LOCUS Exported 8937 bp ds-DNA circular SYN 12-MAY-2021 DEFINITION Expresses eSpCas9(1.1) and two sgRNAs. The first gRNA targets human IFT88 and the second gRNA targets a pDonor-tBFP-NLS-Neo (Universal).. ACCESSION . VERSION . KEYWORDS peSpCas9(1.1)-2×sgRNA (IFT88, donor) SOURCE synthetic DNA construct ORGANISM synthetic DNA construct REFERENCE 1 (bases 1 to 8937) AUTHORS Katoh Y, Michisaka S, Nozaki S, Funabashi T, Hirano T, Takei R, Nakayama K TITLE Practical method for targeted disruption of cilia-related genes by using CRISPR/Cas9-mediated homology-independent knock-in system. JOURNAL Mol Biol Cell. 2017 Feb 8. pii: mbc.E17-01-0051. doi: 10.1091/mbc.E17-01-0051. PUBMED 28179459 REFERENCE 2 (bases 1 to 8937) AUTHORS . TITLE Direct Submission JOURNAL Exported May 12, 2021 from SnapGene Server 1.1.58 http://www.snapgene.com FEATURES Location/Qualifiers source 1..8937 /organism="synthetic DNA construct" /mol_type="other DNA" promoter 1..241 /label=U6 promoter /note="RNA polymerase III promoter for human U6 snRNA" misc_RNA 270..345 /label=gRNA scaffold /note="guide RNA scaffold for the Streptococcus pyogenes CRISPR/Cas9 system" promoter 436..676 /label=U6 promoter /note="RNA polymerase III promoter for human U6 snRNA" misc_RNA 705..780 /label=gRNA scaffold /note="guide RNA scaffold for the Streptococcus pyogenes CRISPR/Cas9 system" enhancer 871..1156 /label=CMV enhancer /note="human cytomegalovirus immediate early enhancer; contains an 18-bp deletion relative to the standard CMV enhancer" promoter 1158..1435 /label=chicken beta-actin promoter intron 1436..1664 /label=hybrid intron /note="hybrid between chicken beta-actin (CBA) and minute virus of mice (MMV) introns (Gray et al., 2011)" regulatory 1676..1685 /regulatory_class="other" /note="vertebrate consensus sequence for strong initiation of translation (Kozak, 1987)" CDS 1685..1750 /codon_start=1 /product="three tandem FLAG(R) epitope tags, followed by an enterokinase cleavage site" /label=3xFLAG /translation="DYKDHDGDYKDHDIDYKDDDDK" CDS 1757..1777 /codon_start=1 /product="nuclear localization signal of SV40 (simian virus 40) large T antigen" /label=SV40 NLS /translation="PKKKRKV" CDS 1802..5902 /codon_start=1 /product="Cas9 endonuclease from the Streptococcus pyogenes Type II CRISPR/Cas system, mutated to improve targeting specificity (Slaymaker et al., 2016)" /label=eSpCas9(1.1) /note="carries the mutations K848A, K1003A, and R1060A" /translation="DKKYSIGLDIGTNSVGWAVITDEYKVPSKKFKVLGNTDRHSIKKN LIGALLFDSGETAEATRLKRTARRRYTRRKNRICYLQEIFSNEMAKVDDSFFHRLEESF LVEEDKKHERHPIFGNIVDEVAYHEKYPTIYHLRKKLVDSTDKADLRLIYLALAHMIKF RGHFLIEGDLNPDNSDVDKLFIQLVQTYNQLFEENPINASGVDAKAILSARLSKSRRLE NLIAQLPGEKKNGLFGNLIALSLGLTPNFKSNFDLAEDAKLQLSKDTYDDDLDNLLAQI GDQYADLFLAAKNLSDAILLSDILRVNTEITKAPLSASMIKRYDEHHQDLTLLKALVRQ QLPEKYKEIFFDQSKNGYAGYIDGGASQEEFYKFIKPILEKMDGTEELLVKLNREDLLR KQRTFDNGSIPHQIHLGELHAILRRQEDFYPFLKDNREKIEKILTFRIPYYVGPLARGN SRFAWMTRKSEETITPWNFEEVVDKGASAQSFIERMTNFDKNLPNEKVLPKHSLLYEYF TVYNELTKVKYVTEGMRKPAFLSGEQKKAIVDLLFKTNRKVTVKQLKEDYFKKIECFDS VEISGVEDRFNASLGTYHDLLKIIKDKDFLDNEENEDILEDIVLTLTLFEDREMIEERL KTYAHLFDDKVMKQLKRRRYTGWGRLSRKLINGIRDKQSGKTILDFLKSDGFANRNFMQ LIHDDSLTFKEDIQKAQVSGQGDSLHEHIANLAGSPAIKKGILQTVKVVDELVKVMGRH KPENIVIEMARENQTTQKGQKNSRERMKRIEEGIKELGSQILKEHPVENTQLQNEKLYL YYLQNGRDMYVDQELDINRLSDYDVDHIVPQSFLADDSIDNKVLTRSDKNRGKSDNVPS EEVVKKMKNYWRQLLNAKLITQRKFDNLTKAERGGLSELDKAGFIKRQLVETRQITKHV AQILDSRMNTKYDENDKLIREVKVITLKSKLVSDFRKDFQFYKVREINNYHHAHDAYLN AVVGTALIKKYPALESEFVYGDYKVYDVRKMIAKSEQEIGKATAKYFFYSNIMNFFKTE ITLANGEIRKAPLIETNGETGEIVWDKGRDFATVRKVLSMPQVNIVKKTEVQTGGFSKE SILPKRNSDKLIARKKDWDPKKYGGFDSPTVAYSVLVVAKVEKGKSKKLKSVKELLGIT IMERSSFEKNPIDFLEAKGYKEVKKDLIIKLPKYSLFELENGRKRMLASAGELQKGNEL ALPSKYVNFLYLASHYEKLKGSPEDNEQKQLFVEQHKHYLDEIIEQISEFSKRVILADA NLDKVLSAYNKHRDKPIREQAENIIHLFTLTNLGAPAAFKYFDTTIDRKRYTSTKEVLD ATLIHQSITGLYETRIDLSQLGGD" CDS 5903..5950 /codon_start=1 /product="bipartite nuclear localization signal from nucleoplasmin" /label=nucleoplasmin NLS /translation="KRPAATKKAGQAKKKK" primer_bind complement(5978..5995) /label=BGH-rev /note="Bovine growth hormone terminator, reverse primer. Also called BGH reverse" polyA_signal 5984..6191 /label=bGH poly(A) signal /note="bovine growth hormone polyadenylation signal" repeat_region 6200..6340 /label=AAV2 ITR /note="inverted terminal repeat of adeno-associated virus serotype 2" repeat_region 6200..6329 /label=AAV2 ITR rep_origin 6415..6870 /direction=RIGHT /label=f1 ori /note="f1 bacteriophage origin of replication; arrow indicates direction of (+) strand synthesis" primer_bind complement(6502..6521) /label=F1ori-R /note="F1 origin, reverse primer" primer_bind 6712..6733 /label=F1ori-F /note="F1 origin, forward primer" primer_bind complement(6887..6906) /label=pRS-marker /note="pRS vectors, use to sequence yeast selectable marker" primer_bind 7006..7028 /label=pGEX 3' /note="pGEX vectors, reverse primer" primer_bind complement(7066..7084) /label=pBRforEco /note="pBR322 vectors, upsteam of EcoRI site, forward primer" promoter 7152..7256 /gene="bla" /label=AmpR promoter CDS 7257..8117 /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(7475..7494) /label=Amp-R /note="Ampicillin resistance gene, reverse primer" rep_origin 8288..8876 /direction=RIGHT /label=ori /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of replication" primer_bind 8777..8796 /label=pBR322ori-F /note="pBR322 origin, forward primer" ORIGIN 1 gagggcctat ttcccatgat tccttcatat ttgcatatac gatacaaggc tgttagagag 61 ataattggaa ttaatttgac tgtaaacaca aagatattag tacaaaatac gtgacgtaga 121 aagtaataat ttcttgggta gtttgcagtt ttaaaattat gttttaaaat ggactatcat 181 atgcttaccg taacttgaaa gtatttcgat ttcttggctt tatatatctt gtggaaagga 241 cgaaacaccg ggccccttga ctgactaagg ttttagagct agaaatagca agttaaaata 301 aggctagtcc gttatcaact tgaaaaagtg gcaccgagtc ggtgcttttt tgttttagag 361 ctagaaatag caagttaaaa taaggctagt ccgtttttag cgcgtgcgcc aattctgcag 421 acaaatggct ctagagaggg cctatttccc atgattcctt catatttgca tatacgatac 481 aaggctgtta gagagataat tggaattaat ttgactgtaa acacaaagat attagtacaa 541 aatacgtgac gtagaaagta ataatttctt gggtagtttg cagttttaaa attatgtttt 601 aaaatggact atcatatgct taccgtaact tgaaagtatt tcgatttctt ggctttatat 661 atcttgtgga aaggacgaaa caccgcatcg tacgcgtacg tgttgtttta gagctagaaa 721 tagcaagtta aaataaggct agtccgttat caacttgaaa aagtggcacc gagtcggtgc 781 ttttttgttt tagagctaga aatagcaagt taaaataagg ctagtccgtt tttagcgcgt 841 gcgccaattc tgcagacaaa tggcggtacc cgttacataa cttacggtaa atggcccgcc 901 tggctgaccg cccaacgacc cccgcccatt gacgtcaata gtaacgccaa tagggacttt 961 ccattgacgt caatgggtgg agtatttacg gtaaactgcc cacttggcag tacatcaagt 1021 gtatcatatg ccaagtacgc cccctattga cgtcaatgac ggtaaatggc ccgcctggca 1081 ttgtgcccag tacatgacct tatgggactt tcctacttgg cagtacatct acgtattagt 1141 catcgctatt accatggtcg aggtgagccc cacgttctgc ttcactctcc ccatctcccc 1201 cccctcccca cccccaattt tgtatttatt tattttttaa ttattttgtg cagcgatggg 1261 ggcggggggg gggggggggc gcgcgccagg cggggcgggg cggggcgagg ggcggggcgg 1321 ggcgaggcgg agaggtgcgg cggcagccaa tcagagcggc gcgctccgaa agtttccttt 1381 tatggcgagg cggcggcggc ggcggcccta taaaaagcga agcgcgcggc gggcgggagt 1441 cgctgcgacg ctgccttcgc cccgtgcccc gctccgccgc cgcctcgcgc cgcccgcccc 1501 ggctctgact gaccgcgtta ctcccacagg tgagcgggcg ggacggccct tctcctccgg 1561 gctgtaatta gctgagcaag aggtaagggt ttaagggatg gttggttggt ggggtattaa 1621 tgtttaatta cctggagcac ctgcctgaaa tcactttttt tcaggttgga ccggtgccac 1681 catggactat aaggaccacg acggagacta caaggatcat gatattgatt acaaagacga 1741 tgacgataag atggccccaa agaagaagcg gaaggtcggt atccacggag tcccagcagc 1801 cgacaagaag tacagcatcg gcctggacat cggcaccaac tctgtgggct gggccgtgat 1861 caccgacgag tacaaggtgc ccagcaagaa attcaaggtg ctgggcaaca ccgaccggca 1921 cagcatcaag aagaacctga tcggagccct gctgttcgac agcggcgaaa cagccgaggc 1981 cacccggctg aagagaaccg ccagaagaag atacaccaga cggaagaacc ggatctgcta 2041 tctgcaagag atcttcagca acgagatggc caaggtggac gacagcttct tccacagact 2101 ggaagagtcc ttcctggtgg aagaggataa gaagcacgag cggcacccca tcttcggcaa 2161 catcgtggac gaggtggcct accacgagaa gtaccccacc atctaccacc tgagaaagaa 2221 actggtggac agcaccgaca aggccgacct gcggctgatc tatctggccc tggcccacat 2281 gatcaagttc cggggccact tcctgatcga gggcgacctg aaccccgaca acagcgacgt 2341 ggacaagctg ttcatccagc tggtgcagac ctacaaccag ctgttcgagg aaaaccccat 2401 caacgccagc ggcgtggacg ccaaggccat cctgtctgcc agactgagca agagcagacg 2461 gctggaaaat ctgatcgccc agctgcccgg cgagaagaag aatggcctgt tcggaaacct 2521 gattgccctg agcctgggcc tgacccccaa cttcaagagc aacttcgacc tggccgagga 2581 tgccaaactg cagctgagca aggacaccta cgacgacgac ctggacaacc tgctggccca 2641 gatcggcgac cagtacgccg acctgtttct ggccgccaag aacctgtccg acgccatcct 2701 gctgagcgac atcctgagag tgaacaccga gatcaccaag gcccccctga gcgcctctat 2761 gatcaagaga tacgacgagc accaccagga cctgaccctg ctgaaagctc tcgtgcggca 2821 gcagctgcct gagaagtaca aagagatttt cttcgaccag agcaagaacg gctacgccgg 2881 ctacattgac ggcggagcca gccaggaaga gttctacaag ttcatcaagc ccatcctgga 2941 aaagatggac ggcaccgagg aactgctcgt gaagctgaac agagaggacc tgctgcggaa 3001 gcagcggacc ttcgacaacg gcagcatccc ccaccagatc cacctgggag agctgcacgc 3061 cattctgcgg cggcaggaag atttttaccc attcctgaag gacaaccggg aaaagatcga 3121 gaagatcctg accttccgca tcccctacta cgtgggccct ctggccaggg gaaacagcag 3181 attcgcctgg atgaccagaa agagcgagga aaccatcacc ccctggaact tcgaggaagt 3241 ggtggacaag ggcgcttccg cccagagctt catcgagcgg atgaccaact tcgataagaa 3301 cctgcccaac gagaaggtgc tgcccaagca cagcctgctg tacgagtact tcaccgtgta 3361 taacgagctg accaaagtga aatacgtgac cgagggaatg agaaagcccg ccttcctgag 3421 cggcgagcag aaaaaggcca tcgtggacct gctgttcaag accaaccgga aagtgaccgt 3481 gaagcagctg aaagaggact acttcaagaa aatcgagtgc ttcgactccg tggaaatctc 3541 cggcgtggaa gatcggttca acgcctccct gggcacatac cacgatctgc tgaaaattat 3601 caaggacaag gacttcctgg acaatgagga aaacgaggac attctggaag atatcgtgct 3661 gaccctgaca ctgtttgagg acagagagat gatcgaggaa cggctgaaaa cctatgccca 3721 cctgttcgac gacaaagtga tgaagcagct gaagcggcgg agatacaccg gctggggcag 3781 gctgagccgg aagctgatca acggcatccg ggacaagcag tccggcaaga caatcctgga 3841 tttcctgaag tccgacggct tcgccaacag aaacttcatg cagctgatcc acgacgacag 3901 cctgaccttt aaagaggaca tccagaaagc ccaggtgtcc ggccagggcg atagcctgca 3961 cgagcacatt gccaatctgg ccggcagccc cgccattaag aagggcatcc tgcagacagt 4021 gaaggtggtg gacgagctcg tgaaagtgat gggccggcac aagcccgaga acatcgtgat 4081 cgaaatggcc agagagaacc agaccaccca gaagggacag aagaacagcc gcgagagaat 4141 gaagcggatc gaagagggca tcaaagagct gggcagccag atcctgaaag aacaccccgt 4201 ggaaaacacc cagctgcaga acgagaagct gtacctgtac tacctgcaga atgggcggga 4261 tatgtacgtg gaccaggaac tggacatcaa ccggctgtcc gactacgatg tggaccatat 4321 cgtgcctcag agctttctgg cggacgactc catcgacaac aaggtgctga ccagaagcga 4381 caagaaccgg ggcaagagcg acaacgtgcc ctccgaagag gtcgtgaaga agatgaagaa 4441 ctactggcgg cagctgctga acgccaagct gattacccag agaaagttcg acaatctgac 4501 caaggccgag agaggcggcc tgagcgaact ggataaggcc ggcttcatca agagacagct 4561 ggtggaaacc cggcagatca caaagcacgt ggcacagatc ctggactccc ggatgaacac 4621 taagtacgac gagaatgaca agctgatccg ggaagtgaaa gtgatcaccc tgaagtccaa 4681 gctggtgtcc gatttccgga aggatttcca gttttacaaa gtgcgcgaga tcaacaacta 4741 ccaccacgcc cacgacgcct acctgaacgc cgtcgtggga accgccctga tcaaaaagta 4801 ccctgcgctg gaaagcgagt tcgtgtacgg cgactacaag gtgtacgacg tgcggaagat 4861 gatcgccaag agcgagcagg aaatcggcaa ggctaccgcc aagtacttct tctacagcaa 4921 catcatgaac tttttcaaga ccgagattac cctggccaac ggcgagatcc ggaaggcgcc 4981 tctgatcgag acaaacggcg aaaccgggga gatcgtgtgg gataagggcc gggattttgc 5041 caccgtgcgg aaagtgctga gcatgcccca agtgaatatc gtgaaaaaga ccgaggtgca 5101 gacaggcggc ttcagcaaag agtctatcct gcccaagagg aacagcgata agctgatcgc 5161 cagaaagaag gactgggacc ctaagaagta cggcggcttc gacagcccca ccgtggccta 5221 ttctgtgctg gtggtggcca aagtggaaaa gggcaagtcc aagaaactga agagtgtgaa 5281 agagctgctg gggatcacca tcatggaaag aagcagcttc gagaagaatc ccatcgactt 5341 tctggaagcc aagggctaca aagaagtgaa aaaggacctg atcatcaagc tgcctaagta 5401 ctccctgttc gagctggaaa acggccggaa gagaatgctg gcctctgccg gcgaactgca 5461 gaagggaaac gaactggccc tgccctccaa atatgtgaac ttcctgtacc tggccagcca 5521 ctatgagaag ctgaagggct cccccgagga taatgagcag aaacagctgt ttgtggaaca 5581 gcacaagcac tacctggacg agatcatcga gcagatcagc gagttctcca agagagtgat 5641 cctggccgac gctaatctgg acaaagtgct gtccgcctac aacaagcacc gggataagcc 5701 catcagagag caggccgaga atatcatcca cctgtttacc ctgaccaatc tgggagcccc 5761 tgccgccttc aagtactttg acaccaccat cgaccggaag aggtacacca gcaccaaaga 5821 ggtgctggac gccaccctga tccaccagag catcaccggc ctgtacgaga cacggatcga 5881 cctgtctcag ctgggaggcg acaaaaggcc ggcggccacg aaaaaggccg gccaggcaaa 5941 aaagaaaaag taagaattcc tagagctcgc tgatcagcct cgactgtgcc ttctagttgc 6001 cagccatctg ttgtttgccc ctcccccgtg ccttccttga ccctggaagg tgccactccc 6061 actgtccttt cctaataaaa tgaggaaatt gcatcgcatt gtctgagtag gtgtcattct 6121 attctggggg gtggggtggg gcaggacagc aagggggagg attgggaaga gaatagcagg 6181 catgctgggg agcggccgca ggaaccccta gtgatggagt tggccactcc ctctctgcgc 6241 gctcgctcgc tcactgaggc cgggcgacca aaggtcgccc gacgcccggg ctttgcccgg 6301 gcggcctcag tgagcgagcg agcgcgcagc tgcctgcagg ggcgcctgat gcggtatttt 6361 ctccttacgc atctgtgcgg tatttcacac cgcatacgtc aaagcaacca tagtacgcgc 6421 cctgtagcgg cgcattaagc gcggcgggtg tggtggttac gcgcagcgtg accgctacac 6481 ttgccagcgc cctagcgccc gctcctttcg ctttcttccc ttcctttctc gccacgttcg 6541 ccggctttcc ccgtcaagct ctaaatcggg ggctcccttt agggttccga tttagtgctt 6601 tacggcacct cgaccccaaa aaacttgatt tgggtgatgg ttcacgtagt gggccatcgc 6661 cctgatagac ggtttttcgc cctttgacgt tggagtccac gttctttaat agtggactct 6721 tgttccaaac tggaacaaca ctcaacccta tctcgggcta ttcttttgat ttataaggga 6781 ttttgccgat ttcggcctat tggttaaaaa atgagctgat ttaacaaaaa tttaacgcga 6841 attttaacaa aatattaacg tttacaattt tatggtgcac tctcagtaca atctgctctg 6901 atgccgcata gttaagccag ccccgacacc cgccaacacc cgctgacgcg ccctgacggg 6961 cttgtctgct cccggcatcc gcttacagac aagctgtgac cgtctccggg agctgcatgt 7021 gtcagaggtt ttcaccgtca tcaccgaaac gcgcgagacg aaagggcctc gtgatacgcc 7081 tatttttata ggttaatgtc atgataataa tggtttctta gacgtcaggt ggcacttttc 7141 ggggaaatgt gcgcggaacc cctatttgtt tatttttcta aatacattca aatatgtatc 7201 cgctcatgag acaataaccc tgataaatgc ttcaataata ttgaaaaagg aagagtatga 7261 gtattcaaca tttccgtgtc gcccttattc ccttttttgc ggcattttgc cttcctgttt 7321 ttgctcaccc agaaacgctg gtgaaagtaa aagatgctga agatcagttg ggtgcacgag 7381 tgggttacat cgaactggat ctcaacagcg gtaagatcct tgagagtttt cgccccgaag 7441 aacgttttcc aatgatgagc acttttaaag ttctgctatg tggcgcggta ttatcccgta 7501 ttgacgccgg gcaagagcaa ctcggtcgcc gcatacacta ttctcagaat gacttggttg 7561 agtactcacc agtcacagaa aagcatctta cggatggcat gacagtaaga gaattatgca 7621 gtgctgccat aaccatgagt gataacactg cggccaactt acttctgaca acgatcggag 7681 gaccgaagga gctaaccgct tttttgcaca acatggggga tcatgtaact cgccttgatc 7741 gttgggaacc ggagctgaat gaagccatac caaacgacga gcgtgacacc acgatgcctg 7801 tagcaatggc aacaacgttg cgcaaactat taactggcga actacttact ctagcttccc 7861 ggcaacaatt aatagactgg atggaggcgg ataaagttgc aggaccactt ctgcgctcgg 7921 cccttccggc tggctggttt attgctgata aatctggagc cggtgagcgt ggaagccgcg 7981 gtatcattgc agcactgggg ccagatggta agccctcccg tatcgtagtt atctacacga 8041 cggggagtca ggcaactatg gatgaacgaa atagacagat cgctgagata ggtgcctcac 8101 tgattaagca ttggtaactg tcagaccaag tttactcata tatactttag attgatttaa 8161 aacttcattt ttaatttaaa aggatctagg tgaagatcct ttttgataat ctcatgacca 8221 aaatccctta acgtgagttt tcgttccact gagcgtcaga ccccgtagaa aagatcaaag 8281 gatcttcttg agatcctttt tttctgcgcg taatctgctg cttgcaaaca aaaaaaccac 8341 cgctaccagc ggtggtttgt ttgccggatc aagagctacc aactcttttt ccgaaggtaa 8401 ctggcttcag cagagcgcag ataccaaata ctgtccttct agtgtagccg tagttaggcc 8461 accacttcaa gaactctgta gcaccgccta catacctcgc tctgctaatc ctgttaccag 8521 tggctgctgc cagtggcgat aagtcgtgtc ttaccgggtt ggactcaaga cgatagttac 8581 cggataaggc gcagcggtcg ggctgaacgg ggggttcgtg cacacagccc agcttggagc 8641 gaacgaccta caccgaactg agatacctac agcgtgagct atgagaaagc gccacgcttc 8701 ccgaagggag aaaggcggac aggtatccgg taagcggcag ggtcggaaca ggagagcgca 8761 cgagggagct tccaggggga aacgcctggt atctttatag tcctgtcggg tttcgccacc 8821 tctgacttga gcgtcgattt ttgtgatgct cgtcaggggg gcggagccta tggaaaaacg 8881 ccagcaacgc ggccttttta cggttcctgg ccttttgctg gccttttgct cacatgt //