LOCUS Exported 7600 bp ds-DNA circular SYN 13-MAY-2021 DEFINITION AAV Vector for expression of N-terminal SpyCas9 fragment with Intein and a U6-driven F+E gRNA scaffold. ACCESSION . VERSION . KEYWORDS AAV N-SpyCas9-Intein, U6-gRNA scaffold (F+E) SOURCE synthetic DNA construct ORGANISM synthetic DNA construct REFERENCE 1 (bases 1 to 7600) AUTHORS Hoffmann MD, Aschenbrenner S, Grosse S, Rapti K, Domenger C, Fakhiri J, Mastel M, Borner K, Eils R, Grimm D, Niopek D TITLE Cell-specific CRISPR-Cas9 activation by microRNA-dependent expression of anti-CRISPR proteins. JOURNAL Nucleic Acids Res. 2019 Apr 15. pii: 5458120. doi: 10.1093/nar/gkz271. PUBMED 30982889 REFERENCE 2 (bases 1 to 7600) AUTHORS . TITLE Direct Submission JOURNAL Exported May 13, 2021 from SnapGene Server 1.1.58 http://www.snapgene.com FEATURES Location/Qualifiers source 1..7600 /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" intron 676..808 /label=chimeric intron /note="chimera between introns from human beta-globin and immunoglobulin heavy chain genes" regulatory 838..847 /regulatory_class="other" /note="vertebrate consensus sequence for strong initiation of translation (Kozak, 1987)" CDS 850..870 /codon_start=1 /product="nuclear localization signal of SV40 (simian virus 40) large T antigen" /label=SV40 NLS /translation="PKKKRKV" CDS 895..2610 /codon_start=1 /product="N-terminal portion of Streptococcus pyogenes Cas9 (Zetsche et al., 2015)" /label=Cas9(N) /translation="DKKYSIGLDIGTNSVGWAVITDEYKVPSKKFKVLGNTDRHSIKKN LIGALLFDSGETAEATRLKRTARRRYTRRKNRICYLQEIFSNEMAKVDDSFFHRLEESF LVEEDKKHERHPIFGNIVDEVAYHEKYPTIYHLRKKLVDSTDKADLRLIYLALAHMIKF RGHFLIEGDLNPDNSDVDKLFIQLVQTYNQLFEENPINASGVDAKAILSARLSKSRRLE NLIAQLPGEKKNGLFGNLIALSLGLTPNFKSNFDLAEDAKLQLSKDTYDDDLDNLLAQI GDQYADLFLAAKNLSDAILLSDILRVNTEITKAPLSASMIKRYDEHHQDLTLLKALVRQ QLPEKYKEIFFDQSKNGYAGYIDGGASQEEFYKFIKPILEKMDGTEELLVKLNREDLLR KQRTFDNGSIPHQIHLGELHAILRRQEDFYPFLKDNREKIEKILTFRIPYYVGPLARGN SRFAWMTRKSEETITPWNFEEVVDKGASAQSFIERMTNFDKNLPNEKVLPKHSLLYEYF TVYNELTKVKYVTEGMRKPAFLSGEQKKAIVDLLFKTNRKVTVKQLKEDYFKKIE" primer_bind complement(3350..3369) /label=EBV-rev /note="SV40 polyA terminator, reverse primer" polyA_signal 3379..3460 /label=SV40 poly(A) signal /note="SV40 polyadenylation signal" primer_bind 3404..3423 /label=SV40pA-R /note="SV40 polyA, reverse primer" promoter 3809..4049 /label=U6 promoter /note="RNA polymerase III promoter for human U6 snRNA" primer_bind 3809..3829 /label=hU6-F /note="Human U6 promoter, forward primer" primer_bind 3980..3999 /label=LKO.1 5' /note="Human U6 promoter, forward primer" repeat_region 4224..4353 /label=AAV2 ITR primer_bind complement(4391..4407) /label=M13 rev /note="common sequencing primer, one of multiple similar variants" primer_bind complement(4391..4407) /label=M13 Reverse /note="In lacZ gene. Also called M13-rev" primer_bind complement(4404..4426) /label=M13/pUC Reverse /note="In lacZ gene" protein_bind 4415..4431 /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(4439..4469) /label=lac promoter /note="promoter for the E. coli lac operon" protein_bind 4484..4505 /label=CAP binding site /bound_moiety="E. coli catabolite activator protein" /note="CAP binding activates transcription in the presence of cAMP." primer_bind complement(4622..4639) /label=L4440 /note="L4440 vector, forward primer" rep_origin complement(4793..5381) /direction=LEFT /label=ori /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of replication" primer_bind complement(4873..4892) /label=pBR322ori-F /note="pBR322 origin, forward primer" CDS complement(5552..6412) /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 6175..6194 /label=Amp-R /note="Ampicillin resistance gene, reverse primer" promoter complement(6413..6517) /gene="bla" /label=AmpR promoter rep_origin complement(6543..6998) /direction=LEFT /label=f1 ori /note="f1 bacteriophage origin of replication; arrow indicates direction of (+) strand synthesis" primer_bind complement(6680..6701) /label=F1ori-F /note="F1 origin, forward primer" primer_bind 6892..6911 /label=F1ori-R /note="F1 origin, reverse primer" primer_bind 7125..7147 /label=M13/pUC Forward /note="In lacZ gene" primer_bind 7139..7156 /label=M13 Forward /note="In lacZ gene. Also called M13-F20 or M13 (-21) Forward" primer_bind 7140..7156 /label=M13 fwd /note="common sequencing primer, one of multiple similar variants" repeat_region 7175..7304 /label=AAV2 ITR primer_bind complement(7351..7371) /label=pBABE 3' /note="SV40 enhancer, reverse primer for pBABE vectors" ORIGIN 1 ggcattgatt 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 ccgcccccta ttgacgtcaa tgacggtaaa tggcccgcct 301 ggcattatgc ccagtacatg accttacggg actttcctac ttggcagtac atctacgtat 361 tagtcatcgc tattaccatg gtgatgcggt tttggcagta caccaatggg cgtggatagc 421 ggtttgactc acggggattt ccaagtctcc accccattga cgtcaatggg agtttgtttt 481 ggcaccaaaa tcaacgggac tttccaaaat gtcgtaataa ccccgccccg ttgacgcaaa 541 tgggcggtag gcgtgtacgg tgggaggtct atataagcag aggtcgttta gtgaaccgtc 601 agatcactag tagctttatt gcggtagttt atcacagtta aattgctaac gcagtcagtg 661 ctcgactgat cacaggtaag tatcaaggtt acaagacagg tttaaggagg ccaatagaaa 721 ctgggcttgt cgagacagag aagattcttg cgtttctgat aggcacctat tggtcttact 781 gacatccact ttgcctttct ctccacaggg gtaccgaagc cgctagcgct accggtcgcc 841 accatggccc caaagaagaa gcggaaggtc ggtatccacg gagtcccagc agccgacaag 901 aagtactcca ttgggctcga tatcggcaca aacagcgtcg gctgggccgt cattacggac 961 gagtacaagg tgccgagcaa aaaattcaaa gttctgggca ataccgatcg ccacagcata 1021 aagaagaacc tcattggcgc cctcctgttc gactccgggg aaacggccga agccacgcgg 1081 ctcaaaagaa cagcacggcg cagatatacc cgcagaaaga atcggatctg ctacctgcag 1141 gagatcttta gtaatgagat ggctaaggtg gatgactctt tcttccatag gctggaggag 1201 tcctttttgg tggaggagga taaaaagcac gagcgccacc caatctttgg caatatcgtg 1261 gacgaggtgg cgtaccatga aaagtaccca accatatatc atctgaggaa gaagcttgta 1321 gacagtactg ataaggctga cttgcggttg atctatctcg cgctggcgca tatgatcaaa 1381 tttcggggac acttcctcat cgagggggac ctgaacccag acaacagcga tgtcgacaaa 1441 ctctttatcc aactggttca gacttacaat cagcttttcg aagagaaccc gatcaacgca 1501 tccggagttg acgccaaagc aatcctgagc gctaggctgt ccaaatcccg gcggctcgaa 1561 aacctcatcg cacagctccc tggggagaag aagaacggcc tgtttggtaa tcttatcgcc 1621 ctgtcactcg ggctgacccc caactttaaa tctaacttcg acctggccga agatgccaag 1681 cttcaactga gcaaagacac ctacgatgat gatctcgaca atctgctggc ccagatcggc 1741 gaccagtacg cagacctttt tttggcggca aagaacctgt cagacgccat tctgctgagt 1801 gatattctgc gagtgaacac ggagatcacc aaagctccgc tgagcgctag tatgatcaag 1861 cgctatgatg agcaccacca agacttgact ttgctgaagg cccttgtcag acagcaactg 1921 cctgagaagt acaaggaaat tttcttcgat cagtctaaaa atggctacgc cggatacatt 1981 gacggcggag caagccagga ggaattttac aaatttatta agcccatctt ggaaaaaatg 2041 gacggcaccg aggagctgct ggtaaagctt aacagagaag atctgttgcg caaacagcgc 2101 actttcgaca atggaagcat cccccaccag attcacctgg gcgaactgca cgctatcctc 2161 aggcggcaag aggatttcta cccctttttg aaagataaca gggaaaagat tgagaaaatc 2221 ctcacatttc ggatacccta ctatgtaggc cccctcgccc ggggaaattc cagattcgcg 2281 tggatgactc gcaaatcaga agagaccatc actccctgga acttcgagga agtcgtggat 2341 aagggggcct ctgcccagtc cttcatcgaa aggatgacta actttgataa aaatctgcct 2401 aacgaaaagg tgcttcctaa acactctctg ctgtacgagt acttcacagt ttataacgag 2461 ctcaccaagg tcaaatacgt cacagaaggg atgagaaagc cagcattcct gtctggagag 2521 cagaagaaag ctatcgtgga cctcctcttc aagacgaacc ggaaagttac cgtgaaacag 2581 ctcaaagaag actatttcaa aaagattgaa tgtttcgact ctgttgaaat cagcggagtg 2641 gaggatcgct tcaacgcatc cctgggaacg tatcacgatc tcctgaaaat cattaaagac 2701 aaggacttcc tggacaatga ggagaacgag gacattcttg aggacattgt cctcaccctt 2761 acgttgtttg aagataggga gatgattgaa gaacgcttga aaacttacgc tcatctcttc 2821 gacgacaaag tcatgaaaca gctcaagagg cgccgatata caggatgggg gcggctgtca 2881 agaaaactga tcaatgggat ccgagacaag cagagtggaa agacaatcct ggattttctt 2941 aagtccgatg gatttgccaa ccggaacttc atgcagttga tccatgatga ctctctcacc 3001 tttaaggagg acatccagaa agcacaagtt tgtctggctg gcgatactct cattaccctg 3061 gccgatggac gacgagtgcc tattagagaa ctggtgtcac agcagaattt ttccgtgtgg 3121 gctctgaatc ctcagactta ccgcctggag agggctagag tgagtagagc tttctgtacc 3181 ggcatcaaac ctgtgtaccg cctcaccact agactgggga gatccattag ggccactgcc 3241 aaccaccgat ttctcacacc tcagggctgg aaacgagtcg atgaactcca gcctggagat 3301 tacctggctc tgcctaggag aatccctact gcctcctgat ctcgagctcg atgagtttgg 3361 acaaaccaca actagaatgc agtgaaaaaa atgctttatt tgtgaaattt gtgatgctat 3421 tgctttattt gtaaccatta taagctgcaa taaacaagtt aacaacaaca attgcattca 3481 ttttatgttt caggttcagg gggaggtgtg ggaggttttt taaagcaagt aaaacctcta 3541 caaatgtggt acttaagagg gggagaccaa agggcacgcg tcgagtccaa cacccgtggg 3601 aatcccatgg gcaccatggc ccctcgctcc aaaaatgctt tcgcgtcgcg cagacactgc 3661 tcggtagttt cggggatcag cgtttgagta agagcccgcg tctgaaccct ccgcgccgcc 3721 ccggccccag tggaaagacg cgcaggcaaa acgcaccacg tgacggagcg tgaccgcgcg 3781 ccgagcgcgc gccaaggtcg ggcaggaaga gggcctattt cccatgattc cttcatattt 3841 gcatatacga tacaaggctg ttagagagat aattagaatt aatttgactg taaacacaaa 3901 gatattagta caaaatacgt gacgtagaaa gtaataattt cttgggtagt ttgcagtttt 3961 aaaattatgt tttaaaatgg actatcatat gcttaccgta acttgaaagt atttcgattt 4021 cttggcttta tatatcttgt ggaaaggacg aaacaccgag tcttcgagga agacaggttt 4081 aagagctatg ctggaaacag catagcaagt ttaaataagg ctagtccgtt atcaacttga 4141 aaaagtggca ccgagtcggt gctttttttc tagagggcgg ccgcaggtag ataagtagca 4201 tggcgggtta atcattaact acaaggaacc cctagtgatg gagttggcca ctccctctct 4261 gcgcgctcgc tcgctcactg aggccgggcg accaaaggtc gcccgacgcc cgggctttgc 4321 ccgggcggcc tcagtgagcg agcgagcgcg cagcctaatt aaggccttaa ttaaatctgg 4381 cgtaatcatg gtcatagctg tttcctgtgt gaaattgtta tccgctcaca attccacaca 4441 acatacgagc cggaagcata aagtgtaaag cctggggtgc ctaatgagtg agctaactca 4501 cattaattgc gttgcgctca ctgcccgctt tccagtcggg aaacctgtcg tgccagctgc 4561 attaatgaat cggccaacgc gcggggagag gcggtttgcg tattgggcgc tcttccgctt 4621 cctcgctcac tgactcgctg cgctcggtcg ttcggctgcg gcgagcggta tcagctcact 4681 caaaggcggt aatacggtta tccacagaat caggggataa cgcaggaaag aacatgtgag 4741 caaaaggcca gcaaaaggcc aggaaccgta aaaaggccgc gttgctggcg tttttccata 4801 ggctccgccc ccctgacgag catcacaaaa atcgacgctc aagtcagagg tggcgaaacc 4861 cgacaggact ataaagatac caggcgtttc cccctggaag ctccctcgtg cgctctcctg 4921 ttccgaccct gccgcttacc ggatacctgt ccgcctttct cccttcggga agcgtggcgc 4981 tttctcatag ctcacgctgt aggtatctca gttcggtgta ggtcgttcgc tccaagctgg 5041 gctgtgtgca cgaacccccc gttcagcccg accgctgcgc cttatccggt aactatcgtc 5101 ttgagtccaa cccggtaaga cacgacttat cgccactggc agcagccact ggtaacagga 5161 ttagcagagc gaggtatgta ggcggtgcta cagagttctt gaagtggtgg cctaactacg 5221 gctacactag aagaacagta tttggtatct gcgctctgct gaagccagtt accttcggaa 5281 aaagagttgg tagctcttga tccggcaaac aaaccaccgc tggtagcggt ggtttttttg 5341 tttgcaagca gcagattacg cgcagaaaaa aaggatctca agaagatcct ttgatctttt 5401 ctacggggtc tgacgctcag tggaacgaaa actcacgtta agggattttg gtcatgagat 5461 tatcaaaaag gatcttcacc tagatccttt taaattaaaa atgaagtttt aaatcaatct 5521 aaagtatata tgagtaaact tggtctgaca gttaccaatg cttaatcagt gaggcaccta 5581 tctcagcgat ctgtctattt cgttcatcca tagttgcctg actccccgtc gtgtagataa 5641 ctacgatacg ggagggctta ccatctggcc ccagtgctgc aatgataccg cgagacccac 5701 gctcaccggc tccagattta tcagcaataa accagccagc cggaagggcc gagcgcagaa 5761 gtggtcctgc aactttatcc gcctccatcc agtctattaa ttgttgccgg gaagctagag 5821 taagtagttc gccagttaat agtttgcgca acgttgttgc cattgctaca ggcatcgtgg 5881 tgtcacgctc gtcgtttggt atggcttcat tcagctccgg ttcccaacga tcaaggcgag 5941 ttacatgatc ccccatgttg tgcaaaaaag cggttagctc cttcggtcct ccgatcgttg 6001 tcagaagtaa gttggccgca gtgttatcac tcatggttat ggcagcactg cataattctc 6061 ttactgtcat gccatccgta agatgctttt ctgtgactgg tgagtactca accaagtcat 6121 tctgagaata gtgtatgcgg cgaccgagtt gctcttgccc ggcgtcaata cgggataata 6181 ccgcgccaca tagcagaact ttaaaagtgc tcatcattgg aaaacgttct tcggggcgaa 6241 aactctcaag gatcttaccg ctgttgagat ccagttcgat gtaacccact cgtgcaccca 6301 actgatcttc agcatctttt actttcacca gcgtttctgg gtgagcaaaa acaggaaggc 6361 aaaatgccgc aaaaaaggga ataagggcga cacggaaatg ttgaatactc atactcttcc 6421 tttttcaata ttattgaagc atttatcagg gttattgtct catgagcgga tacatatttg 6481 aatgtattta gaaaaataaa caaatagggg ttccgcgcac atttccccga aaagtgccac 6541 ctaaattgta agcgttaata ttttgttaaa attcgcgtta aatttttgtt aaatcagctc 6601 attttttaac caataggccg aaatcggcaa aatcccttat aaatcaaaag aatagaccga 6661 gatagggttg agtgttgttc cagtttggaa caagagtcca ctattaaaga acgtggactc 6721 caacgtcaaa gggcgaaaaa ccgtctatca gggcgatggc ccactacgtg aaccatcacc 6781 ctaatcaagt tttttggggt cgaggtgccg taaagcacta aatcggaacc ctaaagggag 6841 cccccgattt agagcttgac ggggaaagcc ggcgaacgtg gcgagaaagg aagggaagaa 6901 agcgaaagga gcgggcgcta gggcgctggc aagtgtagcg gtcacgctgc gcgtaaccac 6961 cacacccgcc gcgcttaatg cgccgctaca gggcgcgtcc cattcgccat tcaggctgcg 7021 caactgttgg gaagggcgat cggtgcgggc ctcttcgcta ttacgccagc tggcgaaagg 7081 gggatgtgct gcaaggcgat taagttgggt aacgccaggg ttttcccagt cacgacgttg 7141 taaaacgacg gccagtgaat taggttaatt aaggctgcgc gctcgctcgc tcactgaggc 7201 cgcccgggca aagcccgggc gtcgggcgac ctttggtcgc ccggcctcag tgagcgagcg 7261 agcgcgcaga gagggagtgg ccaactccat cactaggggt tccttgtagt taatgattaa 7321 cccgccatgc tacttatcta ctgcggccgc ggaatgtgtg tcagttaggg tgtggaaagt 7381 ccccaggctc cccagcaggc agaagtatgc aaagcatgca tctcaattag tcagcaacca 7441 gtcccggtct cctcccatgc atgtcaatat tggccattag ccatattatt cattggttat 7501 atagcataaa tcaatattgg ctattggcca ttgcatacgt tgtatctata tcataatatg 7561 tacatttata ttggctcatg tccaatatga ccgccatgtt //