LOCUS Exported 7604 bp ds-DNA circular SYN 12-MAY-2021 DEFINITION Mammalian expression of wild type Nme Cas9. ACCESSION . VERSION . KEYWORDS pCSDest2-NmeCas9-NLS-3XHA-NLS SOURCE synthetic DNA construct ORGANISM synthetic DNA construct REFERENCE 1 (bases 1 to 7604) AUTHORS Pawluk A, Amrani N, Zhang Y, Garcia B, Hidalgo-Reyes Y, Lee J, Edraki A, Shah M, Sontheimer EJ, Maxwell KL, Davidson AR TITLE Naturally Occurring Off-Switches for CRISPR-Cas9. JOURNAL Cell. 2016 Dec 15;167(7):1829-1838.e9. doi: 10.1016/j.cell.2016.11.017. Epub 2016 Dec 8. PUBMED 27984730 REFERENCE 2 (bases 1 to 7604) AUTHORS . TITLE Direct Submission JOURNAL Exported May 12, 2021 from SnapGene Server 1.1.58 http://www.snapgene.com FEATURES Location/Qualifiers source 1..7604 /organism="synthetic DNA construct" /mol_type="other DNA" regulatory 22..31 /regulatory_class="other" /note="vertebrate consensus sequence for strong initiation of translation (Kozak, 1987)" CDS 28..3273 /codon_start=1 /product="Cas9 endonuclease from the Neisseria meningitidis Type II CRISPR/Cas system (Esvelt et al., 2013)" /label=NmCas9 /note="generates RNA-guided double strand breaks in DNA" /translation="MAAFKPNSINYILGLDIGIASVGWAMVEIDEEENPIRLIDLGVRV FERAEVPKTGDSLAMARRLARSVRRLTRRRAHRLLRTRRLLKREGVLQAANFDENGLIK SLPNTPWQLRAAALDRKLTPLEWSAVLLHLIKHRGYLSQRKNEGETADKELGALLKGVA GNAHALQTGDFRTPAELALNKFEKESGHIRNQRSDYSHTFSRKDLQAELILLFEKQKEF GNPHVSGGLKEGIETLLMTQRPALSGDAVQKMLGHCTFEPAEPKAAKNTYTAERFIWLT KLNNLRILEQGSERPLTDTERATLMDEPYRKSKLTYAQARKLLGLEDTAFFKGLRYGKD NAEASTLMEMKAYHAISRALEKEGLKDKKSPLNLSPELQDEIGTAFSLFKTDEDITGRL KDRIQPEILEALLKHISFDKFVQISLKALRRIVPLMEQGKRYDEACAEIYGDHYGKKNT EEKIYLPPIPADEIRNPVVLRALSQARKVINGVVRRYGSPARIHIETAREVGKSFKDRK EIEKRQEENRKDREKAAAKFREYFPNFVGEPKSKDILKLRLYEQQHGKCLYSGKEINLG RLNEKGYVEIDHALPFSRTWDDSFNNKVLVLGSENQNKGNQTPYEYFNGKDNSREWQEF KARVETSRFPRSKKQRILLQKFDEDGFKERNLNDTRYVNRFLCQFVADRMRLTGKGKKR VFASNGQITNLLRGFWGLRKVRAENDRHHALDAVVVACSTVAMQQKITRFVRYKEMNAF DGKTIDKETGEVLHQKTHFPQPWEFFAQEVMIRVFGKPDGKPEFEEADTLEKLRTLLAE KLSSRPEAVHEYVTPLFVSRAPNRKMSGQGHMETVKSAKRLDEGVSVLRVPLTQLKLKD LEKMVNREREPKLYEALKARLEAHKDDPAKAFAEPFYKYDKAGNRTQQVKAVRVEQVQK TGVWVRNHNGIADNATMVRVDVFEKGDKYYLVPIYSWQVAKGILPDRAVVQGKDEEDWQ LIDDSFNFKFSLHPNDLVEVITKKARMFGYFASCHRGTGNINIRIHDLDHKIGKNGILE GIGVKTALSFQKYQIDELGKEIRPCRLKKRPPVR" CDS 3286..3306 /codon_start=1 /product="nuclear localization signal of SV40 (simian virus 40) large T antigen" /label=SV40 NLS /translation="PKKKRKV" CDS 3307..3396 /codon_start=1 /product="three tandem HA epitope tags" /label=3xHA /translation="YPYDVPDYAGYPYDVPDYAGSYPYDVPDYA" CDS 3409..3435 /codon_start=1 /product="nuclear localization signal (Makkerh et al., 1996)" /label=NLS /translation="PAAKKKKLD" protein_bind complement(3473..3497) /gene="mutant version of attB" /label=attB2 /bound_moiety="BP Clonase(TM)" /note="recombination site for the Gateway(R) BP reaction" promoter complement(3520..3537) /label=T7 promoter /note="promoter for bacteriophage T7 RNA polymerase" polyA_signal 3542..3676 /label=SV40 poly(A) signal /note="SV40 polyadenylation signal" primer_bind complement(3566..3585) /label=EBV-rev /note="SV40 polyA terminator, reverse primer" primer_bind 3620..3639 /label=SV40pA-R /note="SV40 polyA, reverse primer" primer_bind complement(3789..3809) /label=T3 /note="T3 promoter, forward primer" promoter complement(3789..3807) /label=T3 promoter /note="promoter for bacteriophage T3 RNA polymerase" primer_bind complement(3828..3844) /label=M13 rev /note="common sequencing primer, one of multiple similar variants" primer_bind complement(3828..3844) /label=M13 Reverse /note="In lacZ gene. Also called M13-rev" primer_bind complement(3841..3863) /label=M13/pUC Reverse /note="In lacZ gene" protein_bind 3852..3868 /label=lac operator /bound_moiety="lac repressor encoded by lacI" /note="The lac repressor binds to the lac operator to inhibit transcription in E. coli. This inhibition can be relieved by adding lactose or isopropyl-beta-D-thiogalactopyranoside (IPTG)." promoter complement(3876..3906) /label=lac promoter /note="promoter for the E. coli lac operon" protein_bind 3921..3942 /label=CAP binding site /bound_moiety="E. coli catabolite activator protein" /note="CAP binding activates transcription in the presence of cAMP." primer_bind complement(4059..4076) /label=L4440 /note="L4440 vector, forward primer" rep_origin complement(4230..4818) /direction=LEFT /label=ori /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of replication" primer_bind complement(4310..4329) /label=pBR322ori-F /note="pBR322 origin, forward primer" CDS complement(4989..5849) /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 5612..5631 /label=Amp-R /note="Ampicillin resistance gene, reverse primer" promoter complement(5850..5954) /gene="bla" /label=AmpR promoter rep_origin complement(5980..6435) /direction=LEFT /label=f1 ori /note="f1 bacteriophage origin of replication; arrow indicates direction of (+) strand synthesis" primer_bind complement(6117..6138) /label=F1ori-F /note="F1 origin, forward primer" primer_bind 6329..6348 /label=F1ori-R /note="F1 origin, reverse primer" promoter 6513..7490 /label=CMV IE94 promoter /note="enhancer/promoter region of simian cytomegalovirus major immediate early transcription unit IE94" promoter 7525..7543 /label=SP6 promoter /note="promoter for bacteriophage SP6 RNA polymerase" primer_bind 7525..7542 /label=SP6 /note="SP6 promoter, forward primer" protein_bind 7598..18 /gene="mutant version of attB" /label=attB1 /bound_moiety="BP Clonase(TM)" /note="recombination site for the Gateway(R) BP reaction" ORIGIN 1 tgtacaaaaa agcaggctgg cgccaccatg gctgccttca aacctaattc aatcaactac 61 atcctcggcc tcgatatcgg catcgcatcc gtcggctggg cgatggtaga aattgacgaa 121 gaagaaaacc ccatccgcct gattgatttg ggcgtgcgcg tatttgagcg tgccgaagta 181 ccgaaaacag gcgactccct tgccatggca aggcgtttgg cgcgcagtgt tcgccgcctg 241 acccgccgtc gcgcccaccg cctgcttcgg acccgccgcc tattgaaacg cgaaggcgta 301 ttacaagccg ccaattttga cgaaaacggc ttgattaaat ccttaccgaa tacaccatgg 361 caacttcgcg cagccgcatt agaccgcaaa ctgacgcctt tagagtggtc ggcagtcttg 421 ttgcatttaa tcaaacatcg cggctattta tcgcaacgga aaaacgaggg cgaaactgcc 481 gataaggagc ttggcgcttt gcttaaaggc gtagccggca atgcccatgc cttacagaca 541 ggcgatttcc gcacaccggc cgaattggct ttaaataaat ttgagaaaga aagcggccat 601 atccgcaatc agcgcagcga ttattcgcat acgttcagcc gcaaagattt acaggcggag 661 ctgattttgc tgtttgaaaa acaaaaagaa tttggcaatc cgcatgtttc aggcggcctt 721 aaagaaggta ttgaaaccct actgatgacg caacgccctg ccctgtccgg cgatgccgtt 781 caaaaaatgt tggggcattg caccttcgaa ccggcagagc cgaaagccgc taaaaacacc 841 tacacagccg aacgtttcat ctggctgacc aagctgaaca acctgcgtat tttagagcaa 901 ggcagcgagc ggccattgac cgataccgaa cgcgccacgc ttatggacga gccatacaga 961 aaatccaaac tgacttacgc acaagcccgt aagctgctgg gtttagaaga taccgccttt 1021 ttcaaaggct tgcgctatgg taaagacaat gccgaagcct caacattgat ggaaatgaag 1081 gcctaccatg ccatcagccg tgcactggaa aaagaaggat tgaaagacaa aaaatcccca 1141 ttaaaccttt ctcccgaatt acaagacgaa atcggcacgg cattctccct gttcaaaacc 1201 gatgaagaca ttacaggccg tctgaaagac cgtatacagc ccgaaatctt agaagcgctg 1261 ttgaaacaca tcagcttcga taagttcgtc caaatttcct tgaaagcatt gcgccgaatt 1321 gtgcctctaa tggaacaagg caaacgttac gatgaagcct gcgccgaaat ctacggagac 1381 cattacggca agaagaatac ggaagaaaag atttatctgc cgccgattcc cgccgacgaa 1441 atccgcaacc ccgtcgtctt gcgcgcctta tctcaagcac gtaaggtcat taacggcgtg 1501 gtacgccgtt acggctcccc agctcgtatc catattgaaa ctgcaaggga agtaggtaaa 1561 tcgtttaaag accgcaaaga aattgagaaa cgccaagaag aaaaccgcaa agaccgggaa 1621 aaagccgccg ccaaattccg agagtatttc cccaattttg tcggagaacc caaatccaaa 1681 gatattctga aactgcgcct gtacgagcaa caacacggca aatgcctgta ttcgggcaaa 1741 gaaatcaact taggccgtct gaacgaaaaa ggctatgtcg aaatcgacca tgccctgccg 1801 ttctcgcgca catgggacga cagtttcaac aataaagtac tggtattggg cagcgaaaac 1861 caaaacaaag gcaatcaaac cccttacgaa tacttcaacg gcaaagacaa cagccgcgaa 1921 tggcaggaat ttaaagcgcg tgtcgaaacc agccgtttcc cgcgcagtaa aaaacaacgg 1981 attctgctgc aaaaattcga tgaagacggc tttaaagaac gcaatctgaa cgacacgcgc 2041 tacgtcaacc gtttcctgtg tcaatttgtt gccgaccgta tgcggctgac aggtaaaggc 2101 aagaaacgtg tctttgcatc caacggacaa attaccaatc tgttgcgcgg cttttgggga 2161 ttgcgcaaag tgcgtgcgga aaacgaccgc catcacgcct tggacgccgt cgtcgttgcc 2221 tgctcgaccg ttgccatgca gcagaaaatt acccgttttg tacgctataa agagatgaac 2281 gcgtttgacg gtaaaaccat agacaaagaa acaggagaag tgctgcatca aaaaacacac 2341 ttcccacaac cttgggaatt tttcgcacaa gaagtcatga ttcgcgtctt cggcaaaccg 2401 gacggcaaac ccgaattcga agaagccgat accctagaaa aactgcgcac gttgcttgcc 2461 gaaaaattat catctcgccc cgaagccgta cacgaatacg ttacgccact gtttgtttca 2521 cgcgcgccca atcggaagat gagcgggcaa gggcatatgg agaccgtcaa atccgccaaa 2581 cgactggacg aaggcgtcag cgtgttgcgc gtaccgctga cacagttaaa actgaaagac 2641 ttggaaaaaa tggtcaatcg ggagcgcgaa cctaagctat acgaagcact gaaagcacgg 2701 ctggaagcac ataaagacga tcctgccaaa gcctttgccg agccgtttta caaatacgat 2761 aaagcaggca accgcaccca acaggtaaaa gccgtacgcg tagagcaagt acagaaaacc 2821 ggcgtatggg tgcgcaacca taacggtatt gccgacaacg caaccatggt gcgcgtagat 2881 gtgtttgaga aaggcgacaa gtattatctg gtaccgattt acagttggca ggtagcgaaa 2941 gggattttgc cggatagggc tgttgtacaa ggaaaagatg aagaagattg gcaacttatt 3001 gatgatagtt tcaactttaa attctcatta caccctaatg atttagtcga ggttataaca 3061 aaaaaagcta gaatgtttgg ttactttgcc agctgccatc gaggcacagg taatatcaat 3121 atacgcattc atgatcttga tcataaaatt ggcaaaaatg gaatactgga aggtatcggc 3181 gtcaaaaccg ccctttcatt ccaaaaatac caaattgacg aactgggcaa agaaatcaga 3241 ccatgccgtc tgaaaaaacg cccgcctgtc cgtggcaccg gcgggcccaa gaagaagagg 3301 aaggtatacc catacgatgt tcctgactat gcgggctatc cctatgacgt cccggactat 3361 gcaggatcgt atccttatga cgttccagat tacgctggat ccgccgctcc ggcagctaag 3421 aaaaagaaac tggatttcga atccggataa gcggccgcct cgagatatct agacccagct 3481 ttcttgtaca aagtggttga tcctctcgag cctctagaac tatagtgagt cgtattacgt 3541 agatccagac atgataagat acattgatga gtttggacaa accacaacta gaatgcagtg 3601 aaaaaaatgc tttatttgtg aaatttgtga tgctattgct ttatttgtaa ccattataag 3661 ctgcaataaa caagttaaca acaacaattg cattcatttt atgtttcagg ttcaggggga 3721 ggtgtgggag gttttttaat tcgcggccgc ggcgccaatg cattgggccc ggtacccagc 3781 ttttgttccc tttagtgagg gttaattgcg cgcttggcgt aatcatggtc atagctgttt 3841 cctgtgtgaa attgttatcc gctcacaatt ccacacaaca tacgagccgg gagcataaag 3901 tgtaaagcct ggggtgccta atgagtgagc taactcacat taattgcgtt gcgctcactg 3961 cccgctttcc agtcgggaaa cctgtcgtgc cagctgcatt aatgaatcgg ccaacgcgcg 4021 gggagaggcg gtttgcgtat tgggcgctct tccgcttcct cgctcactga ctcgctgcgc 4081 tcggtcgttc ggctgcggcg agcggtatca gctcactcaa aggcggtaat acggttatcc 4141 acagaatcag gggataacgc aggaaagaac atgtgagcaa aaggccagca aaaggccagg 4201 aaccgtaaaa aggccgcgtt gctggcgttt ttccataggc tccgcccccc tgacgagcat 4261 cacaaaaatc gacgctcaag tcagaggtgg cgaaacccga caggactata aagataccag 4321 gcgtttcccc ctggaagctc cctcgtgcgc tctcctgttc cgaccctgcc gcttaccgga 4381 tacctgtccg cctttctccc ttcgggaagc gtggcgcttt ctcatagctc acgctgtagg 4441 tatctcagtt cggtgtaggt cgttcgctcc aagctgggct gtgtgcacga accccccgtt 4501 cagcccgacc gctgcgcctt atccggtaac tatcgtcttg agtccaaccc ggtaagacac 4561 gacttatcgc cactggcagc agccactggt aacaggatta gcagagcgag gtatgtaggc 4621 ggtgctacag agttcttgaa gtggtggcct aactacggct acactagaag aacagtattt 4681 ggtatctgcg ctctgctgaa gccagttacc ttcggaaaaa gagttggtag ctcttgatcc 4741 ggcaaacaaa ccaccgctgg tagcggtggt ttttttgttt gcaagcagca gattacgcgc 4801 agaaaaaaag gatctcaaga agatcctttg atcttttcta cggggtctga cgctcagtgg 4861 aacgaaaact cacgttaagg gattttggtc atgagattat caaaaaggat cttcacctag 4921 atccttttaa attaaaaatg aagttttaaa tcaatctaaa gtatatatga gtaaacttgg 4981 tctgacagtt accaatgctt aatcagtgag gcacctatct cagcgatctg tctatttcgt 5041 tcatccatag ttgcctgact ccccgtcgtg tagataacta cgatacggga gggcttacca 5101 tctggcccca gtgctgcaat gataccgcga gacccacgct caccggctcc agatttatca 5161 gcaataaacc agccagccgg aagggccgag cgcagaagtg gtcctgcaac tttatccgcc 5221 tccatccagt ctattaattg ttgccgggaa gctagagtaa gtagttcgcc agttaatagt 5281 ttgcgcaacg ttgttgccat tgctacaggc atcgtggtgt cacgctcgtc gtttggtatg 5341 gcttcattca gctccggttc ccaacgatca aggcgagtta catgatcccc catgttgtgc 5401 aaaaaagcgg ttagctcctt cggtcctccg atcgttgtca gaagtaagtt ggccgcagtg 5461 ttatcactca tggttatggc agcactgcat aattctctta ctgtcatgcc atccgtaaga 5521 tgcttttctg tgactggtga gtactcaacc aagtcattct gagaatagtg tatgcggcga 5581 ccgagttgct cttgcccggc gtcaatacgg gataataccg cgccacatag cagaacttta 5641 aaagtgctca tcattggaaa acgttcttcg gggcgaaaac tctcaaggat cttaccgctg 5701 ttgagatcca gttcgatgta acccactcgt gcacccaact gatcttcagc atcttttact 5761 ttcaccagcg tttctgggtg agcaaaaaca ggaaggcaaa atgccgcaaa aaagggaata 5821 agggcgacac ggaaatgttg aatactcata ctcttccttt ttcaatatta ttgaagcatt 5881 tatcagggtt attgtctcat gagcggatac atatttgaat gtatttagaa aaataaacaa 5941 ataggggttc cgcgcacatt tccccgaaaa gtgccaccta aattgtaagc gttaatattt 6001 tgttaaaatt cgcgttaaat ttttgttaaa tcagctcatt ttttaaccaa taggccgaaa 6061 tcggcaaaat cccttataaa tcaaaagaat agaccgagat agggttgagt gttgttccag 6121 tttggaacaa gagtccacta ttaaagaacg tggactccaa cgtcaaaggg cgaaaaaccg 6181 tctatcaggg cgatggccca ctacgtgaac catcacccta atcaagtttt ttggggtcga 6241 ggtgccgtaa agcactaaat cggaacccta aagggagccc ccgatttaga gcttgacggg 6301 gaaagccggc gaacgtggcg agaaaggaag ggaagaaagc gaaaggagcg ggcgctaggg 6361 cgctggcaag tgtagcggtc acgctgcgcg taaccaccac acccgccgcg cttaatgcgc 6421 cgctacaggg cgcgtcccat tcgccattca ggctgcgcaa ctgttgggaa gggcgatcgg 6481 tgcgggcctc ttcgctatta cgccagtcga ccgccaattc aatatggcgt atatggactc 6541 atgccaattc aatatggtgg atctggacct gtgccaattc aatatggcgt atatggactc 6601 gtgccaattc aatatggtgg atctggaccc cagccaattc aatatggcgg acttggcacc 6661 atgccaattc aatatggcgg acctggcact gtgccaactg gggaggggtc tacttggcac 6721 ggtgccaagt ttgaggaggg gtcttggccc tgtgccaagt ccgccatatt gaattggcat 6781 ggtgccaata atggcggcca tattggctat atgccaggat caatatatag gcaatatcca 6841 atatggccct atgccaatat ggctattggc caggttcaat actatgtatt ggccctatgc 6901 catatagtat tccatatatg ggttttccta ttgacgtaga tagcccctcc caatgggcgg 6961 tcccatatac catatatggg gcttcctaat accgcccata gccactcccc cattgacgtc 7021 aatggtctct atatatggtc tttcctattg acgtcatatg ggcggtccta ttgacgtata 7081 tggcgcctcc cccattgacg tcaattacgg taaatggccc gcctggctca atgcccattg 7141 acgtcaatag gaccacccac cattgacgtc aatgggatgg ctcattgccc attcatatcc 7201 gttctcacgc cccctattga cgtcaatgac ggtaaatggc ccacttggca gtacatcaat 7261 atctattaat agtaacttgg caagtacatt actattggaa gtacgccagg gtacattggc 7321 agtactccca ttgacgtcaa tggcggtaaa tggcccgcga tggctgccaa gtacatcccc 7381 attgacgtca atggggaggg gcaatgacgc aaatgggcgt tccattgacg taaatgggcg 7441 gtaggcgtgc ctaatgggag gtctatataa gcaatgctcg tttagggaac cgccattctg 7501 cctggggacg tcggagcaag cttgatttag gtgacactat agaatacaag ctacttgttc 7561 tttttgcagg atcccatcga ttcgaattca aggatcaaca agtt //