LOCUS Exported 7238 bp ds-DNA circular SYN 13-MAY-2021 DEFINITION Expresses SaCas9 and a gRNA targeting mouse YAP. ACCESSION . VERSION . KEYWORDS Icam 2 SaCas9 YAP sgRNA1 SOURCE synthetic DNA construct ORGANISM synthetic DNA construct REFERENCE 1 (bases 1 to 7238) AUTHORS Wang L, Luo JY, Li B, Tian XY, Chen LJ, Huang Y, Liu J, Deng D, Lau CW, Wan S, Ai D, Mak KK, Tong KK, Kwan KM, Wang N, Chiu JJ, Zhu Y, Huang Y TITLE Integrin-YAP/TAZ-JNK cascade mediates atheroprotective effect of unidirectional shear flow. JOURNAL Nature. 2016 Dec 7. doi: 10.1038/nature20602. PUBMED 27926730 REFERENCE 2 (bases 1 to 7238) AUTHORS . TITLE Direct Submission JOURNAL Exported May 13, 2021 from SnapGene Server 1.1.58 http://www.snapgene.com FEATURES Location/Qualifiers source 1..7238 /organism="synthetic DNA construct" /mol_type="other DNA" repeat_region 1..130 /label=AAV2 ITR (alternate) /note="Functional equivalent of wild-type AAV2 ITR" regulatory 554..563 /regulatory_class="other" /note="vertebrate consensus sequence for strong initiation of translation (Kozak, 1987)" CDS 566..586 /codon_start=1 /product="nuclear localization signal of SV40 (simian virus 40) large T antigen" /label=SV40 NLS /translation="PKKKRKV" CDS 611..3766 /codon_start=1 /product="Cas9 endonuclease from the Staphylococcus aureus Type II CRISPR/Cas system" /label=SaCas9 /note="generates RNA-guided double strand breaks in DNA" /translation="KRNYILGLDIGITSVGYGIIDYETRDVIDAGVRLFKEANVENNEG RRSKRGARRLKRRRRHRIQRVKKLLFDYNLLTDHSELSGINPYEARVKGLSQKLSEEEF SAALLHLAKRRGVHNVNEVEEDTGNELSTKEQISRNSKALEEKYVAELQLERLKKDGEV RGSINRFKTSDYVKEAKQLLKVQKAYHQLDQSFIDTYIDLLETRRTYYEGPGEGSPFGW KDIKEWYEMLMGHCTYFPEELRSVKYAYNADLYNALNDLNNLVITRDENEKLEYYEKFQ IIENVFKQKKKPTLKQIAKEILVNEEDIKGYRVTSTGKPEFTNLKVYHDIKDITARKEI IENAELLDQIAKILTIYQSSEDIQEELTNLNSELTQEEIEQISNLKGYTGTHNLSLKAI NLILDELWHTNDNQIAIFNRLKLVPKKVDLSQQKEIPTTLVDDFILSPVVKRSFIQSIK VINAIIKKYGLPNDIIIELAREKNSKDAQKMINEMQKRNRQTNERIEEIIRTTGKENAK YLIEKIKLHDMQEGKCLYSLEAIPLEDLLNNPFNYEVDHIIPRSVSFDNSFNNKVLVKQ EENSKKGNRTPFQYLSSSDSKISYETFKKHILNLAKGKGRISKTKKEYLLEERDINRFS VQKDFINRNLVDTRYATRGLMNLLRSYFRVNNLDVKVKSINGGFTSFLRRKWKFKKERN KGYKHHAEDALIIANADFIFKEWKKLDKAKKVMENQMFEEKQAESMPEIETEQEYKEIF ITPHQIKHIKDFKDYKYSHRVDKKPNRELINDTLYSTRKDDKGNTLIVNNLNGLYDKDN DKLKKLINKSPEKLLMYHHDPQTYQKLKLIMEQYGDEKNPLYKYYEETGNYLTKYSKKD NGPVIKKIKYYGNKLNAHLDITDDYPNSRNKVVKLSLKPYRFDVYLDNGVYKFVTVKNL DVIKKENYYEVNSKCYEEAKKLKKISNQAEFIASFYNNDLIKINGELYRVIGVNNDLLN RIEVNMIDITYREYLENMNDKRPPRIIKTIASKTQSIKKYSTDILGNLYEVKSKKHPQI IKKG" CDS 3767..3814 /codon_start=1 /product="bipartite nuclear localization signal from nucleoplasmin" /label=nucleoplasmin NLS /translation="KRPAATKKAGQAKKKK" CDS 3821..3901 /codon_start=1 /product="three tandem HA epitope tags" /label=3xHA /translation="YPYDVPDYAYPYDVPDYAYPYDVPDYA" primer_bind complement(3929..3946) /label=BGH-rev /note="Bovine growth hormone terminator, reverse primer. Also called BGH reverse" polyA_signal 3935..4046 /label=bGH poly(A) signal /note="bovine growth hormone polyadenylation signal" promoter 4141..4381 /label=U6 promoter /note="RNA polymerase III promoter for human U6 snRNA" primer_bind 4141..4161 /label=hU6-F /note="Human U6 promoter, forward primer" primer_bind 4312..4331 /label=LKO.1 5' /note="Human U6 promoter, forward primer" misc_RNA 4412..4487 /label=Sa gRNA scaffold /note="guide RNA scaffold for the Staphylococcus aureus CRISPR/Cas9 system" repeat_region 4501..4641 /label=AAV2 ITR /note="inverted terminal repeat of adeno-associated virus serotype 2" repeat_region 4501..4630 /label=AAV2 ITR rep_origin 4716..5171 /direction=RIGHT /label=f1 ori /note="f1 bacteriophage origin of replication; arrow indicates direction of (+) strand synthesis" primer_bind complement(4803..4822) /label=F1ori-R /note="F1 origin, reverse primer" primer_bind 5013..5034 /label=F1ori-F /note="F1 origin, forward primer" primer_bind complement(5188..5207) /label=pRS-marker /note="pRS vectors, use to sequence yeast selectable marker" primer_bind 5307..5329 /label=pGEX 3' /note="pGEX vectors, reverse primer" primer_bind complement(5367..5385) /label=pBRforEco /note="pBR322 vectors, upsteam of EcoRI site, forward primer" promoter 5453..5557 /gene="bla" /label=AmpR promoter CDS 5558..6418 /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(5776..5795) /label=Amp-R /note="Ampicillin resistance gene, reverse primer" rep_origin 6589..7177 /direction=RIGHT /label=ori /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of replication" primer_bind 7078..7097 /label=pBR322ori-F /note="pBR322 origin, forward primer" ORIGIN 1 cctgcaggca gctgcgcgct cgctcgctca ctgaggccgc ccgggcgtcg ggcgaccttt 61 ggtcgcccgg cctcagtgag cgagcgagcg cgcagagagg gagtggccaa ctccatcact 121 aggggttcct gcggcctcta gactcgaggt ctcccaggca tgactccaac aatgcatccc 181 atgggatttg gggttcccca gatctggggc ttgtaggcct gactctcccc tgtgcacacg 241 tctcatacac gcatgcgtgc acccattgcc tgccccgccc cttgcacagg gagtcagcag 301 ggaggactgg gttatgccct gcttatcagc agcttcccag cttcctctgc ctggattctt 361 agaggcctgg ggtcctagaa cgagctggtg cacgtggctt cccaaagatc tctcagataa 421 tgagaggaaa tgcagtcatc agtttgcaga aggctaggga ttctgggcca tagctcagac 481 ctgcgcccac catctccctc caggcagccc ttggctggtc cctgcgagcc cgtggagact 541 gccagtcacc ggtgccacca tggccccaaa gaagaagcgg aaggtcggta tccacggagt 601 cccagcagcc aagcggaact acatcctggg cctggacatc ggcatcacca gcgtgggcta 661 cggcatcatc gactacgaga cacgggacgt gatcgatgcc ggcgtgcggc tgttcaaaga 721 ggccaacgtg gaaaacaacg agggcaggcg gagcaagaga ggcgccagaa ggctgaagcg 781 gcggaggcgg catagaatcc agagagtgaa gaagctgctg ttcgactaca acctgctgac 841 cgaccacagc gagctgagcg gcatcaaccc ctacgaggcc agagtgaagg gcctgagcca 901 gaagctgagc gaggaagagt tctctgccgc cctgctgcac ctggccaaga gaagaggcgt 961 gcacaacgtg aacgaggtgg aagaggacac cggcaacgag ctgtccacca aagagcagat 1021 cagccggaac agcaaggccc tggaagagaa atacgtggcc gaactgcagc tggaacggct 1081 gaagaaagac ggcgaagtgc ggggcagcat caacagattc aagaccagcg actacgtgaa 1141 agaagccaaa cagctgctga aggtgcagaa ggcctaccac cagctggacc agagcttcat 1201 cgacacctac atcgacctgc tggaaacccg gcggacctac tatgagggac ctggcgaggg 1261 cagccccttc ggctggaagg acatcaaaga atggtacgag atgctgatgg gccactgcac 1321 ctacttcccc gaggaactgc ggagcgtgaa gtacgcctac aacgccgacc tgtacaacgc 1381 cctgaacgac ctgaacaatc tcgtgatcac cagggacgag aacgagaagc tggaatatta 1441 cgagaagttc cagatcatcg agaacgtgtt caagcagaag aagaagccca ccctgaagca 1501 gatcgccaaa gaaatcctcg tgaacgaaga ggatattaag ggctacagag tgaccagcac 1561 cggcaagccc gagttcacca acctgaaggt gtaccacgac atcaaggaca ttaccgcccg 1621 gaaagagatt attgagaacg ccgagctgct ggatcagatt gccaagatcc tgaccatcta 1681 ccagagcagc gaggacatcc aggaagaact gaccaatctg aactccgagc tgacccagga 1741 agagatcgag cagatctcta atctgaaggg ctataccggc acccacaacc tgagcctgaa 1801 ggccatcaac ctgatcctgg acgagctgtg gcacaccaac gacaaccaga tcgctatctt 1861 caaccggctg aagctggtgc ccaagaaggt ggacctgtcc cagcagaaag agatccccac 1921 caccctggtg gacgacttca tcctgagccc cgtcgtgaag agaagcttca tccagagcat 1981 caaagtgatc aacgccatca tcaagaagta cggcctgccc aacgacatca ttatcgagct 2041 ggcccgcgag aagaactcca aggacgccca gaaaatgatc aacgagatgc agaagcggaa 2101 ccggcagacc aacgagcgga tcgaggaaat catccggacc accggcaaag agaacgccaa 2161 gtacctgatc gagaagatca agctgcacga catgcaggaa ggcaagtgcc tgtacagcct 2221 ggaagccatc cctctggaag atctgctgaa caaccccttc aactatgagg tggaccacat 2281 catccccaga agcgtgtcct tcgacaacag cttcaacaac aaggtgctcg tgaagcagga 2341 agaaaacagc aagaagggca accggacccc attccagtac ctgagcagca gcgacagcaa 2401 gatcagctac gaaaccttca agaagcacat cctgaatctg gccaagggca agggcagaat 2461 cagcaagacc aagaaagagt atctgctgga agaacgggac atcaacaggt tctccgtgca 2521 gaaagacttc atcaaccgga acctggtgga taccagatac gccaccagag gcctgatgaa 2581 cctgctgcgg agctacttca gagtgaacaa cctggacgtg aaagtgaagt ccatcaatgg 2641 cggcttcacc agctttctgc ggcggaagtg gaagtttaag aaagagcgga acaaggggta 2701 caagcaccac gccgaggacg ccctgatcat tgccaacgcc gatttcatct tcaaagagtg 2761 gaagaaactg gacaaggcca aaaaagtgat ggaaaaccag atgttcgagg aaaagcaggc 2821 cgagagcatg cccgagatcg aaaccgagca ggagtacaaa gagatcttca tcacccccca 2881 ccagatcaag cacattaagg acttcaagga ctacaagtac agccaccggg tggacaagaa 2941 gcctaataga gagctgatta acgacaccct gtactccacc cggaaggacg acaagggcaa 3001 caccctgatc gtgaacaatc tgaacggcct gtacgacaag gacaatgaca agctgaaaaa 3061 gctgatcaac aagagccccg aaaagctgct gatgtaccac cacgaccccc agacctacca 3121 gaaactgaag ctgattatgg aacagtacgg cgacgagaag aatcccctgt acaagtacta 3181 cgaggaaacc gggaactacc tgaccaagta ctccaaaaag gacaacggcc ccgtgatcaa 3241 gaagattaag tattacggca acaaactgaa cgcccatctg gacatcaccg acgactaccc 3301 caacagcaga aacaaggtcg tgaagctgtc cctgaagccc tacagattcg acgtgtacct 3361 ggacaatggc gtgtacaagt tcgtgaccgt gaagaatctg gatgtgatca aaaaagaaaa 3421 ctactacgaa gtgaatagca agtgctatga ggaagctaag aagctgaaga agatcagcaa 3481 ccaggccgag tttatcgcct ccttctacaa caacgatctg atcaagatca acggcgagct 3541 gtatagagtg atcggcgtga acaacgacct gctgaaccgg atcgaagtga acatgatcga 3601 catcacctac cgcgagtacc tggaaaacat gaacgacaag aggcccccca ggatcattaa 3661 gacaatcgcc tccaagaccc agagcattaa gaagtacagc acagacattc tgggcaacct 3721 gtatgaagtg aaatctaaga agcaccctca gatcatcaaa aagggcaaaa ggccggcggc 3781 cacgaaaaag gccggccagg caaaaaagaa aaagggatcc tacccatacg atgttccaga 3841 ttacgcttac ccatacgatg ttccagatta cgcttaccca tacgatgttc cagattacgc 3901 ttaagaattc ctagagctcg ctgatcagcc tcgactgtgc cttctagttg ccagccatct 3961 gttgtttgcc cctcccccgt gccttccttg accctggaag gtgccactcc cactgtcctt 4021 tcctaataaa atgaggaaat tgcatcgcat tgtctgagta ggtgtcattc tattctgggg 4081 ggtggggtgg ggcaggacag caagggggag gattgggaag agaatagctg gggaggtacc 4141 gagggcctat ttcccatgat tccttcatat ttgcatatac gatacaaggc tgttagagag 4201 ataattggaa ttaatttgac tgtaaacaca aagatattag tacaaaatac gtgacgtaga 4261 aagtaataat ttcttgggta gtttgcagtt ttaaaattat gttttaaaat ggactatcat 4321 atgcttaccg taacttgaaa gtatttcgat ttcttggctt tatatatctt gtggaaagga 4381 cgaaacaccg tctgaggcac gttggccgtc tgttttagta ctctggaaac agaatctact 4441 aaaacaaggc aaaatgccgt gtttatctcg tcaacttgtt ggcgagattt ttgcggccgc 4501 aggaacccct agtgatggag ttggccactc cctctctgcg cgctcgctcg ctcactgagg 4561 ccgggcgacc aaaggtcgcc cgacgcccgg gctttgcccg ggcggcctca gtgagcgagc 4621 gagcgcgcag ctgcctgcag gggcgcctga tgcggtattt tctccttacg catctgtgcg 4681 gtatttcaca ccgcatacgt caaagcaacc atagtacgcg ccctgtagcg gcgcattaag 4741 cgcggcgggt gtggtggtta cgcgcagcgt gaccgctaca cttgccagcg ccttagcgcc 4801 cgctcctttc gctttcttcc cttcctttct cgccacgttc gccggctttc cccgtcaagc 4861 tctaaatcgg gggctccctt tagggttccg atttagtgct ttacggcacc tcgaccccaa 4921 aaaacttgat ttgggtgatg gttcacgtag tgggccatcg ccctgataga cggtttttcg 4981 ccctttgacg ttggagtcca cgttctttaa tagtggactc ttgttccaaa ctggaacaac 5041 actcaactct atctcgggct attcttttga tttataaggg attttgccga tttcggtcta 5101 ttggttaaaa aatgagctga tttaacaaaa atttaacgcg aattttaaca aaatattaac 5161 gtttacaatt ttatggtgca ctctcagtac aatctgctct gatgccgcat agttaagcca 5221 gccccgacac ccgccaacac ccgctgacgc gccctgacgg gcttgtctgc tcccggcatc 5281 cgcttacaga caagctgtga ccgtctccgg gagctgcatg tgtcagaggt tttcaccgtc 5341 atcaccgaaa cgcgcgagac gaaagggcct cgtgatacgc ctatttttat aggttaatgt 5401 catgataata atggtttctt agacgtcagg tggcactttt cggggaaatg tgcgcggaac 5461 ccctatttgt ttatttttct aaatacattc aaatatgtat ccgctcatga gacaataacc 5521 ctgataaatg cttcaataat attgaaaaag gaagagtatg agtattcaac atttccgtgt 5581 cgcccttatt cccttttttg cggcattttg ccttcctgtt tttgctcacc cagaaacgct 5641 ggtgaaagta aaagatgctg aagatcagtt gggtgcacga gtgggttaca tcgaactgga 5701 tctcaacagc ggtaagatcc ttgagagttt tcgccccgaa gaacgttttc caatgatgag 5761 cacttttaaa gttctgctat gtggcgcggt attatcccgt attgacgccg ggcaagagca 5821 actcggtcgc cgcatacact attctcagaa tgacttggtt gagtactcac cagtcacaga 5881 aaagcatctt acggatggca tgacagtaag agaattatgc agtgctgcca taaccatgag 5941 tgataacact gcggccaact tacttctgac aacgatcgga ggaccgaagg agctaaccgc 6001 ttttttgcac aacatggggg atcatgtaac tcgccttgat cgttgggaac cggagctgaa 6061 tgaagccata ccaaacgacg agcgtgacac cacgatgcct gtagcaatgg caacaacgtt 6121 gcgcaaacta ttaactggcg aactacttac tctagcttcc cggcaacaat taatagactg 6181 gatggaggcg gataaagttg caggaccact tctgcgctcg gcccttccgg ctggctggtt 6241 tattgctgat aaatctggag ccggtgagcg tggaagccgc ggtatcattg cagcactggg 6301 gccagatggt aagccctccc gtatcgtagt tatctacacg acggggagtc aggcaactat 6361 ggatgaacga aatagacaga tcgctgagat aggtgcctca ctgattaagc attggtaact 6421 gtcagaccaa gtttactcat atatacttta gattgattta aaacttcatt tttaatttaa 6481 aaggatctag gtgaagatcc tttttgataa tctcatgacc aaaatccctt aacgtgagtt 6541 ttcgttccac tgagcgtcag accccgtaga aaagatcaaa ggatcttctt gagatccttt 6601 ttttctgcgc gtaatctgct gcttgcaaac aaaaaaacca ccgctaccag cggtggtttg 6661 tttgccggat caagagctac caactctttt tccgaaggta actggcttca gcagagcgca 6721 gataccaaat actgttcttc tagtgtagcc gtagttaggc caccacttca agaactctgt 6781 agcaccgcct acatacctcg ctctgctaat cctgttacca gtggctgctg ccagtggcga 6841 taagtcgtgt cttaccgggt tggactcaag acgatagtta ccggataagg cgcagcggtc 6901 gggctgaacg gggggttcgt gcacacagcc cagcttggag cgaacgacct acaccgaact 6961 gagataccta cagcgtgagc tatgagaaag cgccacgctt cccgaaggga gaaaggcgga 7021 caggtatccg gtaagcggca gggtcggaac aggagagcgc acgagggagc ttccaggggg 7081 aaacgcctgg tatctttata gtcctgtcgg gtttcgccac ctctgacttg agcgtcgatt 7141 tttgtgatgc tcgtcagggg ggcggagcct atggaaaaac gccagcaacg cggccttttt 7201 acggttcctg gccttttgct ggccttttgc tcacatgt //