LOCUS pGG-C-dCas9-CDA-UL-D_ 8052 bp DNA circular UNA 22-MAR-2023 DEFINITION . ACCESSION urn.local...oh-9s73oy9 VERSION urn.local...oh-9s73oy9 KEYWORDS . SOURCE . ORGANISM . FEATURES Location/Qualifiers GG 1..11 /modified_by="wacae" /note="Derived using Geneious Prime 2020.2.2 'Annotate from Database' based on nucleotide similarity" /Transferred_From="C" /Transferred_Similarity="100.00%" /Primary_Match="100.00%; Adjusted-Interval-0:0/0; C" /label="C" CDS 14..4117 /product="nickase mutant of the Cas9 endonuclease from the Streptococcus pyogenes Type II CRISPR/Cas system" /note="generates RNA-guided single strand nicks in DNA due to the D10A mutation in the RuvC catalytic domain" /Original_Translation="DKKYSIGLAIGTNSVGWAVITDEYKVPSKKFKVLG NTDRHSIKKNLIGALLFDSGETAEATRLKRTARRRYTRRKNRICYLQEIFSNEMAKVD DSFFHRLEESFLVEEDKKHERHPIFGNIVDEVAYHEKYPTIYHLRKKLVDSTDKADLR LIYLALAHMIKFRGHFLIEGDLNPDNSDVDKLFIQLVQTYNQLFEENPINASGVDAKA ILSARLSKSRRLENLIAQLPGEKKNGLFGNLIALSLGLTPNFKSNFDLAEDAKLQLSK DTYDDDLDNLLAQIGDQYADLFLAAKNLSDAILLSDILRVNTEITKAPLSASMIKRYD EHHQDLTLLKALVRQQLPEKYKEIFFDQSKNGYAGYIDGGASQEEFYKFIKPILEKMD GTEELLVKLNREDLLRKQRTFDNGSIPHQIHLGELHAILRRQEDFYPFLKDNREKIEK ILTFRIPYYVGPLARGNSRFAWMTRKSEETITPWNFEEVVDKGASAQSFIERMTNFDK NLPNEKVLPKHSLLYEYFTVYNELTKVKYVTEGMRKPAFLSGEQKKAIVDLLFKTNRK VTVKQLKEDYFKKIECFDSVEISGVEDRFNASLGTYHDLLKIIKDKDFLDNEENEDIL EDIVLTLTLFEDREMIEERLKTYAHLFDDKVMKQLKRRRYTGWGRLSRKLINGIRDKQ SGKTILDFLKSDGFANRNFMQLIHDDSLTFKEDIQKAQVSGQGDSLHEHIANLAGSPA IKKGILQTVKVVDELVKVMGRHKPENIVIEMARENQTTQKGQKNSRERMKRIEEGIKE LGSQILKEHPVENTQLQNEKLYLYYLQNGRDMYVDQELDINRLSDYDVDHIVPQSFLK DDSIDNKVLTRSDKNRGKSDNVPSEEVVKKMKNYWRQLLNAKLITQRKFDNLTKAERG GLSELDKAGFIKRQLVETRQITKHVAQILDSRMNTKYDENDKLIREVKVITLKSKLVS DFRKDFQFYKVREINNYHHAHDAYLNAVVGTALIKKYPKLESEFVYGDYKVYDVRKMI AKSEQEIGKATAKYFFYSNIMNFFKTEITLANGEIRKRPLIETNGETGEIVWDKGRDF ATVRKVLSMPQVNIVKKTEVQTGGFSKESILPKRNSDKLIARKKDWDPKKYGGFDSPT VAYSVLVVAKVEKGKSKKLKSVKELLGITIMERSSFEKNPIDFLEAKGYKEVKKDLII KLPKYSLFELENGRKRMLASAGELQKGNELALPSKYVNFLYLASHYEKLKGSPEDNEQ KQLFVEQHKHYLDEIIEQISEFSKRVILADANLDKVLSAYNKHRDKPIREQAENIIHL FTLTNLGAPAAFKYFDTTIDRKRYTSTKEVLDATLIHQSITGLYETRIDLSQLGGD" /note="Derived using Geneious Prime 2020.2.2 'Annotate from Database' based on protein similarity" /X-Transferred_From="SpCas9(D10A,H840A)" /X-Transferred_Translation_Similarity="100.00%" /X-Primary_Match="100.00%; Adjusted-Interval-0:0/0; SpCas9(D10A,H840A)" /modified_by="mamee" /note="Derived using Geneious Prime 2020.2.2 'Annotate from Database' based on nucleotide similarity" /Transferred_From="SpCas9-D10A,H840A" /Transferred_Similarity="100.00%" /Primary_Match="100.00%; Adjusted-Interval-0:0/0; SpCas9-D10A,H840A" /label="SpCas9-D10A,H840A" modified_base 41..43 /created_by="mamee" /label="D10A" modified_base 2531..2533 /created_by="mamee" /label="H840A" Linker 4133..4324 /created_by="warddecaestecker" /note="Derived using Geneious Prime 2020.2.4 'Annotate from Database' based on protein similarity" /Transferred_From="64-aa linker" /Transferred_Translation_Similarity="95.31%" /Primary_Match="95.31%; Adjusted-Interval-0:0/0; 64-aa linker" /label="64-aa linker" tag 4325..4390 /codon_start=1 /product="three tandem FLAG(R) epitope tags, followed by an enterokinase cleavage site" /Transferred_Translation="DYKDHDGDYKDHDIDYKDDDDK" /modified_by="mamee" /note="Derived using Geneious Prime 2023.0.1 'Annotate from Database' based on protein similarity" /Transferred_From="3xFLAG" /Transferred_Translation_Similarity="100.00%" /Primary_Match="100.00%; Adjusted-Interval-0:0/0; 3xFLAG" /label="3xFLAG" tag 4409..4474 /codon_start=1 /product="three tandem FLAG(R) epitope tags, followed by an enterokinase cleavage site" /Transferred_Translation="DYKDHDGDYKDHDIDYKDDDDK" /modified_by="mamee" /note="Derived using Geneious Prime 2023.0.1 'Annotate from Database' based on protein similarity" /Transferred_From="3xFLAG" /Transferred_Translation_Similarity="100.00%" /Primary_Match="100.00%; Adjusted-Interval-0:0/0; 3xFLAG" /label="3xFLAG" CDS 4481..5104 /created_by="lapau" /note="Derived using Geneious Prime 2020.2.4 'Annotate from Database' based on nucleotide similarity" /Transferred_From="PmCDA1" /Transferred_Similarity="100.00%" /Primary_Match="100.00%; Adjusted-Interval-0:0/0; PmCDA1" /Alternative_Match="100.00%; Adjusted-Interval-0:0/0; PmCDA1" /label="PmCDA1" CDS 5111..5362 /created_by="lapau" /label="UGI" tag 5366..5374 /created_by="lapau" /modified_by="mamee" /label="LVA-tag" GG complement(5378..5388) /created_by="thjac" /modified_by="thjac" /note="Derived using Geneious Prime 2020.2.4 'Annotate from Database' based on nucleotide similarity" /Transferred_From="D" /Transferred_Similarity="100.00%" /Primary_Match="100.00%; Adjusted-Interval-0:0/0; D" /label="D" primer complement(5408..5428) /note="Derived using Geneious Prime 2020.2.2 'Annotate from Database' based on nucleotide similarity" /Transferred_From="pGG-B-HA-TurboID-linker-C - GG_SEQ_rev" /Transferred_Similarity="100.00%" /Primary_Match="100.00%; Adjusted-Interval-0:0/0; GG_SEQ_rev (pGG-B-HA-TurboID-linker-C - GG_SEQ_rev)" /label="GG_SEQ_rev" promoter 5908..6012 /created_by="mamee" /modified_by="mamee" /note="Derived using Geneious Prime 2023.0.1 'Annotate from Database' based on nucleotide similarity" /Transferred_From="AmpR" /Transferred_Similarity="100.00%" /Primary_Match="100.00%; Adjusted-Interval-0:0/0; amp (AmpR)" /label="amp" misc_feature 6013..6873 /X-Transferred_From="AmpR" /X-Transferred_Similarity="100.00%" /modified_by="mamee" /note="Derived using Geneious Prime 2023.0.1 'Annotate from Database' based on nucleotide similarity" /Transferred_From="AmpR" /Transferred_Similarity="100.00%" /Primary_Match="100.00%; Adjusted-Interval-0:0/0; AmpR" /label="AmpR" /note="Geneious type: Bacterial Selection" rep_origin 7044..7632 /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="100.00%" /Primary_Match="100.00%; 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 7996..8017 /note="Derived using Geneious Prime 2020.2.2 'Annotate from Database' based on nucleotide similarity" /Transferred_From="pGG-B-HA-TurboID-linker-C - GG_SEQ_fw" /Transferred_Similarity="100.00%" /Primary_Match="100.00%; Adjusted-Interval-0:0/0; GG_SEQ_fw (pGG-B-HA-TurboID-linker-C - GG_SEQ_fw)" /label="GG_SEQ_fw" ORIGIN 1 ggtctcaggc tccatggata agaaatactc aataggctta gctatcggca caaatagcgt 61 cggatgggcg gtgatcactg atgaatataa ggttccgtct aaaaagttca aggttctggg 121 aaatacagac cgccacagta tcaaaaaaaa tcttataggg gctcttttat ttgacagtgg 181 agagacagcg gaagcgactc gtctcaaacg gacagctcgt agaaggtata cacgtcggaa 241 gaatcgtatt tgttatctac aggagatttt ttcaaatgag atggcgaaag tagatgatag 301 tttctttcat cgacttgaag agtctttttt ggtggaagaa gacaagaagc atgaacgtca 361 tcctattttt ggaaatatag tagatgaagt tgcttatcat gagaaatatc caactatcta 421 tcatctgcga aaaaaattgg tagattctac tgataaagcg gatttgcgct taatctattt 481 ggccttagcg catatgatta agtttcgtgg tcattttttg attgagggag atttaaatcc 541 tgataatagt gatgtggaca aactatttat ccagttggta caaacctaca atcaattatt 601 tgaagaaaac cctattaacg caagtggagt agatgctaaa gcgattcttt ctgcacgatt 661 gagtaaatca agacgattag aaaatctcat tgctcagctc cccggtgaga agaaaaatgg 721 cttatttggg aatctcattg ctttgtcatt gggtttgacc cctaatttta aatcaaattt 781 tgatttggca gaagatgcta aattacagct ttcaaaagat acttacgatg atgatttaga 841 taatttattg gcgcaaattg gagatcaata tgctgatttg tttttggcag ctaagaattt 901 atcagatgct attttacttt cagatatcct aagagtaaat actgaaataa ctaaggctcc 961 cctatcagct tcaatgatta aacgctacga tgaacatcat caagacttga ctcttttaaa 1021 agctttagtt cgacaacaac ttccagaaaa gtataaagaa atcttttttg atcaatcaaa 1081 aaacggatat gcaggttata ttgatggggg agctagccaa gaagaatttt ataaatttat 1141 caaaccaatt ttagaaaaaa tggatggtac tgaggaatta ttggtgaaac taaatcgtga 1201 agatttgctg cgcaagcaac ggacctttga caacggctct attccccatc aaattcactt 1261 gggtgagctg catgctattt tgagaagaca agaagacttt tatccatttt taaaagacaa 1321 tcgtgagaag attgaaaaaa tcttgacttt tcgaattcct tattatgttg gtccattggc 1381 gcgtggcaat agtcgttttg catggatgac tcggaagtct gaagaaacaa ttaccccatg 1441 gaattttgaa gaagttgtcg ataaaggtgc ttcagctcaa tcatttattg aacgcatgac 1501 aaactttgat aaaaatcttc caaatgaaaa agtactacca aaacatagtt tgctttatga 1561 gtattttacg gtttataacg aattgacaaa ggtcaaatat gttactgaag gaatgcgaaa 1621 accagcattt ctttcaggtg aacagaagaa agccattgtt gatttactct tcaaaacaaa 1681 tcgaaaagta accgttaagc aattaaaaga agattatttc aaaaaaatag aatgttttga 1741 tagtgttgaa atttcaggag ttgaagatag atttaatgct tcattaggta cctaccatga 1801 tttgctaaaa attattaaag ataaagattt tttggataat gaagaaaatg aagatatctt 1861 agaggatatt gttttaacat tgaccttatt tgaagatagg gagatgattg aggaaagact 1921 taaaacatat gctcacctct ttgatgataa ggtgatgaaa cagcttaaac gtcgccgtta 1981 tactggttgg ggacgtttgt ctcgaaaatt gattaatggt attagggata agcaatctgg 2041 caaaacaata ttagattttt tgaaatcaga tggttttgcc aatcgcaatt ttatgcagct 2101 gatccatgat gatagtttga catttaaaga agacattcaa aaagcacaag tgtctggaca 2161 aggcgatagt ttacatgaac atattgcaaa tttagctggt agccctgcta ttaaaaaagg 2221 tattttacag actgtaaaag ttgttgatga attggtcaaa gtaatggggc ggcataagcc 2281 agaaaatatc gttattgaaa tggcacgtga aaatcagaca actcaaaagg gccagaaaaa 2341 ttcgcgagag cgtatgaaac gaatcgaaga aggtatcaaa gaattaggaa gtcagattct 2401 taaagagcat cctgttgaaa atactcaatt gcaaaatgaa aagctctatc tctattatct 2461 ccaaaatgga agagacatgt atgtggacca agaattagat attaatcgtt taagtgatta 2521 tgatgtcgat gccattgttc cacaaagttt ccttaaagac gattcaatag acaataaggt 2581 cttaacgcgt tctgataaaa atcgtggtaa atcggataac gttccaagtg aagaagtagt 2641 caaaaagatg aaaaactatt ggagacaact tctaaacgcc aagttaatca ctcaacgtaa 2701 gtttgataat ttaacgaaag ctgaacgtgg aggtttgagt gaacttgata aagctggttt 2761 tatcaaacgc caattggttg aaactcgcca aatcactaag catgtggcac aaattttgga 2821 tagtcgcatg aatactaaat acgatgaaaa tgataaactt attcgagagg ttaaagtgat 2881 taccttaaaa tctaaattag tttctgactt ccgaaaagat ttccaattct ataaagtacg 2941 tgagattaac aattaccatc atgcccatga tgcgtatcta aatgccgtcg ttggaactgc 3001 tttgattaag aaatatccaa aacttgaatc ggagtttgtc tatggtgatt ataaagttta 3061 tgatgttcgt aaaatgattg ctaagtctga gcaagaaata ggcaaagcaa ccgcaaaata 3121 tttcttttac tctaatatca tgaacttctt caaaacagaa attacacttg caaatggaga 3181 gattcgcaaa cgccctctaa tcgaaactaa tggggaaact ggagaaattg tctgggataa 3241 agggcgagat tttgccacag tgcgcaaagt attgtccatg ccccaagtca atattgtcaa 3301 gaaaacagaa gtacagacag gcggattctc caaggagtca attttaccaa aaagaaattc 3361 ggacaagctt attgctcgta aaaaagactg ggatccaaaa aaatatggtg gttttgatag 3421 tccaacggta gcttattcag tcctagtggt tgctaaggtg gaaaaaggga aatcgaagaa 3481 gttaaaatcc gttaaagagt tactagggat cacaattatg gaaagaagtt cctttgaaaa 3541 aaatccgatt gactttttag aagctaaagg atataaggaa gttaaaaaag acttaatcat 3601 taaactacct aaatatagtc tttttgagtt agaaaacggt cgtaaacgga tgctggctag 3661 tgccggagaa ttacaaaaag gaaatgagct ggctctgcca agcaaatatg tgaatttttt 3721 atatttagct agtcattatg aaaagttgaa gggtagtcca gaagataacg aacaaaaaca 3781 attgtttgtg gagcagcata agcattattt agatgagatt attgagcaaa tcagtgaatt 3841 ttctaagcgt gttattttag cagatgccaa tttagataaa gttcttagtg catataacaa 3901 acatagagac aaaccaatac gtgaacaagc agaaaatatt attcatttat ttacgttgac 3961 gaatcttgga gctcccgctg cttttaaata ttttgataca acaattgatc gtaaacgata 4021 tacgtctaca aaagaagttt tagatgccac tcttatccat caatccatca ctggtcttta 4081 tgaaacacgc attgatttga gtcagctagg aggtgacggt ggaggaggtt ctggaggtgg 4141 aggttctgct gagtatgtgc gagccctctt tgactttaat gggaatgatg aagaggatct 4201 tccctttaag aaaggagaca tcctgagaat ccgggataag cctgaggagc agtggtggaa 4261 tgcagaggac agcgaaggaa agagggggat gattcctgtc ccttacgtgg agaagtattc 4321 cggagactat aaggaccacg acggagacta caaggatcat gatattgatt acaaagacga 4381 tgacgataag tctaggctcg agtccggaga ctataaggac cacgacggag actacaagga 4441 tcatgatatt gattacaaag acgatgacga taagtctagg atgaccgacg ctgagtacgt 4501 gagaatccat gagaagttgg acatctacac gtttaagaaa cagtttttca acaacaaaaa 4561 atccgtgtcg catagatgct acgttctctt tgaattaaaa cgacggggtg aacgtagagc 4621 gtgtttttgg ggctatgctg tgaataaacc acagagcggg acagaacgtg gcattcacgc 4681 cgaaatcttt agcattagaa aagtcgaaga atacctgcgc gacaaccccg gacaattcac 4741 gataaattgg tactcatcct ggagtccttg tgcagattgc gctgaaaaga tcttagaatg 4801 gtataaccag gagctgcggg ggaacggcca cactttgaaa atctgggctt gcaaactcta 4861 ttacgagaaa aatgcgagga atcaaattgg gctgtggaat ctcagagata acggggttgg 4921 gttgaatgta atggtaagtg aacactacca atgttgcagg aaaatattca tccaatcgtc 4981 gcacaatcaa ttgaatgaga atagatggct tgagaagact ttgaagcgag ctgaaaaacg 5041 acggagcgag ttgtccatta tgattcaggt aaaaatactc cacaccacta agagtcctgc 5101 tgtttctaga atgaccaacc tttccgacat catagagaag gaaacaggca aacagttggt 5161 catccaagag tcgatactca tgcttcctga agaagttgag gaggtcattg ggaataagcc 5221 ggaaagtgac attctcgtac acactgcgta tgatgagagc accgatgaga acgtgatgct 5281 gctcacgtca gatgccccag agtacaaacc ctgggctctg gtgattcagg actctaatgg 5341 agagaacaag atcaagatgc taggactggt tgcataatca gtgagaccga attctcgccc 5401 tatagtgagt cgtattacct ggccgtcgtt ttacaacgtc gtgactggga aaaccctggc 5461 gttacccaac ttaatcgcct tgcagcacat ccccctttcg ccagctggcg taatagcgaa 5521 gaggcccgca ccgatcgccc ttcccaacag ttgcgcagcc tgaatggcga atggcgcctg 5581 atgcggtatt ttctccttac gcatctgtgc ggtatttcac accgcatatg gtgcactctc 5641 agtacaatct gctctgatgc cgcatagtta agccagcccc gacacccgcc aacacccgct 5701 gacgcgccct gacgggcttg tctgctcccg gcatccgctt acagacaagc tgtgaccgtc 5761 tccgggagct gcatgtgtca gaggttttca ccgtcatcac cgaaacgcgc gagacgaaag 5821 ggcctcgtga tacgcctatt tttataggtt aatgtcatga taataatggt ttcttagacg 5881 tcaggtggca cttttcgggg aaatgtgcgc ggaaccccta tttgtttatt tttctaaata 5941 cattcaaata tgtatccgct catgagacaa taaccctgat aaatgcttca ataatattga 6001 aaaaggaaga gtatgagtat tcaacatttc cgtgtcgccc ttattccctt ttttgcggca 6061 ttttgccttc ctgtttttgc tcacccagaa acgctggtga aagtaaaaga tgctgaagat 6121 cagttgggtg cacgagtggg ttacatcgaa ctggatctca acagcggtaa gatccttgag 6181 agttttcgcc ccgaagaacg ttttccaatg atgagcactt ttaaagttct gctatgtggc 6241 gcggtattat cccgtattga cgccgggcaa gagcaactcg gtcgccgcat acactattct 6301 cagaatgact tggttgagta ctcaccagtc acagaaaagc atcttacgga tggcatgaca 6361 gtaagagaat tatgcagtgc tgccataacc atgagtgata acactgcggc caacttactt 6421 ctgacaacga tcggaggacc gaaggagcta accgcttttt tgcacaacat gggggatcat 6481 gtaactcgcc ttgatcgttg ggaaccggag ctgaatgaag ccataccaaa cgacgagcgt 6541 gacaccacga tgcctgtagc aatggcaaca acgttgcgca aactattaac tggcgaacta 6601 cttactctag cttcccggca acaattaata gactggatgg aggcggataa agttgcagga 6661 ccacttctgc gctcggccct tccggctggc tggtttattg ctgataaatc tggagccggt 6721 gagcgtggct ctcgaggtat cattgcagca ctggggccag atggtaagcc ctcccgtatc 6781 gtagttatct acacgacggg gagtcaggca actatggatg aacgaaatag acagatcgct 6841 gagataggtg cctcactgat taagcattgg taactgtcag accaagttta ctcatatata 6901 ctttagattg atttaaaact tcatttttaa tttaaaagga tctaggtgaa gatccttttt 6961 gataatctca tgaccaaaat cccttaacgt gagttttcgt tccactgagc gtcagacccc 7021 gtagaaaaga tcaaaggatc ttcttgagat cctttttttc tgcgcgtaat ctgctgcttg 7081 caaacaaaaa aaccaccgct accagcggtg gtttgtttgc cggatcaaga gctaccaact 7141 ctttttccga aggtaactgg cttcagcaga gcgcagatac caaatactgt tcttctagtg 7201 tagccgtagt taggccacca cttcaagaac tctgtagcac cgcctacata cctcgctctg 7261 ctaatcctgt taccagtggc tgctgccagt ggcgataagt cgtgtcttac cgggttggac 7321 tcaagacgat agttaccgga taaggcgcag cggtcgggct gaacgggggg ttcgtgcaca 7381 cagcccagct tggagcgaac gacctacacc gaactgagat acctacagcg tgagctatga 7441 gaaagcgcca cgcttcccga agggagaaag gcggacaggt atccggtaag cggcagggtc 7501 ggaacaggag agcgcacgag ggagcttcca gggggaaacg cctggtatct ttatagtcct 7561 gtcgggtttc gccacctctg acttgagcgt cgatttttgt gatgctcgtc aggggggcgg 7621 agcctatgga aaaacgccag caacgcggcc tttttacggt tcctggcctt ttgctggcct 7681 tttgctcaca tgttctttcc tgcgttatcc cctgattctg tggataaccg tattaccgcc 7741 tttgagtgag ctgataccgc tcgccgcagc cgaacgaccg agcgcagcga gtcagtgagc 7801 gaggaagcgg aagagcgccc aatacgcaaa ccgcctctcc ccgcgcgttg gccgattcat 7861 taatgcagct ggcacgacag gtttcccgac tggaaagcgg gcagtgagcg caacgcaatt 7921 aatgtgagtt agctcactca ttaggcaccc caggctttac actttatgct tccggctcgt 7981 atgttgtgtg gaattgtgag cggataacaa tttcacacga gctatttagg tgacactata 8041 gaagtgaagc tt //