LOCUS Exported 8014 bp ds-DNA circular SYN 13-MAY-2021 DEFINITION Expression plasmid for human codon-optimized dead/inactive SpCas9 (without U6-sgRNA coding sequence). ACCESSION . VERSION . KEYWORDS pX330-HA-dSpCas9 SOURCE synthetic DNA construct ORGANISM synthetic DNA construct REFERENCE 1 (bases 1 to 8014) AUTHORS Kulcsar PI, Talas A, Huszar K, Ligeti Z, Toth E, Weinhardt N, Fodor E, Welker E TITLE Crossing enhanced and high fidelity SpCas9 nucleases to optimize specificity and cleavage. JOURNAL Genome Biol. 2017 Oct 6;18(1):190. doi: 10.1186/s13059-017-1318-8. PUBMED 28985763 REFERENCE 2 (bases 1 to 8014) AUTHORS . TITLE Direct Submission JOURNAL Exported May 13, 2021 from SnapGene Server 1.1.58 http://www.snapgene.com FEATURES Location/Qualifiers source 1..8014 /organism="synthetic DNA construct" /mol_type="other DNA" enhancer 1..286 /label=CMV enhancer /note="human cytomegalovirus immediate early enhancer; contains an 18-bp deletion relative to the standard CMV enhancer" promoter 288..563 /label=chicken beta-actin promoter intron 564..791 /label=hybrid intron /note="hybrid between chicken beta-actin (CBA) and minute virus of mice (MMV) introns (Gray et al., 2011)" CDS 812..838 /codon_start=1 /product="HA (human influenza hemagglutinin) epitope tag" /label=HA /translation="YPYDVPDYA" CDS 842..862 /codon_start=1 /product="nuclear localization signal of SV40 (simian virus 40) large T antigen" /label=SV40 NLS /translation="PKKKRKV" CDS 872..4972 /codon_start=1 /product="catalytically dead mutant of the Cas9 endonuclease from the Streptococcus pyogenes Type II CRISPR/Cas system" /label=dCas9 /note="RNA-guided DNA-binding protein that lacks endonuclease activity due to the D10A mutation in the RuvC catalytic domain and the H840A mutation in the HNH catalytic domain" /translation="DKKYSIGLAIGTNSVGWAVITDEYKVPSKKFKVLGNTDRHSIKKN LIGALLFDSGETAEATRLKRTARRRYTRRKNRICYLQEIFSNEMAKVDDSFFHRLEESF LVEEDKKHERHPIFGNIVDEVAYHEKYPTIYHLRKKLVDSTDKADLRLIYLALAHMIKF RGHFLIEGDLNPDNSDVDKLFIQLVQTYNQLFEENPINASGVDAKAILSARLSKSRRLE NLIAQLPGEKKNGLFGNLIALSLGLTPNFKSNFDLAEDAKLQLSKDTYDDDLDNLLAQI GDQYADLFLAAKNLSDAILLSDILRVNTEITKAPLSASMIKRYDEHHQDLTLLKALVRQ QLPEKYKEIFFDQSKNGYAGYIDGGASQEEFYKFIKPILEKMDGTEELLVKLNREDLLR KQRTFDNGSIPHQIHLGELHAILRRQEDFYPFLKDNREKIEKILTFRIPYYVGPLARGN SRFAWMTRKSEETITPWNFEEVVDKGASAQSFIERMTNFDKNLPNEKVLPKHSLLYEYF TVYNELTKVKYVTEGMRKPAFLSGEQKKAIVDLLFKTNRKVTVKQLKEDYFKKIECFDS VEISGVEDRFNASLGTYHDLLKIIKDKDFLDNEENEDILEDIVLTLTLFEDREMIEERL KTYAHLFDDKVMKQLKRRRYTGWGRLSRKLINGIRDKQSGKTILDFLKSDGFANRNFMQ LIHDDSLTFKEDIQKAQVSGQGDSLHEHIANLAGSPAIKKGILQTVKVVDELVKVMGRH KPENIVIEMARENQTTQKGQKNSRERMKRIEEGIKELGSQILKEHPVENTQLQNEKLYL YYLQNGRDMYVDQELDINRLSDYDVDAIVPQSFLKDDSIDNKVLTRSDKNRGKSDNVPS EEVVKKMKNYWRQLLNAKLITQRKFDNLTKAERGGLSELDKAGFIKRQLVETRQITKHV AQILDSRMNTKYDENDKLIREVKVITLKSKLVSDFRKDFQFYKVREINNYHHAHDAYLN AVVGTALIKKYPKLESEFVYGDYKVYDVRKMIAKSEQEIGKATAKYFFYSNIMNFFKTE ITLANGEIRKRPLIETNGETGEIVWDKGRDFATVRKVLSMPQVNIVKKTEVQTGGFSKE SILPKRNSDKLIARKKDWDPKKYGGFDSPTVAYSVLVVAKVEKGKSKKLKSVKELLGIT IMERSSFEKNPIDFLEAKGYKEVKKDLIIKLPKYSLFELENGRKRMLASAGELQKGNEL ALPSKYVNFLYLASHYEKLKGSPEDNEQKQLFVEQHKHYLDEIIEQISEFSKRVILADA NLDKVLSAYNKHRDKPIREQAENIIHLFTLTNLGAPAAFKYFDTTIDRKRYTSTKEVLD ATLIHQSITGLYETRIDLSQLGGD" CDS 4973..5020 /codon_start=1 /product="bipartite nuclear localization signal from nucleoplasmin" /label=nucleoplasmin NLS /translation="KRPAATKKAGQAKKKK" primer_bind complement(5045..5062) /label=BGH-rev /note="Bovine growth hormone terminator, reverse primer. Also called BGH reverse" polyA_signal 5051..5258 /label=bGH poly(A) signal /note="bovine growth hormone polyadenylation signal" repeat_region 5267..5407 /label=AAV2 ITR /note="inverted terminal repeat of adeno-associated virus serotype 2" repeat_region 5267..5396 /label=AAV2 ITR rep_origin 5482..5937 /direction=RIGHT /label=f1 ori /note="f1 bacteriophage origin of replication; arrow indicates direction of (+) strand synthesis" primer_bind complement(5569..5588) /label=F1ori-R /note="F1 origin, reverse primer" primer_bind 5779..5800 /label=F1ori-F /note="F1 origin, forward primer" primer_bind complement(5954..5973) /label=pRS-marker /note="pRS vectors, use to sequence yeast selectable marker" primer_bind 6073..6095 /label=pGEX 3' /note="pGEX vectors, reverse primer" primer_bind complement(6133..6151) /label=pBRforEco /note="pBR322 vectors, upsteam of EcoRI site, forward primer" promoter 6219..6323 /gene="bla" /label=AmpR promoter CDS 6324..7184 /codon_start=1 /gene="bla" /product="beta-lactamase" /label=AmpR /note="confers resistance to ampicillin, carbenicillin, and related antibiotics" /translation="MSIQHFRVALIPFFAAFCLPVFAHPETLVKVKDAEDQLGARVGYI ELDLNSGKILESFRPEERFPMMSTFKVLLCGAVLSRIDAGQEQLGRRIHYSQNDLVEYS PVTEKHLTDGMTVRELCSAAITMSDNTAANLLLTTIGGPKELTAFLHNMGDHVTRLDRW EPELNEAIPNDERDTTMPVAMATTLRKLLTGELLTLASRQQLIDWMEADKVAGPLLRSA LPAGWFIADKSGAGERGSRGIIAALGPDGKPSRIVVIYTTGSQATMDERNRQIAEIGAS LIKHW" primer_bind complement(6542..6561) /label=Amp-R /note="Ampicillin resistance gene, reverse primer" rep_origin 7355..7943 /direction=RIGHT /label=ori /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of replication" primer_bind 7844..7863 /label=pBR322ori-F /note="pBR322 origin, forward primer" ORIGIN 1 cgttacataa cttacggtaa atggcccgcc tggctgaccg cccaacgacc cccgcccatt 61 gacgtcaata gtaacgccaa tagggacttt ccattgacgt caatgggtgg agtatttacg 121 gtaaactgcc cacttggcag tacatcaagt gtatcatatg ccaagtacgc cccctattga 181 cgtcaatgac ggtaaatggc ccgcctggca ttgtgcccag tacatgacct tatgggactt 241 tcctacttgg cagtacatct acgtattagt catcgctatt accatggtcg aggtgagccc 301 cacgttctgc ttcactctcc ccatctcccc cccctcccca cccccaattt tgtatttatt 361 tattttttaa ttattttgtg cagcgatggg ggcggggggg gggggggcgc gcgccaggcg 421 gggcggggcg gggcgagggg cggggcgggg cgaggcggag aggtgcggcg gcagccaatc 481 agagcggcgc gctccgaaag tttcctttta tggcgaggcg gcggcggcgg cggccctata 541 aaaagcgaag cgcgcggcgg gcgggagtcg ctgcgcgctg ccttcgcccc gtgccccgct 601 ccgccgccgc ctcgcgccgc ccgccccggc tctgactgac cgcgttactc ccacaggtga 661 gcgggcggga cggcccttct cctccgggct gtaattagct gagcaagagg taagggttta 721 agggatggtt ggttggtggg gtattaatgt ttaattacct ggagcacctg cctgaaatca 781 ctttttttca ggttggaccg gtgccaccat gtacccatac gatgttccag attacgcttc 841 gccgaagaaa aagcgcaagg tcgaagcgtc cgacaagaag tacagcatcg gcctggccat 901 cggcaccaac tctgtgggct gggccgtgat caccgacgag tacaaggtgc ccagcaagaa 961 attcaaggtg ctgggcaaca ccgaccggca cagcatcaag aagaacctga tcggagccct 1021 gctgttcgac agcggcgaaa cagccgaggc cacccggctg aagagaaccg ccagaagaag 1081 atacaccaga cggaagaacc ggatctgcta tctgcaagag atcttcagca acgagatggc 1141 caaggtggac gacagcttct tccacagact ggaagagtcc ttcctggtgg aagaggataa 1201 gaagcacgag cggcacccca tcttcggcaa catcgtggac gaggtggcct accacgagaa 1261 gtaccccacc atctaccacc tgagaaagaa actggtggac agcaccgaca aggccgacct 1321 gcggctgatc tatctggccc tggcccacat gatcaagttc cggggccact tcctgatcga 1381 gggcgacctg aaccccgaca acagcgacgt ggacaagctg ttcatccagc tggtgcagac 1441 ctacaaccag ctgttcgagg aaaaccccat caacgccagc ggcgtggacg ccaaggccat 1501 cctgtctgcc agactgagca agagcagacg gctggaaaat ctgatcgccc agctgcccgg 1561 cgagaagaag aatggcctgt tcggcaacct gattgccctg agcctgggcc tgacccccaa 1621 cttcaagagc aacttcgacc tggccgagga tgccaaactg cagctgagca aggacaccta 1681 cgacgacgac ctggacaacc tgctggccca gatcggcgac cagtacgccg acctgtttct 1741 ggccgccaag aacctgtccg acgccatcct gctgagcgac atcctgagag tgaacaccga 1801 gatcaccaag gcccccctga gcgcctctat gatcaagaga tacgacgagc accaccagga 1861 cctgaccctg ctgaaagctc tcgtgcggca gcagctgcct gagaagtaca aagagatttt 1921 cttcgaccag agcaagaacg gctacgccgg ctacattgac ggcggagcca gccaggaaga 1981 gttctacaag ttcatcaagc ccatcctgga aaagatggac ggcaccgagg aactgctcgt 2041 gaagctgaac agagaggacc tgctgcggaa gcagcggacc ttcgacaacg gcagcatccc 2101 ccaccagatc cacctgggag agctgcacgc cattctgcgg cggcaggaag atttttaccc 2161 attcctgaag gacaaccggg aaaagatcga gaagatcctg accttccgca tcccctacta 2221 cgtgggccct ctggccaggg gaaacagcag attcgcctgg atgaccagaa agagcgagga 2281 aaccatcacc ccctggaact tcgaggaagt ggtggacaag ggcgcttccg cccagagctt 2341 catcgagcgg atgaccaact tcgataagaa cctgcccaac gagaaggtgc tgcccaagca 2401 cagcctgctg tacgagtact tcaccgtgta taacgagctg accaaagtga aatacgtgac 2461 cgagggaatg agaaagcccg ccttcctgag cggcgagcag aaaaaggcca tcgtggacct 2521 gctgttcaag accaaccgga aagtgaccgt gaagcagctg aaagaggact acttcaagaa 2581 aatcgagtgc ttcgactccg tggaaatctc cggcgtggaa gatcggttca acgcctccct 2641 gggcacatac cacgatctgc tgaaaattat caaggacaag gacttcctgg acaatgagga 2701 aaacgaggac attctggaag atatcgtgct gaccctgaca ctgtttgagg acagagagat 2761 gatcgaggaa cggctgaaaa cctatgccca cctgttcgac gacaaagtga tgaagcagct 2821 gaagcggcgg agatacaccg gctggggcag gctgagccgg aagctgatca acggcatccg 2881 ggacaagcag tccggcaaga caatcctgga tttcctgaag tccgacggct tcgccaacag 2941 aaacttcatg cagctgatcc acgacgacag cctgaccttt aaagaggaca tccagaaagc 3001 ccaggtgtcc ggccagggcg atagcctgca cgagcacatt gccaatctgg ccggcagccc 3061 cgccattaag aagggcatcc tgcagacagt gaaggtggtg gacgagctcg tgaaagtgat 3121 gggccggcac aagcccgaga acatcgtgat cgaaatggcc agagagaacc agaccaccca 3181 gaagggacag aagaacagcc gcgagagaat gaagcggatc gaagagggca tcaaagagct 3241 gggcagccag atcctgaaag aacaccccgt ggaaaacacc cagctgcaga acgagaagct 3301 gtacctgtac tacctgcaga atgggcggga tatgtacgtg gaccaggaac tggacatcaa 3361 ccggctgtcc gactacgatg tggacgccat cgtgcctcag agctttctga aggacgactc 3421 catcgacaac aaggtgctga ccagaagcga caagaaccgg ggcaagagcg acaacgtgcc 3481 ctccgaagag gtcgtgaaga agatgaagaa ctactggcgg cagctgctga acgccaagct 3541 gattacccag agaaagttcg acaatctgac caaggccgag agaggcggcc tgagcgaact 3601 ggataaggcc ggcttcatca agagacagct ggtggaaacc cggcagatca caaagcacgt 3661 ggcacagatc ctggactccc ggatgaacac taagtacgac gagaatgaca agctgatccg 3721 ggaagtgaaa gtgatcaccc tgaagtccaa gctggtgtcc gatttccgga aggatttcca 3781 gttttacaaa gtgcgcgaga tcaacaacta ccaccacgcc cacgacgcct acctgaacgc 3841 cgtcgtggga accgccctga tcaaaaagta ccctaagctg gaaagcgagt tcgtgtacgg 3901 cgactacaag gtgtacgacg tgcggaagat gatcgccaag agcgagcagg aaatcggcaa 3961 ggctaccgcc aagtacttct tctacagcaa catcatgaac tttttcaaga ccgagattac 4021 cctggccaac ggcgagatcc ggaagcggcc tctgatcgag acaaacggcg aaaccgggga 4081 gatcgtgtgg gataagggcc gggattttgc caccgtgcgg aaagtgctga gcatgcccca 4141 agtgaatatc gtgaaaaaga ccgaggtgca gacaggcggc ttcagcaaag agtctatcct 4201 gcccaagagg aacagcgata agctgatcgc cagaaagaag gactgggacc ctaagaagta 4261 cggcggcttc gacagcccca ccgtggccta ttctgtgctg gtggtggcca aagtggaaaa 4321 gggcaagtcc aagaaactga agagtgtgaa agagctgctg gggatcacca tcatggaaag 4381 aagcagcttc gagaagaatc ccatcgactt tctggaagcc aagggctaca aagaagtgaa 4441 aaaggacctg atcatcaagc tgcctaagta ctccctgttc gagctggaaa acggccggaa 4501 gagaatgctg gcctctgccg gcgaactgca gaagggaaac gaactggccc tgccctccaa 4561 atatgtgaac ttcctgtacc tggccagcca ctatgagaag ctgaagggct cccccgagga 4621 taatgagcag aaacagctgt ttgtggaaca gcacaagcac tacctggacg agatcatcga 4681 gcagatcagc gagttctcca agagagtgat cctggccgac gctaatctgg acaaagtgct 4741 gtccgcctac aacaagcacc gggataagcc catcagagag caggccgaga atatcatcca 4801 cctgtttacc ctgaccaatc tgggagcccc tgccgccttc aagtactttg acaccaccat 4861 cgaccggaag aggtacacca gcaccaaaga ggtgctggac gccaccctga tccaccagag 4921 catcaccggc ctgtacgaga cacggatcga cctgtctcag ctgggaggcg acaaaaggcc 4981 ggcggccacg aaaaaggccg gccaggcaaa aaagaaaaag taattcctag agctcgctga 5041 tcagcctcga ctgtgccttc tagttgccag ccatctgttg tttgcccctc ccccgtgcct 5101 tccttgaccc tggaaggtgc cactcccact gtcctttcct aataaaatga ggaaattgca 5161 tcgcattgtc tgagtaggtg tcattctatt ctggggggtg gggtggggca ggacagcaag 5221 ggggaggatt gggaagagaa tagcaggcat gctggggagc ggccgcagga acccctagtg 5281 atggagttgg ccactccctc tctgcgcgct cgctcgctca ctgaggccgg gcgaccaaag 5341 gtcgcccgac gcccgggctt tgcccgggcg gcctcagtga gcgagcgagc gcgcagctgc 5401 ctgcaggggc gcctgatgcg gtattttctc cttacgcatc tgtgcggtat ttcacaccgc 5461 atacgtcaaa gcaaccatag tacgcgccct gtagcggcgc attaagcgcg gcgggtgtgg 5521 tggttacgcg cagcgtgacc gctacacttg ccagcgcctt agcgcccgct cctttcgctt 5581 tcttcccttc ctttctcgcc acgttcgccg gctttccccg tcaagctcta aatcgggggc 5641 tccctttagg gttccgattt agtgctttac ggcacctcga ccccaaaaaa cttgatttgg 5701 gtgatggttc acgtagtggg ccatcgccct gatagacggt ttttcgccct ttgacgttgg 5761 agtccacgtt ctttaatagt ggactcttgt tccaaactgg aacaacactc aactctatct 5821 cgggctattc ttttgattta taagggattt tgccgatttc ggtctattgg ttaaaaaatg 5881 agctgattta acaaaaattt aacgcgaatt ttaacaaaat attaacgttt acaattttat 5941 ggtgcactct cagtacaatc tgctctgatg ccgcatagtt aagccagccc cgacacccgc 6001 caacacccgc tgacgcgccc tgacgggctt gtctgctccc ggcatccgct tacagacaag 6061 ctgtgaccgt ctccgggagc tgcatgtgtc agaggttttc accgtcatca ccgaaacgcg 6121 cgagacgaaa gggcctcgtg atacgcctat ttttataggt taatgtcatg ataataatgg 6181 tttcttagac gtcaggtggc acttttcggg gaaatgtgcg cggaacccct atttgtttat 6241 ttttctaaat acattcaaat atgtatccgc tcatgagaca ataaccctga taaatgcttc 6301 aataatattg aaaaaggaag agtatgagta ttcaacattt ccgtgtcgcc cttattccct 6361 tttttgcggc attttgcctt cctgtttttg ctcacccaga aacgctggtg aaagtaaaag 6421 atgctgaaga tcagttgggt gcacgagtgg gttacatcga actggatctc aacagcggta 6481 agatccttga gagttttcgc cccgaagaac gttttccaat gatgagcact tttaaagttc 6541 tgctatgtgg cgcggtatta tcccgtattg acgccgggca agagcaactc ggtcgccgca 6601 tacactattc tcagaatgac ttggttgagt actcaccagt cacagaaaag catcttacgg 6661 atggcatgac agtaagagaa ttatgcagtg ctgccataac catgagtgat aacactgcgg 6721 ccaacttact tctgacaacg atcggaggac cgaaggagct aaccgctttt ttgcacaaca 6781 tgggggatca tgtaactcgc cttgatcgtt gggaaccgga gctgaatgaa gccataccaa 6841 acgacgagcg tgacaccacg atgcctgtag caatggcaac aacgttgcgc aaactattaa 6901 ctggcgaact acttactcta gcttcccggc aacaattaat agactggatg gaggcggata 6961 aagttgcagg accacttctg cgctcggccc ttccggctgg ctggtttatt gctgataaat 7021 ctggagccgg tgagcgtgga agccgcggta tcattgcagc actggggcca gatggtaagc 7081 cctcccgtat cgtagttatc tacacgacgg ggagtcaggc aactatggat gaacgaaata 7141 gacagatcgc tgagataggt gcctcactga ttaagcattg gtaactgtca gaccaagttt 7201 actcatatat actttagatt gatttaaaac ttcattttta atttaaaagg atctaggtga 7261 agatcctttt tgataatctc atgaccaaaa tcccttaacg tgagttttcg ttccactgag 7321 cgtcagaccc cgtagaaaag atcaaaggat cttcttgaga tccttttttt ctgcgcgtaa 7381 tctgctgctt gcaaacaaaa aaaccaccgc taccagcggt ggtttgtttg ccggatcaag 7441 agctaccaac tctttttccg aaggtaactg gcttcagcag agcgcagata ccaaatactg 7501 ttcttctagt gtagccgtag ttaggccacc acttcaagaa ctctgtagca ccgcctacat 7561 acctcgctct gctaatcctg ttaccagtgg ctgctgccag tggcgataag tcgtgtctta 7621 ccgggttgga ctcaagacga tagttaccgg ataaggcgca gcggtcgggc tgaacggggg 7681 gttcgtgcac acagcccagc ttggagcgaa cgacctacac cgaactgaga tacctacagc 7741 gtgagctatg agaaagcgcc acgcttcccg aagggagaaa ggcggacagg tatccggtaa 7801 gcggcagggt cggaacagga gagcgcacga gggagcttcc agggggaaac gcctggtatc 7861 tttatagtcc tgtcgggttt cgccacctct gacttgagcg tcgatttttg tgatgctcgt 7921 caggggggcg gagcctatgg aaaaacgcca gcaacgcggc ctttttacgg ttcctggcct 7981 tttgctggcc ttttgctcac atgctagagg tacc //