LOCUS Exported 6118 bp ds-DNA circular SYN 12-MAY-2021 DEFINITION AAV plasmid expressing CjCas9 under control of miniCMV promoter. ACCESSION . VERSION . KEYWORDS pX551-miniCMV-CjCas9 SOURCE synthetic DNA construct ORGANISM synthetic DNA construct REFERENCE 1 (bases 1 to 6118) TITLE Alex Hewitt Lab plasmids REFERENCE 2 (bases 1 to 6118) AUTHORS . TITLE Direct Submission JOURNAL Exported May 12, 2021 from SnapGene Server 1.1.58 http://www.snapgene.com FEATURES Location/Qualifiers source 1..6118 /organism="synthetic DNA construct" /mol_type="other DNA" primer_bind 128..147 /label=T7 /note="T7 promoter, forward primer" promoter 128..146 /label=T7 promoter /note="promoter for bacteriophage T7 RNA polymerase" promoter 165..203 /label=minimal CMV promoter /note="human cytomegalovirus (CMV) immediate early promoter" primer_bind 200..224 /label=LNCX /note="Human CMV promoter, forward primer" CDS 259..285 /codon_start=1 /product="HA (human influenza hemagglutinin) epitope tag" /label=HA /translation="YPYDVPDYA" CDS 289..309 /codon_start=1 /product="nuclear localization signal of SV40 (simian virus 40) large T antigen" /label=SV40 NLS /translation="PKKKRKV" CDS 319..3267 /codon_start=1 /product="Cas9 endonuclease from the Campylobacter jejuni Type II CRISPR/Cas system (Kim et al., 2016)" /label=CjCas9 /translation="ARILAFDIGISSIGWAFSENDELKDCGVRIFTKVENPKTGESLAL PRRLARSARKRLARRKARLNHLKHLIANEFKLNYEDYQSFDESLAKAYKGSLISPYELR FRALNELLSKQDFARVILHIAKRRGYDDIKNSDDKEKGAILKAIKQNEEKLANYQSVGE YLYKEYFQKFKENSKEFTNVRNKKESYERCIAQSFLKDELKLIFKKQREFGFSFSKKFE EEVLSVAFYKRALKDFSHLVGNCSFFTDEKRAPKNSPLAFMFVALTRIINLLNNLKNTE GILYTKDDLNALLNEVLKNGTLTYKQTKKLLGLSDDYEFKGEKGTYFIEFKKYKEFIKA LGEHNLSQDDLNEIAKDITLIKDEIKLKKALAKYDLNQNQIDSLSKLEFKDHLNISFKA LKLVTPLMLEGKKYDEACNELNLKVAINEDKKDFLPAFNETYYKDEVTNPVVLRAIKEY RKVLNALLKKYGKVHKINIELAREVGKNHSQRAKIEKEQNENYKAKKDAELECEKLGLK INSKNILKLRLFKEQKEFCAYSGEKIKISDLQDEKMLEIDHIYPYSRSFDDSYMNKVLV FTKQNQEKLNQTPFEAFGNDSAKWQKIEVLAKNLPTKKQKRILDKNYKDKEQKNFKDRN LNDTRYIARLVLNYTKDYLDFLPLSDDENTKLNDTQKGSKVHVEAKSGMLTSALRHTWG FSAKDRNNHLHHAIDAVIIAYANNSIVKAFSDFKKEQESNSAELYAKKISELDYKNKRK FFEPFSGFRQKVLDKIDEIFVSKPERKKPSGALHEETFRKEEEFYQSYGGKEGVLKALE LGKIRKVNGKIVKNGDMFRVDIFKHKKTNKFYAVPIYTMDFALKVLPNKAVARSKKGEI KDWILMDENYEFCFSLYKDSLILIQTKDMQEPEFVYYNAFTSSTVSLIVSKHDNKFETL SKNQKILFKNANEKEVIAKSIGIQNLKVFEKYIVSALGEVTKAEFRQREDFKK" CDS 3271..3291 /codon_start=1 /product="nuclear localization signal of SV40 (simian virus 40) large T antigen" /label=SV40 NLS /translation="PKKKRKV" polyA_signal 3310..3358 /note="synthetic polyadenylation signal" repeat_region 3366..3506 /label=AAV2 ITR /note="inverted terminal repeat of adeno-associated virus serotype 2" repeat_region 3366..3495 /label=AAV2 ITR rep_origin 3581..4036 /direction=RIGHT /label=f1 ori /note="f1 bacteriophage origin of replication; arrow indicates direction of (+) strand synthesis" primer_bind complement(3668..3687) /label=F1ori-R /note="F1 origin, reverse primer" primer_bind 3878..3899 /label=F1ori-F /note="F1 origin, forward primer" primer_bind complement(4053..4072) /label=pRS-marker /note="pRS vectors, use to sequence yeast selectable marker" primer_bind 4172..4194 /label=pGEX 3' /note="pGEX vectors, reverse primer" primer_bind complement(4232..4250) /label=pBRforEco /note="pBR322 vectors, upsteam of EcoRI site, forward primer" promoter 4318..4422 /gene="bla" /label=AmpR promoter CDS 4423..5283 /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(4641..4660) /label=Amp-R /note="Ampicillin resistance gene, reverse primer" rep_origin 5454..6042 /direction=RIGHT /label=ori /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of replication" primer_bind 5943..5962 /label=pBR322ori-F /note="pBR322 origin, forward primer" repeat_region 6104..115 /label=AAV2 ITR (alternate) /note="Functional equivalent of wild-type AAV2 ITR" ORIGIN 1 gcgctcgctc gctcactgag gccgcccggg cgtcgggcga cctttggtcg cccggcctca 61 gtgagcgagc gagcgcgcag agagggagtg gccaactcca tcactagggg ttcctgcggc 121 ctctagataa tacgactcac tataggggga tccacgtatg tcgaggtagg cgtgtacggt 181 gggaggccta tataagcaga gctcgtttag tgaaccgtca gatcgcctgg aggtaccgcc 241 accaccggtg ccaccatgta cccatacgat gttccagatt acgcttcgcc gaagaaaaag 301 cgcaaggtcg aagcgtccgc ccgcatcctc gctttcgaca tcggaatctc tagtatcgga 361 tgggccttct ctgaaaacga cgaactgaaa gactgcggcg tgagaatctt cacaaaggtt 421 gaaaacccta aaacaggcga gtctttagct ctgccacgta ggttggcccg ctccgcccga 481 aaaaggctgg ctcggcggaa ggctcgcctc aaccacttga agcatttgat agctaatgag 541 ttcaaactga actacgaaga ttaccagtcc ttcgacgagt cattggcaaa agcctacaaa 601 ggcagcctta tcagtcctta tgagttgaga tttcgcgcac tcaacgaact gctttctaag 661 caagactttg ctagggtcat tctgcacatc gcaaaacggc gaggttatga cgatatcaag 721 aactccgacg ataaagaaaa gggagccatt ctcaaggcga tcaaacagaa tgaggaaaaa 781 ttggcaaact accagagtgt gggcgagtat ctgtataaag agtatttcca gaagtttaag 841 gaaaacagca aggagtttac aaacgtcaga aataaaaagg agtcttacga gagatgcatc 901 gcgcagtcat tcctcaaaga tgagctgaag ctgatattta agaagcaacg cgaatttggt 961 ttctcattct ctaagaagtt cgaagaggag gttctttccg tggcgtttta caagagggcg 1021 ctcaaagact tctcccacct ggttggtaac tgtagtttct tcacggatga gaagcgagct 1081 cccaaaaatt ctcccctggc tttcatgttt gttgccctga ctcggatcat taacctgctg 1141 aacaacctga aaaatactga agggatcttg tatacgaagg acgacctaaa tgcactcctg 1201 aatgaagtgc tcaaaaacgg aactctaacc tataaacaga ccaagaaatt actggggctc 1261 tctgacgact acgagttcaa gggcgagaag ggtacttatt ttatcgaatt caaaaagtat 1321 aaggagttca ttaaagcatt gggggaacac aacctcagcc aggacgatct caatgaaatt 1381 gccaaggaca tcacgctgat taaagacgag ataaaactga aaaaggcact ggccaagtat 1441 gacctcaacc agaaccagat cgactctctg tccaagctgg agttcaaaga ccacctaaac 1501 atatccttca aagccctgaa actggtcacc cctctaatgc tcgaaggaaa aaaatacgac 1561 gaggcgtgta atgaactgaa tcttaaggtg gccatcaatg aggataagaa ggactttctt 1621 ccagccttta acgagacata ttacaaagac gaggtcacaa acccggttgt gctgagggcc 1681 ataaaagagt atcggaaggt tctgaatgcc ctcctgaaga agtacggcaa agtgcacaaa 1741 ataaatatcg aattggctag ggaggtgggg aagaaccatt ctcagcgagc aaagatcgag 1801 aaagagcaga atgagaacta caaagccaag aaagacgccg aactggagtg cgaaaagctg 1861 gggcttaaaa taaacagtaa aaacatcctg aaattaagat tgttcaaaga gcaaaaggag 1921 ttttgcgcct actcagggga aaaaatcaaa atatcagacc tgcaggacga gaaaatgctg 1981 gagatcgacc acatctatcc gtatagcagg tcatttgacg attcctacat gaacaaagtg 2041 cttgtgttta ccaaacagaa ccaagaaaag ctgaaccaaa ccccctttga ggctttcgga 2101 aacgactcag ccaagtggca gaaaatcgaa gtcctagcca agaatctgcc tacaaaaaaa 2161 caaaagagga ttcttgataa gaactataag gacaaggaac agaaaaactt taaagacagg 2221 aacctgaatg acacgaggta cattgcgcga ctggttctaa actataccaa agactacctg 2281 gatttcctcc ctctgagcga cgacgagaat actaaactga atgataccca gaaaggctca 2341 aaggtccacg ttgaggctaa gtccgggatg ctgactagcg ccctccgcca cacgtggggc 2401 ttcagcgcca aagatcggaa taatcatctt catcacgcta ttgatgcagt aatcatagcc 2461 tacgctaaca acagcatcgt gaaagccttc tccgatttca agaaagaaca ggagtctaat 2521 agcgccgagt tgtacgccaa gaaaatttcc gaattggact ataaaaataa gagaaaattc 2581 ttcgaaccct tctccgggtt tcgccaaaag gtcttagata agatcgacga gattttcgtt 2641 tccaagcccg aaagaaaaaa gccttcaggg gcactgcacg aagagacatt ccgcaaggaa 2701 gaggaatttt accaatctta cggtggtaaa gagggagttc tgaaggctct ggagcttggg 2761 aagatccgca aggtaaacgg gaaaatcgtg aaaaacgggg acatgttcag ggtggatatc 2821 ttcaagcaca aaaagaccaa caagttctac gcagtaccca tctacactat ggatttcgct 2881 ttaaaggttc tcccaaataa ggcggtggct cgatcgaaga aaggagagat caaggactgg 2941 atcttaatgg atgaaaatta cgagttttgc ttctcgctct acaaagatag cctgattctg 3001 atccagacaa aagacatgca ggaaccagaa tttgtttatt ataacgcctt cacgagcagt 3061 acagtgtccc tgattgtgag caagcatgat aacaagttcg agactctgtc taagaatcag 3121 aaaatccttt tcaagaacgc caacgagaag gaggtcatcg caaagtcaat tggcatccaa 3181 aacctgaagg tgttcgagaa atacatagtg tccgcactcg gtgaagtaac taaagccgaa 3241 tttcgacagc gcgaggattt taagaaaagc cccaagaaga agagaaaggt ggaggccagc 3301 taagaattca ataaaagatc tttgttttca ttagatctgt gtgttggttt tttgtgtgcg 3361 gccgcaggaa cccctagtga tggagttggc cactccctct ctgcgcgctc gctcgctcac 3421 tgaggccggg cgaccaaagg tcgcccgacg cccgggcttt gcccgggcgg cctcagtgag 3481 cgagcgagcg cgcagctgcc tgcaggggcg cctgatgcgg tattttctcc ttacgcatct 3541 gtgcggtatt tcacaccgca tacgtcaaag caaccatagt acgcgccctg tagcggcgca 3601 ttaagcgcgg cgggtgtggt ggttacgcgc agcgtgaccg ctacacttgc cagcgcctta 3661 gcgcccgctc ctttcgcttt cttcccttcc tttctcgcca cgttcgccgg ctttccccgt 3721 caagctctaa atcgggggct ccctttaggg ttccgattta gtgctttacg gcacctcgac 3781 cccaaaaaac ttgatttggg tgatggttca cgtagtgggc catcgccctg atagacggtt 3841 tttcgccctt tgacgttgga gtccacgttc tttaatagtg gactcttgtt ccaaactgga 3901 acaacactca actctatctc gggctattct tttgatttat aagggatttt gccgatttcg 3961 gtctattggt taaaaaatga gctgatttaa caaaaattta acgcgaattt taacaaaata 4021 ttaacgttta caattttatg gtgcactctc agtacaatct gctctgatgc cgcatagtta 4081 agccagcccc gacacccgcc aacacccgct gacgcgccct gacgggcttg tctgctcccg 4141 gcatccgctt acagacaagc tgtgaccgtc tccgggagct gcatgtgtca gaggttttca 4201 ccgtcatcac cgaaacgcgc gagacgaaag ggcctcgtga tacgcctatt tttataggtt 4261 aatgtcatga taataatggt ttcttagacg tcaggtggca cttttcgggg aaatgtgcgc 4321 ggaaccccta tttgtttatt tttctaaata cattcaaata tgtatccgct catgagacaa 4381 taaccctgat aaatgcttca ataatattga aaaaggaaga gtatgagtat tcaacatttc 4441 cgtgtcgccc ttattccctt ttttgcggca ttttgccttc ctgtttttgc tcacccagaa 4501 acgctggtga aagtaaaaga tgctgaagat cagttgggtg cacgagtggg ttacatcgaa 4561 ctggatctca acagcggtaa gatccttgag agttttcgcc ccgaagaacg ttttccaatg 4621 atgagcactt ttaaagttct gctatgtggc gcggtattat cccgtattga cgccgggcaa 4681 gagcaactcg gtcgccgcat acactattct cagaatgact tggttgagta ctcaccagtc 4741 acagaaaagc atcttacgga tggcatgaca gtaagagaat tatgcagtgc tgccataacc 4801 atgagtgata acactgcggc caacttactt ctgacaacga tcggaggacc gaaggagcta 4861 accgcttttt tgcacaacat gggggatcat gtaactcgcc ttgatcgttg ggaaccggag 4921 ctgaatgaag ccataccaaa cgacgagcgt gacaccacga tgcctgtagc aatggcaaca 4981 acgttgcgca aactattaac tggcgaacta cttactctag cttcccggca acaattaata 5041 gactggatgg aggcggataa agttgcagga ccacttctgc gctcggccct tccggctggc 5101 tggtttattg ctgataaatc tggagccggt gagcgtggaa gccgcggtat cattgcagca 5161 ctggggccag atggtaagcc ctcccgtatc gtagttatct acacgacggg gagtcaggca 5221 actatggatg aacgaaatag acagatcgct gagataggtg cctcactgat taagcattgg 5281 taactgtcag accaagttta ctcatatata ctttagattg atttaaaact tcatttttaa 5341 tttaaaagga tctaggtgaa gatccttttt gataatctca tgaccaaaat cccttaacgt 5401 gagttttcgt tccactgagc gtcagacccc gtagaaaaga tcaaaggatc ttcttgagat 5461 cctttttttc tgcgcgtaat ctgctgcttg caaacaaaaa aaccaccgct accagcggtg 5521 gtttgtttgc cggatcaaga gctaccaact ctttttccga aggtaactgg cttcagcaga 5581 gcgcagatac caaatactgt tcttctagtg tagccgtagt taggccacca cttcaagaac 5641 tctgtagcac cgcctacata cctcgctctg ctaatcctgt taccagtggc tgctgccagt 5701 ggcgataagt cgtgtcttac cgggttggac tcaagacgat agttaccgga taaggcgcag 5761 cggtcgggct gaacgggggg ttcgtgcaca cagcccagct tggagcgaac gacctacacc 5821 gaactgagat acctacagcg tgagctatga gaaagcgcca cgcttcccga agggagaaag 5881 gcggacaggt atccggtaag cggcagggtc ggaacaggag agcgcacgag ggagcttcca 5941 gggggaaacg cctggtatct ttatagtcct gtcgggtttc gccacctctg acttgagcgt 6001 cgatttttgt gatgctcgtc aggggggcgg agcctatgga aaaacgccag caacgcggcc 6061 tttttacggt tcctggcctt ttgctggcct tttgctcaca tgtcctgcag gcagctgc //