_bind 4313..4332 /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 tcgggccctg cagctctaga gctcgaattc tacaggtcac 601 taataccatc taagtagttg gttcatagtg actgcatatg ttgtgtttta cagtattatg 661 tagtctgttt tttatgcaaa atctaattta atatattgat atttatatca ttttacgttt 721 ctcgttcaac tttcttgtac aaagtggctg ggcgcgccgg tggaggcggt tcaggaggtg 781 gcatgggcaa gcccatcccc aaccccctgc tgggcctgga cagcaccgct agcaaagaca 841 atactgtgcc tctgaagctg atcgctctcc tggctaatgg cgagttccat agtggcgaac 901 agctgggaga aaccctgggc atgtccaggg ccgctatcaa caagcacatt cagactctgc 961 gcgactgggg cgtggacgtg ttcaccgtgc ccggaaaggg ctactctctg cccgagccta 1021 tcccgctgct gaacgctaaa cagattctgg gacagctgga cggcgggagc gtggcagtcc 1081 tgcctgtggt cgactccacc aatcagtacc tgctggatcg aatcggcgag ctgaagagtg 1141 gggatgcttg cattgcagaa tatcagcagg cagggagagg aagcagaggg aggaaatggt 1201 tctctccttt tggagctaac ctgtacctga gtatgttttg gcgcctgaag cggggaccag 1261 cagcaatcgg cctgggcccg gtcatcggaa ttgtcatggc agaagcgctg cgaaagctgg 1321 gagcagacaa ggtgcgagtc aaatggccca atgacctgta tctgcaggat agaaagctgg 1381 caggcatcct ggtggagctg gccggaataa caggcgatgc tgcacagatc gtcattggcg 1441 ccgggattaa cgtggctatg aggcgcgtgg aggaaagcgt ggtcaatcag ggctggatca 1501 cactgcagga agcagggatt aacctggaca ggaatactct ggccgctacg ctgatccgag 1561 agctgcgggc agccctggaa ctgttcgagc aggaaggcct ggctccatat ctgccacggt 1621 gggagaagct ggataacttc atcaatagac ccgtgaagct gatcattggg gacaaagaga 1681 ttttcgggat tagccggggg attgataaac agggagccct gctgctggaa caggacggag 1741 ttatcaaacc ctggatgggc ggagaaatca gtctgcggtc tgccgaaaag ctgcagctgc 1801 ctcccctgga gcgcctgacc ctggacggag gcggtggaag cggcggaggt gggcgcgcct 1861 ccatggtgag caagggcgag gagctgttca ccggggtggt gcccatcctg gtcgagctgg 1921 acggcgacgt aaacggccac aagttcagcg tgtccggcga gggcgagggc gatgccacct 1981 acggcaagct gaccctgaag ctgatctgca ccaccggcaa gctgcccgtg ccctggccca 2041 ccctcgtgac caccctgggc tacggcctgc agtgcttcgc ccgctacccc gaccacatga 2101 agcagcacga cttcttcaag tccgccatgc ccgaaggcta cgtccaggag cgcaccatct 2161 tcttcaagga cgacggcaac tacaagaccc gcgccgaggt gaagttcgag ggcgacaccc 2221 tggtgaaccg catcgagctg aagggcatcg acttcaagga ggacggcaac atcctggggc 2281 acaagctgga gtacaactac aacagccaca acgtctatat caccgccgac aagcagaaga 2341 acggcatcaa ggccaacttc aagatccgcc acaacatcga ggacggcggc gtgcagctcg 2401 ccgaccacta ccagcagaac acccccatcg gcgacggccc cgtgctgctg cccgacaacc 2461 actacctgag ctaccagtcc gccctgagca aagaccccaa cgagaagcgc gatcacatgg 2521 tcctgctgga gttcgtgacc gccgccggga tcactctcgg catggacgag ctgtacaagt 2581 gacaacttta ttatacaaag ttggcattat aaaaaagcat tgcttatcaa tttgttgcaa 2641 cgaacaggtc actatcagtc aaaataaaat cattatttgg agctccatgg tagcgttaac 2701 gcggccgcga tatcccctat agtgagtcgt attacatggt catagctgtt tcctggcagc 2761 tctggcccgt gtctcaaaat ctctgatgtt acattgcaca agataaaaat atatcatcat 2821 gaacaataaa actgtctgct tacataaaca gtaatacaag gggtgttatg agccatattc 2881 aacgggaaac gtcgaggccg cgattaaatt ccaacatgga tgctgattta tatgggtata 2941 aatgggctcg cgataatgtc gggcaatcag gtgcgacaat ctatcgcttg tatgggaagc 3001 ccgatgcgcc agagttgttt ctgaaacatg gcaaaggtag cgttgccaat gatgttacag 3061 atgagatggt cagactaaac tggctgacgg aatttatgcc tcttccgacc atcaagcatt 3121 ttatccgtac tcctgatgat gcatggttac tcaccactgc gatccccgga aaaacagcat 3181 tccaggtatt agaagaatat cctgattcag gtgaaaatat tgttgatgcg ctggcagtgt 3241 tcctgcgccg gttgcattcg attcctgttt gtaattgtcc ttttaacagc gatcgcgtat 3301 ttcgtctcgc tcaggcgcaa tcacgaatga ataacggttt ggttgatgcg agtgattttg 3361 atgacgagcg taatggctgg cctgttgaac aagtctggaa agaaatgcat aaacttttgc 3421 cattctcacc ggattcagtc gtcactcatg gtgatttctc acttgataac cttatttttg 3481 acgaggggaa attaataggt tgtattgatg ttggacgagt cggaatcgca gaccgatacc 3541 aggatcttgc catcctatgg aactgcctcg gtgagttttc tccttcatta cagaaacggc 3601 tttttcaaaa atatggtatt gataatcctg atatgaataa attgcagttt catttgatgc 3661 tcgatgagtt tttctaatca gaattggtta attggttgta acactggcag agcattacgc 3721 tgacttgacg ggacggcgca agctcatgac caaaatccct taacgtgagt tacgcgtcgt 3781 tccactgagc gtcagacccc gtagaaaaga tcaaaggatc ttcttgagat cctttttttc 3841 tgcgcgtaat ctgctgcttg caaacaaaaa aaccaccgct accagcggtg gtttgtttgc 3901 cggatcaaga gctaccaact ctttttccga aggtaactgg cttcagcaga gcgcagatac 3961 caaatactgt ccttctagtg tagccgtagt taggccacca cttcaagaac tctgtagcac 4021 cgcctacata cctcgctctg ctaatcctgt taccagtggc tgctgccagt ggcgataagt 4081 cgtgtcttac cgggttggac tcaagacgat agttaccgga taaggcgcag cggtcgggct 4141 gaacgggggg ttcgtgcaca cagcccagct tggagcgaac gacctacacc gaactgagat 4201 acctacagcg tgagcattga gaaagcgcca cgcttcccga agggagaaag gcggacaggt 4261 atccggtaag cggcagggtc ggaacaggag agcgcacgag ggagcttcca gggggaaacg 4321 cctggtatct ttatagtcct gtcgggtttc gccacctctg acttgagcgt cgatttttgt 4381 gatgctcgtc aggggggcgg agcctatgga aaaacgccag caacgcggcc tttttacggt 4441 tcctggcctt ttgctggcct tttgctcaca tgtt //