LOCUS Exported 7729 bp ds-DNA circular SYN 13-MAY-2021 DEFINITION Mutagenesis of Kcnn3. ACCESSION . VERSION . KEYWORDS pAAV-CMV-FLEX-SaCas9-U6-sgKcnn3 SOURCE synthetic DNA construct ORGANISM synthetic DNA construct REFERENCE 1 (bases 1 to 7729) TITLE Zweifel CRISPR AAV plasmids REFERENCE 2 (bases 1 to 7729) AUTHORS . TITLE Direct Submission JOURNAL Exported May 13, 2021 from SnapGene Server 1.1.58 http://www.snapgene.com FEATURES Location/Qualifiers source 1..7729 /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 621..654 /label=lox2272 /bound_moiety="Cre recombinase" /note="Cre-mediated recombination occurs in the 8-bp core sequence (GGATACTT). lox2272 sites are compatible with each other, but incompatible with loxP or loxN sites (Lee and Saito, 1988)." protein_bind 705..738 /label=loxP /bound_moiety="Cre recombinase" /note="Cre-mediated recombination occurs in the 8-bp core sequence (GCATACAT)." CDS complement(750..830) /codon_start=1 /product="three tandem HA epitope tags" /label=3xHA /translation="YPYDVPDYAYPYDVPDYAYPYDVPDYA" CDS complement(837..884) /codon_start=1 /product="bipartite nuclear localization signal from nucleoplasmin" /label=nucleoplasmin NLS /translation="KRPAATKKAGQAKKKK" CDS complement(885..4040) /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(4065..4085) /codon_start=1 /product="nuclear localization signal of SV40 (simian virus 40) large T antigen" /label=SV40 NLS /translation="PKKKRKV" protein_bind complement(4117..4150) /label=lox2272 /bound_moiety="Cre recombinase" /note="Cre-mediated recombination occurs in the 8-bp core sequence (GGATACTT). lox2272 sites are compatible with each other, but incompatible with loxP or loxN sites (Lee and Saito, 1988)." protein_bind complement(4201..4234) /label=loxP /bound_moiety="Cre recombinase" /note="Cre-mediated recombination occurs in the 8-bp core sequence (GCATACAT)." primer_bind complement(4259..4276) /label=BGH-rev /note="Bovine growth hormone terminator, reverse primer. Also called BGH reverse" polyA_signal 4265..4472 /label=bGH poly(A) signal /note="bovine growth hormone polyadenylation signal" promoter 4479..4719 /label=U6 promoter /note="RNA polymerase III promoter for human U6 snRNA" primer_bind 4479..4499 /label=hU6-F /note="Human U6 promoter, forward primer" primer_bind 4650..4669 /label=LKO.1 5' /note="Human U6 promoter, forward primer" misc_RNA 4750..4825 /label=Sa gRNA scaffold /note="guide RNA scaffold for the Staphylococcus aureus CRISPR/Cas9 system" repeat_region 4839..4979 /label=AAV2 ITR /note="inverted terminal repeat of adeno-associated virus serotype 2" repeat_region 4839..4968 /label=AAV2 ITR rep_origin 5054..5509 /direction=RIGHT /label=f1 ori /note="f1 bacteriophage origin of replication; arrow indicates direction of (+) strand synthesis" primer_bind complement(5141..5160) /label=F1ori-R /note="F1 origin, reverse primer" primer_bind 5351..5372 /label=F1ori-F /note="F1 origin, forward primer" primer_bind complement(5526..5545) /label=pRS-marker /note="pRS vectors, use to sequence yeast selectable marker" primer_bind 5645..5667 /label=pGEX 3' /note="pGEX vectors, reverse primer" primer_bind complement(5705..5723) /label=pBRforEco /note="pBR322 vectors, upsteam of EcoRI site, forward primer" promoter 5791..5895 /gene="bla" /label=AmpR promoter CDS 5896..6756 /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(6114..6133) /label=Amp-R /note="Ampicillin resistance gene, reverse primer" rep_origin 6927..7515 /direction=RIGHT /label=ori /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of replication" primer_bind 7416..7435 /label=pBR322ori-F /note="pBR322 origin, forward primer" repeat_region 7577..7706 /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 cgactccgga ataacttcgt ataggatact ttatacgaag ttatgcagaa 661 tggtagctgg attgtagctg ctattagcaa tatgaaacct cttaataact tcgtatagca 721 tacattatac gaagttatgg cgcgccttaa gcgtaatctg gaacatcgta tgggtaagcg 781 taatctggaa catcgtatgg gtaagcgtaa tctggaacat cgtatgggta ggatcccttt 841 ttcttttttg cctggccggc ctttttcgtg gccgccggcc ttttgccctt tttgatgatc 901 tgagggtgct tcttagattt cacttcatac aggttgccca gaatgtctgt gctgtacttc 961 ttaatgctct gggtcttgga ggcgattgtc ttaatgatcc tggggggcct cttgtcgttc 1021 atgttttcca ggtactcgcg gtaggtgatg tcgatcatgt tcacttcgat ccggttcagc 1081 aggtcgttgt tcacgccgat cactctatac agctcgccgt tgatcttgat cagatcgttg 1141 ttgtagaagg aggcgataaa ctcggcctgg ttgctgatct tcttcagctt cttagcttcc 1201 tcatagcact tgctattcac ttcgtagtag ttttcttttt tgatcacatc cagattcttc 1261 acggtcacga acttgtacac gccattgtcc aggtacacgt cgaatctgta gggcttcagg 1321 gacagcttca cgaccttgtt tctgctgttg gggtagtcgt cggtgatgtc cagatgggcg 1381 ttcagtttgt tgccgtaata cttaatcttc ttgatcacgg ggccgttgtc ctttttggag 1441 tacttggtca ggtagttccc ggtttcctcg tagtacttgt acaggggatt cttctcgtcg 1501 ccgtactgtt ccataatcag cttcagtttc tggtaggtct gggggtcgtg gtggtacatc 1561 agcagctttt cggggctctt gttgatcagc tttttcagct tgtcattgtc cttgtcgtac 1621 aggccgttca gattgttcac gatcagggtg ttgcccttgt cgtccttccg ggtggagtac 1681 agggtgtcgt taatcagctc tctattaggc ttcttgtcca cccggtggct gtacttgtag 1741 tccttgaagt ccttaatgtg cttgatctgg tggggggtga tgaagatctc tttgtactcc 1801 tgctcggttt cgatctcggg catgctctcg gcctgctttt cctcgaacat ctggttttcc 1861 atcacttttt tggccttgtc cagtttcttc cactctttga agatgaaatc ggcgttggca 1921 atgatcaggg cgtcctcggc gtggtgcttg taccccttgt tccgctcttt cttaaacttc 1981 cacttccgcc gcagaaagct ggtgaagccg ccattgatgg acttcacttt cacgtccagg 2041 ttgttcactc tgaagtagct ccgcagcagg ttcatcaggc ctctggtggc gtatctggta 2101 tccaccaggt tccggttgat gaagtctttc tgcacggaga acctgttgat gtcccgttct 2161 tccagcagat actctttctt ggtcttgctg attctgccct tgcccttggc cagattcagg 2221 atgtgcttct tgaaggtttc gtagctgatc ttgctgtcgc tgctgctcag gtactggaat 2281 ggggtccggt tgcccttctt gctgttttct tcctgcttca cgagcacctt gttgttgaag 2341 ctgttgtcga aggacacgct tctggggatg atgtggtcca cctcatagtt gaaggggttg 2401 ttcagcagat cttccagagg gatggcttcc aggctgtaca ggcacttgcc ttcctgcatg 2461 tcgtgcagct tgatcttctc gatcaggtac ttggcgttct ctttgccggt ggtccggatg 2521 atttcctcga tccgctcgtt ggtctgccgg ttccgcttct gcatctcgtt gatcattttc 2581 tgggcgtcct tggagttctt ctcgcgggcc agctcgataa tgatgtcgtt gggcaggccg 2641 tacttcttga tgatggcgtt gatcactttg atgctctgga tgaagcttct cttcacgacg 2701 gggctcagga tgaagtcgtc caccagggtg gtggggatct ctttctgctg ggacaggtcc 2761 accttcttgg gcaccagctt cagccggttg aagatagcga tctggttgtc gttggtgtgc 2821 cacagctcgt ccaggatcag gttgatggcc ttcaggctca ggttgtgggt gccggtatag 2881 cccttcagat tagagatctg ctcgatctct tcctgggtca gctcggagtt cagattggtc 2941 agttcttcct ggatgtcctc gctgctctgg tagatggtca ggatcttggc aatctgatcc 3001 agcagctcgg cgttctcaat aatctctttc cgggcggtaa tgtccttgat gtcgtggtac 3061 accttcaggt tggtgaactc gggcttgccg gtgctggtca ctctgtagcc cttaatatcc 3121 tcttcgttca cgaggatttc tttggcgatc tgcttcaggg tgggcttctt cttctgcttg 3181 aacacgttct cgatgatctg gaacttctcg taatattcca gcttctcgtt ctcgtccctg 3241 gtgatcacga gattgttcag gtcgttcagg gcgttgtaca ggtcggcgtt gtaggcgtac 3301 ttcacgctcc gcagttcctc ggggaagtag gtgcagtggc ccatcagcat ctcgtaccat 3361 tctttgatgt ccttccagcc gaaggggctg ccctcgccag gtccctcata gtaggtccgc 3421 cgggtttcca gcaggtcgat gtaggtgtcg atgaagctct ggtccagctg gtggtaggcc 3481 ttctgcacct tcagcagctg tttggcttct ttcacgtagt cgctggtctt gaatctgttg 3541 atgctgcccc gcacttcgcc gtctttcttc agccgttcca gctgcagttc ggccacgtat 3601 ttctcttcca gggccttgct gttccggctg atctgctctt tggtggacag ctcgttgccg 3661 gtgtcctctt ccacctcgtt cacgttgtgc acgcctcttc tcttggccag gtgcagcagg 3721 gcggcagaga actcttcctc gctcagcttc tggctcaggc ccttcactct ggcctcgtag 3781 gggttgatgc cgctcagctc gctgtggtcg gtcagcaggt tgtagtcgaa cagcagcttc 3841 ttcactctct ggattctatg ccgcctccgc cgcttcagcc ttctggcgcc tctcttgctc 3901 cgcctgccct cgttgttttc cacgttggcc tctttgaaca gccgcacgcc ggcatcgatc 3961 acgtcccgtg tctcgtagtc gatgatgccg tagcccacgc tggtgatgcc gatgtccagg 4021 cccaggatgt agttccgctt ggctgctggg actccgtgga taccgacctt ccgcttcttc 4081 tttggggcca tgcggccgct gcagaccggt gctagcataa cttcgtataa agtatcctat 4141 acgaagttat ttgccttaac ccagaaatta tcactgttat tctttagaat ggtgcaaaga 4201 ataacttcgt ataatgtatg ctatacgaag ttatgaattc ctagagctcg ctgatcagcc 4261 tcgactgtgc cttctagttg ccagccatct gttgtttgcc cctcccccgt gccttccttg 4321 accctggaag gtgccactcc cactgtcctt tcctaataaa atgaggaaat tgcatcgcat 4381 tgtctgagta ggtgtcattc tattctgggg ggtggggtgg ggcaggacag caagggggag 4441 gattgggaag agaatagcag gcatgctggg gaggtaccga gggcctattt cccatgattc 4501 cttcatattt gcatatacga tacaaggctg ttagagagat aattggaatt aatttgactg 4561 taaacacaaa gatattagta caaaatacgt gacgtagaaa gtaataattt cttgggtagt 4621 ttgcagtttt aaaattatgt tttaaaatgg actatcatat gcttaccgta acttgaaagt 4681 atttcgattt cttggcttta tatatcttgt ggaaaggacg aaacaccgtt ctgggcactt 4741 ccatgactcg ttttagtact ctggaaacag aatctactaa aacaaggcaa aatgccgtgt 4801 ttatctcgtc aacttgttgg cgagattttt gcggccgcag gaacccctag tgatggagtt 4861 ggccactccc tctctgcgcg ctcgctcgct cactgaggcc gggcgaccaa aggtcgcccg 4921 acgcccgggc tttgcccggg cggcctcagt gagcgagcga gcgcgcagct gcctgcaggg 4981 gcgcctgatg cggtattttc tccttacgca tctgtgcggt atttcacacc gcatacgtca 5041 aagcaaccat agtacgcgcc ctgtagcggc gcattaagcg cggcgggtgt ggtggttacg 5101 cgcagcgtga ccgctacact tgccagcgcc ttagcgcccg ctcctttcgc tttcttccct 5161 tcctttctcg ccacgttcgc cggctttccc cgtcaagctc taaatcgggg gctcccttta 5221 gggttccgat ttagtgcttt acggcacctc gaccccaaaa aacttgattt gggtgatggt 5281 tcacgtagtg ggccatcgcc ctgatagacg gtttttcgcc ctttgacgtt ggagtccacg 5341 ttctttaata gtggactctt gttccaaact ggaacaacac tcaactctat ctcgggctat 5401 tcttttgatt tataagggat tttgccgatt tcggtctatt ggttaaaaaa tgagctgatt 5461 taacaaaaat ttaacgcgaa ttttaacaaa atattaacgt ttacaatttt atggtgcact 5521 ctcagtacaa tctgctctga tgccgcatag ttaagccagc cccgacaccc gccaacaccc 5581 gctgacgcgc cctgacgggc ttgtctgctc ccggcatccg cttacagaca agctgtgacc 5641 gtctccggga gctgcatgtg tcagaggttt tcaccgtcat caccgaaacg cgcgagacga 5701 aagggcctcg tgatacgcct atttttatag gttaatgtca tgataataat ggtttcttag 5761 acgtcaggtg gcacttttcg gggaaatgtg cgcggaaccc ctatttgttt atttttctaa 5821 atacattcaa atatgtatcc gctcatgaga caataaccct gataaatgct tcaataatat 5881 tgaaaaagga agagtatgag tattcaacat ttccgtgtcg cccttattcc cttttttgcg 5941 gcattttgcc ttcctgtttt tgctcaccca gaaacgctgg tgaaagtaaa agatgctgaa 6001 gatcagttgg gtgcacgagt gggttacatc gaactggatc tcaacagcgg taagatcctt 6061 gagagttttc gccccgaaga acgttttcca atgatgagca cttttaaagt tctgctatgt 6121 ggcgcggtat tatcccgtat tgacgccggg caagagcaac tcggtcgccg catacactat 6181 tctcagaatg acttggttga gtactcacca gtcacagaaa agcatcttac ggatggcatg 6241 acagtaagag aattatgcag tgctgccata accatgagtg ataacactgc ggccaactta 6301 cttctgacaa cgatcggagg accgaaggag ctaaccgctt ttttgcacaa catgggggat 6361 catgtaactc gccttgatcg ttgggaaccg gagctgaatg aagccatacc aaacgacgag 6421 cgtgacacca cgatgcctgt agcaatggca acaacgttgc gcaaactatt aactggcgaa 6481 ctacttactc tagcttcccg gcaacaatta atagactgga tggaggcgga taaagttgca 6541 ggaccacttc tgcgctcggc ccttccggct ggctggttta ttgctgataa atctggagcc 6601 ggtgagcgtg gaagccgcgg tatcattgca gcactggggc cagatggtaa gccctcccgt 6661 atcgtagtta tctacacgac ggggagtcag gcaactatgg atgaacgaaa tagacagatc 6721 gctgagatag gtgcctcact gattaagcat tggtaactgt cagaccaagt ttactcatat 6781 atactttaga ttgatttaaa acttcatttt taatttaaaa ggatctaggt gaagatcctt 6841 tttgataatc tcatgaccaa aatcccttaa cgtgagtttt cgttccactg agcgtcagac 6901 cccgtagaaa agatcaaagg atcttcttga gatccttttt ttctgcgcgt aatctgctgc 6961 ttgcaaacaa aaaaaccacc gctaccagcg gtggtttgtt tgccggatca agagctacca 7021 actctttttc cgaaggtaac tggcttcagc agagcgcaga taccaaatac tgttcttcta 7081 gtgtagccgt agttaggcca ccacttcaag aactctgtag caccgcctac atacctcgct 7141 ctgctaatcc tgttaccagt ggctgctgcc agtggcgata agtcgtgtct taccgggttg 7201 gactcaagac gatagttacc ggataaggcg cagcggtcgg gctgaacggg gggttcgtgc 7261 acacagccca gcttggagcg aacgacctac accgaactga gatacctaca gcgtgagcta 7321 tgagaaagcg ccacgcttcc cgaagggaga aaggcggaca ggtatccggt aagcggcagg 7381 gtcggaacag gagagcgcac gagggagctt ccagggggaa acgcctggta tctttatagt 7441 cctgtcgggt ttcgccacct ctgacttgag cgtcgatttt tgtgatgctc gtcagggggg 7501 cggagcctat ggaaaaacgc cagcaacgcg gcctttttac ggttcctggc cttttgctgg 7561 ccttttgctc acatgtcctg caggcagctg cgcgctcgct cgctcactga ggccgcccgg 7621 gcgtcgggcg acctttggtc gcccggcctc agtgagcgag cgagcgcgca gagagggagt 7681 ggccaactcc atcactaggg gttcctgcgg cctctagact cgaggcgtt //