LOCUS pEN-R2-GGGGS-Apex2-FLAG-L3 3430 bp ds-DNA circular SYN 20-MAR-2023 DEFINITION . ACCESSION VERSION . KEYWORDS . SOURCE synthetic DNA construct ORGANISM recombinant plasmid. REFERENCE 1 (bases 1 to 3430) AUTHORS VIB Network TITLE Direct Submission JOURNAL Exported Sep 10, 2020 from SnapGene 4.2.0 http://www.snapgene.com FEATURES Location/Qualifiers attR2 complement(1..126) /created_by="mamee" /Color="#00cccc" /modified_by="mamee" /note="Derived using Geneious Prime 2023.0.1 'Annotate from Database' based on nucleotide similarity" /Transferred_From="attR2" /Transferred_Similarity="99.21%" /Primary_Match="99.21%; Adjusted-Interval-0:0/0; attR2" CDS 128..142 /codon_start=1 /translation="GGGGS" /label="GGGGS" CDS 143..889 /codon_start=1 /product="soybean ascorbate peroxidase, engineered to serve as a monomeric tag for electron microscopy and live-cell proteomics (Lam et al., 2015)" /note="more active than the original APEX due to the A134P mutation" /translation="GKSYPTVSADYQDAVEKAKKKLRGFIAEKRCAPLMLRLAFHSAG TFDKGTKTGGPFGTIKHPAELAHSANNGLDIAVRLLEPLKAEFPILSYADFYQLAGVV AVEVTGGPKVPFHPGREDKPEPPPEGRLPDPTKGSDHLRDVFGKAMGLTDQDIVALSG GHTIGAAHKERSGFEGPWTSNPLIFDNSYFTELLSGEKEGLLQLPSDKALLSDPVFRP LVDKYAADEDAFFADYAEAHQKLSELGFADA" /label="APEX2" tag 890..913 /codon_start=1 /product="FLAG(R) epitope tag, followed by an enterokinase cleavage site" /translation="DYKDDDDK" /modified_by="mamee" /label="FLAG" attL3 complement(917..1012) /created_by="mamee" /Color="#00cccc" /modified_by="mamee" /note="Derived using Geneious Prime 2023.0.1 'Annotate from Database' based on nucleotide similarity" /Transferred_From="attL3" /Transferred_Similarity="100.00%" /Primary_Match="100.00%; Adjusted-Interval-0:0/0; attL3" primer complement(1070..1090) /note="Derived using Geneious Prime 2020.2.2 'Annotate from Database' based on nucleotide similarity" /Transferred_From="pCDB-Cas9 - GWM13R" /Transferred_Similarity="100.00%" /Primary_Match="100.00%; Adjusted-Interval-0:0/0; GWM13R (pCDB-Cas9 - GWM13R)" /label="GWM13R" promoter 1151..1201 /created_by="chgai" /note="Derived using Geneious Prime 2022.1.1 'Annotate from Database' based on nucleotide similarity" /X-Transferred_From="pCG034_pKan_Kan-CBE" /X-Transferred_Similarity="100.00%" /X-Primary_Match="100.00%; Adjusted-Interval-0:0/0; Kan_promoter (pCG034_pKan_Kan-CBE)" /X-Alternative_Match="100.00%; Adjusted-Interval-0:0/0; Kan_promoter (pCG040_ABE-Nuc-NLS_Kan -reporter_control)" /X-Alternative_Match="100.00%; Adjusted-Interval-0:0/0; Kan_promoter (pCG043_Kan-ABE-Dest_co ntrol_ccdB)" /X-Alternative_Match="100.00%; Adjusted-Interval-0:0/0; Kan_promoter (pCG046_ABE-LbCas12a-NL S_Kan_control)" /X-Alternative_Match="100.00%; Adjusted-Interval-0:0/0; Kan_promoter (pCG005_pGGD-pKan-KanR- E)" /X-Alternative_Match="100.00%; Adjusted-Interval-0:0/0; Kan_promoter (pCG006_pGGA-Kan_promot er-B)" /X-Alternative_Match="100.00%; Adjusted-Interval-0:0/0; Kan_promoter (pCG035_pKan_Kan)" /X-Alternative_Match="100.00%; Adjusted-Interval-0:0/0; Kan_promoter (pCG039_ABE-N7-NLS_Kan- reporter_control)" /X-Alternative_Match="100.00%; Adjusted-Interval-0:0/0; Kan_promoter (pCG041_ABE-SV40-NLS_Ka n-reporter_control)" /X-Alternative_Match="100.00%; Adjusted-Interval-0:0/0; Kan_promoter (pCG054_Kan_CBE_control )" /modified_by="mamee" /note="Derived using Geneious Prime 2023.0.1 'Annotate from Database' based on nucleotide similarity" /Transferred_From="KanR" /Transferred_Similarity="100.00%" /Primary_Match="100.00%; Adjusted-Interval-0:0/0; kan (KanR)" /label="kan" misc_feature 1202..2011 /modified_by="wacae" /note="Derived using Geneious Prime 2020.2.2 'Annotate from Database' based on nucleotide similarity" /Transferred_From="KanR" /Transferred_Similarity="100.00%" /Primary_Match="100.00%; Adjusted-Interval-0:0/0; KanR" /label="KanR" /note="Geneious type: Bacterial Selection" rep_origin 2158..2746 /created_by="thjac" /modified_by="mamee" /note="Derived using Geneious Prime 2020.2.2 'Annotate from Database' based on nucleotide similarity" /Transferred_From="high-copy-number ColE1/pMB1/pBR322/pUC origin of replication" /Transferred_Similarity="99.49%" /Primary_Match="99.49%; Adjusted-Interval-0:0/0; ColE1/pMB1/pBR322/pUC Ori (high-copy-number ColE1/pMB1/pBR322/pUC origin of replication)" /label="ColE1/pMB1/pBR322/pUC Ori" primer 3345..3365 /note="Derived using Geneious Prime 2020.2.2 'Annotate from Database' based on nucleotide similarity" /Transferred_From="pCDB-Cas9 - GWM13F" /Transferred_Similarity="100.00%" /Primary_Match="100.00%; Adjusted-Interval-0:0/0; GWM13F (pCDB-Cas9 - GWM13F)" /label="GWM13F" ORIGIN 1 tcatagtgac tgcatatgtt gtgttttaca gtattatgta gtctgttttt tatgcaaaat 61 ctaatttaat atattgatat ttatatcatt ttacgtttct cgttcaactt tcttgtacaa 121 agtggccgga ggcggtggat cgggaaagtc ttacccaact gtgagtgctg attaccagga 181 cgccgttgag aaggcgaaga agaagctcag aggcttcatc gctgagaaga gatgcgctcc 241 tctaatgctc cgtttggcat tccactctgc tggaaccttt gacaagggca cgaagaccgg 301 tggacccttc ggaaccatca agcaccctgc cgaactggct cacagcgcta acaacggtct 361 tgacatcgct gttaggcttt tggagccact caaggcggag ttccctattt tgagctacgc 421 cgatttctac cagttggctg gcgttgttgc cgttgaggtc acgggtggac ctaaggttcc 481 attccaccct ggaagagagg acaagcctga gccaccacca gagggtcgct tgcccgatcc 541 cactaagggt tctgaccatt tgagagatgt gtttggcaaa gctatggggc ttactgacca 601 agatatcgtt gctctatctg ggggtcacac tattggagct gcacacaagg agcgttctgg 661 atttgagggt ccctggacct ctaatcctct tattttcgac aactcatact tcacggagtt 721 gttgagtggt gagaaggaag gtctccttca gctaccttct gacaaggctc ttttgtctga 781 ccctgtattc cgccctctcg ttgacaaata tgcagcggac gaagatgcct tctttgctga 841 ttacgctgag gctcaccaaa agctttccga gcttgggttt gctgatgccg actacaaaga 901 cgatgacgat aaatgacaac tttattatac aaagttggca ttataaaaaa gcattgctta 961 tcaatttgtt gcaacgaaca ggtcactatc agtcaaaata aaatcattat ttggagctcc 1021 atggtagcgt taacgcggcc gcgatatccc ctatagtgag tcgtattaca tggtcatagc 1081 tgtttcctgg cagctctggc ccgtgtctca aaatctctga tgttacattg cacaagataa 1141 aaatatatca tcatgaacaa taaaactgtc tgcttacata aacagtaata caaggggtgt 1201 tatgagccat attcaacggg aaacgtcgag gccgcgatta aattccaaca tggatgctga 1261 tttatatggg tataaatggg ctcgcgataa tgtcgggcaa tcaggtgcga caatctatcg 1321 cttgtatggg aagcccgatg cgccagagtt gtttctgaaa catggcaaag gtagcgttgc 1381 caatgatgtt acagatgaga tggtcagact aaactggctg acggaattta tgcctcttcc 1441 gaccatcaag cattttatcc gtactcctga tgatgcatgg ttactcacca ctgcgatccc 1501 cggaaaaaca gcattccagg tattagaaga atatcctgat tcaggtgaaa atattgttga 1561 tgcgctggca gtgttcctgc gccggttgca ttcgattcct gtttgtaatt gtccttttaa 1621 cagcgatcgc gtatttcgtc tcgctcaggc gcaatcacga atgaataacg gtttggttga 1681 tgcgagtgat tttgatgacg agcgtaatgg ctggcctgtt gaacaagtct ggaaagaaat 1741 gcataaactt ttgccattct caccggattc agtcgtcact catggtgatt tctcacttga 1801 taaccttatt tttgacgagg ggaaattaat aggttgtatt gatgttggac gagtcggaat 1861 cgcagaccga taccaggatc ttgccatcct atggaactgc ctcggtgagt tttctccttc 1921 attacagaaa cggctttttc aaaaatatgg tattgataat cctgatatga ataaattgca 1981 gtttcatttg atgctcgatg agtttttcta atcagaattg gttaattggt tgtaacactg 2041 gcagagcatt acgctgactt gacgggacgg cgcaagctca tgaccaaaat cccttaacgt 2101 gagttacgcg tcgttccact gagcgtcaga ccccgtagaa aagatcaaag gatcttcttg 2161 agatcctttt tttctgcgcg taatctgctg cttgcaaaca aaaaaaccac cgctaccagc 2221 ggtggtttgt ttgccggatc aagagctacc aactcttttt ccgaaggtaa ctggcttcag 2281 cagagcgcag ataccaaata ctgtccttct agtgtagccg tagttaggcc accacttcaa 2341 gaactctgta gcaccgccta catacctcgc tctgctaatc ctgttaccag tggctgctgc 2401 cagtggcgat aagtcgtgtc ttaccgggtt ggactcaaga cgatagttac cggataaggc 2461 gcagcggtcg ggctgaacgg ggggttcgtg cacacagccc agcttggagc gaacgaccta 2521 caccgaactg agatacctac agcgtgagca ttgagaaagc gccacgcttc ccgaagggag 2581 aaaggcggac aggtatccgg taagcggcag ggtcggaaca ggagagcgca cgagggagct 2641 tccaggggga aacgcctggt atctttatag tcctgtcggg tttcgccacc tctgacttga 2701 gcgtcgattt ttgtgatgct cgtcaggggg gcggagccta tggaaaaacg ccagcaacgc 2761 ggccttttta cggttcctgg ccttttgctg gccttttgct cacatgttct ttcctgcgtt 2821 atcccctgat tctgtggata accgtattac cgcctttgag tgagctgata ccgctcgccg 2881 cagccgaacg accgagcgca gcgagtcagt gagcgaggaa gcggaagagc gcccaatacg 2941 caaaccgcct ctccccgcgc gttggccgat tcattaatgc agctggcacg acaggtttcc 3001 cgactggaaa gcgggcagtg agcgcaacgc aattaatacg cgtaccgcta gccaggaaga 3061 gtttgtagaa acgcaaaaag gccatccgtc aggatggcct tctgcttagt ttgatgcctg 3121 gcagtttatg gcgggcgtcc tgcccgccac cctccgggcc gttgcttcac aacgttcaaa 3181 tccgctcccg gcggatttgt cctactcagg agagcgttca ccgacaaaca acagataaaa 3241 cgaaaggccc agtcttccga ctgagccttt cgttttattt gatgcctggc agttccctac 3301 tctcgcgtta acgctagcat ggatgttttc ccagtcacga cgttgtaaaa cgacggccag 3361 tcttaagctc gggccctgca gctctagagc tcgaattcta caggtcacta ataccatcta 3421 agtagttgat //