LOCUS Exported 7726 bp ds-DNA circular SYN 13-MAY-2021 DEFINITION Mutagenesis of Ntsr1. ACCESSION . VERSION . KEYWORDS pAAV-CMV-FLEXfrt-SaCas9-U6-sgNtsr1 SOURCE synthetic DNA construct ORGANISM synthetic DNA construct REFERENCE 1 (bases 1 to 7726) TITLE Zweifel CRISPR AAV plasmids REFERENCE 2 (bases 1 to 7726) AUTHORS . TITLE Direct Submission JOURNAL Exported May 13, 2021 from SnapGene Server 1.1.58 http://www.snapgene.com FEATURES Location/Qualifiers source 1..7726 /organism="synthetic DNA construct" /mol_type="other DNA" enhancer 1..380 /label=CMV enhancer /note="human cytomegalovirus immediate early enhancer" promoter 381..584 /label=CMV promoter /note="human cytomegalovirus (CMV) immediate early promoter" primer_bind 535..555 /label=CMV-F /note="Human CMV immediate early promoter, forward primer" protein_bind 627..660 /label=FRT (minimal) /bound_moiety="FLP recombinase from the Saccharomyces cerevisiae 2u plasmid" /note="supports FLP-mediated excision but not integration (Turan and Bode, 2011)" CDS complement(732..812) /codon_start=1 /product="three tandem HA epitope tags" /label=3xHA /translation="YPYDVPDYAYPYDVPDYAYPYDVPDYA" CDS complement(819..866) /codon_start=1 /product="bipartite nuclear localization signal from nucleoplasmin" /label=nucleoplasmin NLS /translation="KRPAATRKAGQAKKKK" CDS complement(867..4022) /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 complement(4047..4067) /codon_start=1 /product="nuclear localization signal of SV40 (simian virus 40) large T antigen" /label=SV40 NLS /translation="PKKKRKV" primer_bind complement(4257..4274) /label=BGH-rev /note="Bovine growth hormone terminator, reverse primer. Also called BGH reverse" polyA_signal 4263..4470 /label=bGH poly(A) signal /note="bovine growth hormone polyadenylation signal" promoter 4477..4717 /label=U6 promoter /note="RNA polymerase III promoter for human U6 snRNA" primer_bind 4477..4497 /label=hU6-F /note="Human U6 promoter, forward primer" primer_bind 4648..4667 /label=LKO.1 5' /note="Human U6 promoter, forward primer" misc_RNA 4747..4822 /label=Sa gRNA scaffold /note="guide RNA scaffold for the Staphylococcus aureus CRISPR/Cas9 system" repeat_region 4836..4976 /label=AAV2 ITR /note="inverted terminal repeat of adeno-associated virus serotype 2" repeat_region 4836..4965 /label=AAV2 ITR rep_origin 5051..5506 /direction=RIGHT /label=f1 ori /note="f1 bacteriophage origin of replication; arrow indicates direction of (+) strand synthesis" primer_bind complement(5138..5157) /label=F1ori-R /note="F1 origin, reverse primer" primer_bind 5348..5369 /label=F1ori-F /note="F1 origin, forward primer" primer_bind complement(5523..5542) /label=pRS-marker /note="pRS vectors, use to sequence yeast selectable marker" primer_bind 5642..5664 /label=pGEX 3' /note="pGEX vectors, reverse primer" primer_bind complement(5702..5720) /label=pBRforEco /note="pBR322 vectors, upsteam of EcoRI site, forward primer" promoter 5788..5892 /gene="bla" /label=AmpR promoter CDS 5893..6753 /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(6111..6130) /label=Amp-R /note="Ampicillin resistance gene, reverse primer" rep_origin 6924..7512 /direction=RIGHT /label=ori /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of replication" primer_bind 7413..7432 /label=pBR322ori-F /note="pBR322 origin, forward primer" repeat_region 7574..7703 /label=AAV2 ITR (alternate) /note="Functional equivalent of wild-type AAV2 ITR" ORIGIN 1 gacattgatt attgactagt tattaatagt aatcaattac ggggtcatta gttcatagcc 61 catatatgga gttccgcgtt acataactta cggtaaatgg cccgcctggc tgaccgccca 121 acgacccccg cccattgacg tcaataatga cgtatgttcc catagtaacg ccaataggga 181 ctttccattg acgtcaatgg gtggagtatt tacggtaaac tgcccacttg gcagtacatc 241 aagtgtatca tatgccaagt acgcccccta ttgacgtcaa tgacggtaaa tggcccgcct 301 ggcattatgc ccagtacatg accttatggg actttcctac ttggcagtac atctacgtat 361 tagtcatcgc tattaccatg gtgatgcggt tttggcagta catcaatggg cgtggatagc 421 ggtttgactc acggggattt ccaagtctcc accccattga cgtcaatggg agtttgtttt 481 ggcaccaaaa tcaacgggac tttccaaaat gtcgtaacaa ctccgcccca ttgacgcaaa 541 tgggcggtag gcgtgtacgg tgggaggtct atataagcag agctctctgg ctaactaccg 601 gtgctagcgt cgacagaatt ggatccgaag ttcctattct ctagaaagta taggaacttc 661 caaggcctga agttcctatt cttcaaaagg tataggaact tcgcggccgc ttgatatctt 721 ggcgcgcctt aagcgtaatc tggaacatcg tatgggtaag cgtaatctgg aacatcgtat 781 gggtaagcgt aatctggaac atcgtatggg taggatccct ttttcttttt tgcctggccg 841 gcctttctcg tggccgccgg ccttttgccc tttttgatga tctgagggtg cttcttagat 901 ttcacttcat acaggttgcc cagaatgtct gtgctgtact tcttaatgct ctgggtcttg 961 gaggcgattg tcttaatgat cctggggggc ctcttgtcgt tcatgttttc caggtactcg 1021 cggtaggtga tgtcgatcat gttcacttcg atccggttca gcaggtcgtt gttcacgccg 1081 atcactctat acagctcgcc gttgatcttg atcagatcgt tgttgtagaa ggaggcgata 1141 aactcggcct ggttgctgat cttcttcagc ttcttagctt cctcatagca cttgctattc 1201 acttcgtagt agttttcttt tttgatcaca tccagattct tcacggtcac gaacttgtac 1261 acgccattgt ccaggtacac gtcgaatctg tagggcttca gggacagctt cacgaccttg 1321 tttctgctgt tggggtagtc gtcggtgatg tccagatggg cgttcagttt gttgccgtaa 1381 tacttaatct tcttgatcac ggggccgttg tcctttttgg agtacttggt caggtagttc 1441 ccggtttcct cgtagtactt gtacagggga ttcttctcgt cgccgtactg ttccataatc 1501 agcttcagtt tctggtaggt ctgggggtcg tggtggtaca tcagcagctt ttcggggctc 1561 ttgttgatca gctttttcag cttgtcattg tccttgtcgt acaggccgtt cagattgttc 1621 acgatcaggg tgttgccctt gtcgtccttc cgggtggagt acagggtgtc gttaatcagc 1681 tctctattag gcttcttgtc cacccggtgg ctgtacttgt agtccttgaa gtccttaatg 1741 tgcttgatct ggtggggggt gatgaagatc tctttgtact cctgctcggt ttcgatctcg 1801 ggcatgctct cggcctgctt ttcctcgaac atctggtttt ccatcacttt tttggccttg 1861 tccagtttct tccactcttt gaagatgaaa tcggcgttgg caatgatcag ggcgtcctcg 1921 gcgtggtgct tgtacccctt gttccgctct ttcttaaact tccacttccg ccgcagaaag 1981 ctggtgaagc cgccattgat ggacttcact ttcacgtcca ggttgttcac tctgaagtag 2041 ctccgcagca ggttcatcag gcctctggtg gcgtatctgg tatccaccag gttccggttg 2101 atgaagtctt tctgcacgga gaacctgttg atgtcccgtt cttccagcag atactctttc 2161 ttggtcttgc tgattctgcc cttgcccttg gccagattca ggatgtgctt cttgaaggtt 2221 tcgtagctga tcttgctgtc gctgctgctc aggtactgga atggggtccg gttgcccttc 2281 ttgctgtttt cttcctgctt cacgagcacc ttgttgttga agctgttgtc gaaggacacg 2341 cttctgggga tgatgtggtc cacctcatag ttgaaggggt tgttcagcag atcttccaga 2401 gggatggctt ccaggctgta caggcacttg ccttcctgca tgtcgtgcag cttgatcttc 2461 tcgatcaggt acttggcgtt ctctttgccg gtggtccgga tgatttcctc gatccgctcg 2521 ttggtctgcc ggttccgctt ctgcatctcg ttgatcattt tctgggcgtc cttggagttc 2581 ttctcgcggg ccagctcgat aatgatgtcg ttgggcaggc cgtacttctt gatgatggcg 2641 ttgatcactt tgatgctctg gatgaagctt ctcttcacga cggggctcag gatgaagtcg 2701 tccaccaggg tggtggggat ctctttctgc tgggacaggt ccaccttctt gggcaccagc 2761 ttcagccggt tgaagatagc gatctggttg tcgttggtgt gccacagctc gtccaggatc 2821 aggttgatgg ccttcaggct caggttgtgg gtgccggtat agcccttcag attagagatc 2881 tgctcgatct cttcctgggt cagctcggag ttcagattgg tcagttcttc ctggatgtcc 2941 tcgctgctct ggtagatggt caggatcttg gcaatctgat ccagcagctc ggcgttctca 3001 ataatctctt tccgggcggt aatgtccttg atgtcgtggt acaccttcag gttggtgaac 3061 tcgggcttgc cggtgctggt cactctgtag cccttaatat cctcttcgtt cacgaggatt 3121 tctttggcga tctgcttcag ggtgggcttc ttcttctgct tgaacacgtt ctcgatgatc 3181 tggaacttct cgtaatattc cagcttctcg ttctcgtccc tggtgatcac gagattgttc 3241 aggtcgttca gggcgttgta caggtcggcg ttgtaggcgt acttcacgct ccgcagttcc 3301 tcggggaagt aggtgcagtg gcccatcagc atctcgtacc attctttgat gtccttccag 3361 ccgaaggggc tgccctcgcc aggtccctca tagtaggtcc gccgggtttc cagcaggtcg 3421 atgtaggtgt cgatgaagct ctggtccagc tggtggtagg ccttctgcac cttcagcagc 3481 tgtttggctt ctttcacgta gtcgctggtc ttgaatctgt tgatgctgcc ccgcacttcg 3541 ccgtctttct tcagccgttc cagctgcagt tcggccacgt atttctcttc cagggccttg 3601 ctgttccggc tgatctgctc tttggtggac agctcgttgc cggtgtcctc ttccacctcg 3661 ttcacgttgt gcacgcctct tctcttggcc aggtgcagca gggcggcaga gaactcttcc 3721 tcgctcagct tctggctcag gcccttcact ctggcctcgt aggggttgat gccgctcagc 3781 tcgctgtggt cggtcagcag gttgtagtcg aacagcagct tcttcactct ctggattcta 3841 tgccgcctcc gccgcttcag ccttctggcg cctctcttgc tccgcctgcc ctcgttgttt 3901 tccacgttgg cctctttgaa cagccgcacg ccggcatcga tcacgtcccg tgtctcgtag 3961 tcgatgatgc cgtagcccac gctggtgatg ccgatgtcca ggcccaggat gtagttccgc 4021 ttggctgctg ggactccgtg gataccgacc ttccgcttct tctttggggc catgcggccg 4081 ctgcagaccg gtttagatct ttactagttt aagcttcaat tggaagttcc tattctttct 4141 agagtatagg aacttcctgc agtaggctag cgaagttcct attccttttg aagtatagga 4201 acttcgtcga gggggggccc gagcttatcg atgaattcct agagctcgct gatcagcctc 4261 gactgtgcct tctagttgcc agccatctgt tgtttgcccc tcccccgtgc cttccttgac 4321 cctggaaggt gccactccca ctgtcctttc ctaataaaat gaggaaattg catcgcattg 4381 tctgagtagg tgtcattcta ttctgggggg tggggtgggg caggacagca agggggagga 4441 ttgggaagag aatagcaggc atgctgggga ggtaccgagg gcctatttcc catgattcct 4501 tcatatttgc atatacgata caaggctgtt agagagataa ttggaattaa tttgactgta 4561 aacacaaaga tattagtaca aaatacgtga cgtagaaagt aataatttct tgggtagttt 4621 gcagttttaa aattatgttt taaaatggac tatcatatgc ttaccgtaac ttgaaagtat 4681 ttcgatttct tggctttata tatcttgtgg aaaggacgaa acaccgcatc gtctccagtc 4741 cgaactgttt tagtactctg gaaacagaat ctactaaaac aaggcaaaat gccgtgttta 4801 tctcgtcaac ttgttggcga gatttttgcg gccgcaggaa cccctagtga tggagttggc 4861 cactccctct ctgcgcgctc gctcgctcac tgaggccggg cgaccaaagg tcgcccgacg 4921 cccgggcttt gcccgggcgg cctcagtgag cgagcgagcg cgcagctgcc tgcaggggcg 4981 cctgatgcgg tattttctcc ttacgcatct gtgcggtatt tcacaccgca tacgtcaaag 5041 caaccatagt acgcgccctg tagcggcgca ttaagcgcgg cgggtgtggt ggttacgcgc 5101 agcgtgaccg ctacacttgc cagcgcctta gcgcccgctc ctttcgcttt cttcccttcc 5161 tttctcgcca cgttcgccgg ctttccccgt caagctctaa atcgggggct ccctttaggg 5221 ttccgattta gtgctttacg gcacctcgac cccaaaaaac ttgatttggg tgatggttca 5281 cgtagtgggc catcgccctg atagacggtt tttcgccctt tgacgttgga gtccacgttc 5341 tttaatagtg gactcttgtt ccaaactgga acaacactca actctatctc gggctattct 5401 tttgatttat aagggatttt gccgatttcg gtctattggt taaaaaatga gctgatttaa 5461 caaaaattta acgcgaattt taacaaaata ttaacgttta caattttatg gtgcactctc 5521 agtacaatct gctctgatgc cgcatagtta agccagcccc gacacccgcc aacacccgct 5581 gacgcgccct gacgggcttg tctgctcccg gcatccgctt acagacaagc tgtgaccgtc 5641 tccgggagct gcatgtgtca gaggttttca ccgtcatcac cgaaacgcgc gagacgaaag 5701 ggcctcgtga tacgcctatt tttataggtt aatgtcatga taataatggt ttcttagacg 5761 tcaggtggca cttttcgggg aaatgtgcgc ggaaccccta tttgtttatt tttctaaata 5821 cattcaaata tgtatccgct catgagacaa taaccctgat aaatgcttca ataatattga 5881 aaaaggaaga gtatgagtat tcaacatttc cgtgtcgccc ttattccctt ttttgcggca 5941 ttttgccttc ctgtttttgc tcacccagaa acgctggtga aagtaaaaga tgctgaagat 6001 cagttgggtg cacgagtggg ttacatcgaa ctggatctca acagcggtaa gatccttgag 6061 agttttcgcc ccgaagaacg ttttccaatg atgagcactt ttaaagttct gctatgtggc 6121 gcggtattat cccgtattga cgccgggcaa gagcaactcg gtcgccgcat acactattct 6181 cagaatgact tggttgagta ctcaccagtc acagaaaagc atcttacgga tggcatgaca 6241 gtaagagaat tatgcagtgc tgccataacc atgagtgata acactgcggc caacttactt 6301 ctgacaacga tcggaggacc gaaggagcta accgcttttt tgcacaacat gggggatcat 6361 gtaactcgcc ttgatcgttg ggaaccggag ctgaatgaag ccataccaaa cgacgagcgt 6421 gacaccacga tgcctgtagc aatggcaaca acgttgcgca aactattaac tggcgaacta 6481 cttactctag cttcccggca acaattaata gactggatgg aggcggataa agttgcagga 6541 ccacttctgc gctcggccct tccggctggc tggtttattg ctgataaatc tggagccggt 6601 gagcgtggaa gccgcggtat cattgcagca ctggggccag atggtaagcc ctcccgtatc 6661 gtagttatct acacgacggg gagtcaggca actatggatg aacgaaatag acagatcgct 6721 gagataggtg cctcactgat taagcattgg taactgtcag accaagttta ctcatatata 6781 ctttagattg atttaaaact tcatttttaa tttaaaagga tctaggtgaa gatccttttt 6841 gataatctca tgaccaaaat cccttaacgt gagttttcgt tccactgagc gtcagacccc 6901 gtagaaaaga tcaaaggatc ttcttgagat cctttttttc tgcgcgtaat ctgctgcttg 6961 caaacaaaaa aaccaccgct accagcggtg gtttgtttgc cggatcaaga gctaccaact 7021 ctttttccga aggtaactgg cttcagcaga gcgcagatac caaatactgt tcttctagtg 7081 tagccgtagt taggccacca cttcaagaac tctgtagcac cgcctacata cctcgctctg 7141 ctaatcctgt taccagtggc tgctgccagt ggcgataagt cgtgtcttac cgggttggac 7201 tcaagacgat agttaccgga taaggcgcag cggtcgggct gaacgggggg ttcgtgcaca 7261 cagcccagct tggagcgaac gacctacacc gaactgagat acctacagcg tgagctatga 7321 gaaagcgcca cgcttcccga agggagaaag gcggacaggt atccggtaag cggcagggtc 7381 ggaacaggag agcgcacgag ggagcttcca gggggaaacg cctggtatct ttatagtcct 7441 gtcgggtttc gccacctctg acttgagcgt cgatttttgt gatgctcgtc aggggggcgg 7501 agcctatgga aaaacgccag caacgcggcc tttttacggt tcctggcctt ttgctggcct 7561 tttgctcaca tgtcctgcag gcagctgcgc gctcgctcgc tcactgaggc cgcccgggcg 7621 tcgggcgacc tttggtcgcc cggcctcagt gagcgagcga gcgcgcagag agggagtggc 7681 caactccatc actaggggtt cctgcggcct ctagactcga ggcgtt //