LOCUS Exported 5029 bp ds-DNA circular SYN 13-MAY-2021 DEFINITION Self-complementary AAV vector expressing Nme2Cas9 sgRNA targeting Rosa26 and AcrIIC3Nme. ACCESSION . VERSION . KEYWORDS p1192-scAAV-U6-sgRNA_Nme2Cas9_Rosa26-CB-PI-AcrIIC3Nme_FLAG_NLS SOURCE synthetic DNA construct ORGANISM synthetic DNA construct REFERENCE 1 (bases 1 to 5029) AUTHORS Lee J, Mou H, Ibraheim R, Liang SQ, Liu P, Xue W, Sontheimer EJ TITLE Tissue-restricted Genome Editing in vivo Specified by MicroRNA-repressible Anti-CRISPR Proteins. JOURNAL RNA. 2019 Aug 22. pii: rna.071704.119. doi: 10.1261/rna.071704.119. PUBMED 31439808 REFERENCE 2 (bases 1 to 5029) AUTHORS . TITLE Direct Submission JOURNAL Exported May 13, 2021 from SnapGene Server 1.1.58 http://www.snapgene.com FEATURES Location/Qualifiers source 1..5029 /organism="synthetic DNA construct" /mol_type="other DNA" misc_RNA complement(156..248) /label=Nm tracrRNA /note="trans-activating CRISPR RNA for the Neisseria meningitidis CRISPR/Cas9 system (Hou et al., 2013)" promoter complement(309..549) /label=U6 promoter /note="RNA polymerase III promoter for human U6 snRNA" primer_bind complement(359..378) /label=LKO.1 5' /note="Human U6 promoter, forward primer" primer_bind complement(529..549) /label=hU6-F /note="Human U6 promoter, forward primer" intron 1209..1305 /label=SV40 intron /note="modified SV40 intron with splice donor and acceptor sites" CDS 1733..1756 /codon_start=1 /product="FLAG(R) epitope tag, followed by an enterokinase cleavage site" /label=FLAG /translation="DYKDDDDK" CDS 1763..1783 /codon_start=1 /product="nuclear localization signal of SV40 (simian virus 40) large T antigen" /label=SV40 NLS /translation="PKKKRKV" primer_bind complement(1838..1855) /label=BGH-rev /note="Bovine growth hormone terminator, reverse primer. Also called BGH reverse" polyA_signal 1844..1955 /label=bGH poly(A) signal /note="bovine growth hormone polyadenylation signal" repeat_region 2079..2208 /label=AAV2 ITR primer_bind complement(2227..2244) /label=M13 Forward /note="In lacZ gene. Also called M13-F20 or M13 (-21) Forward" primer_bind complement(2227..2243) /label=M13 fwd /note="common sequencing primer, one of multiple similar variants" primer_bind complement(2236..2258) /label=M13/pUC Forward /note="In lacZ gene" rep_origin 2385..2840 /direction=RIGHT /label=f1 ori /note="f1 bacteriophage origin of replication; arrow indicates direction of (+) strand synthesis" primer_bind complement(2472..2491) /label=F1ori-R /note="F1 origin, reverse primer" primer_bind 2682..2703 /label=F1ori-F /note="F1 origin, forward primer" promoter 2866..2970 /gene="bla" /label=AmpR promoter CDS 2971..3831 /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(3189..3208) /label=Amp-R /note="Ampicillin resistance gene, reverse primer" rep_origin 4002..4590 /direction=RIGHT /label=ori /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of replication" primer_bind 4491..4510 /label=pBR322ori-F /note="pBR322 origin, forward primer" primer_bind 4744..4761 /label=L4440 /note="L4440 vector, forward primer" protein_bind 4878..4899 /label=CAP binding site /bound_moiety="E. coli catabolite activator protein" /note="CAP binding activates transcription in the presence of cAMP." promoter 4914..4944 /label=lac promoter /note="promoter for the E. coli lac operon" protein_bind 4952..4968 /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)." primer_bind 4957..4979 /label=M13/pUC Reverse /note="In lacZ gene" primer_bind 4976..4992 /label=M13 rev /note="common sequencing primer, one of multiple similar variants" primer_bind 4976..4992 /label=M13 Reverse /note="In lacZ gene. Also called M13-rev" ORIGIN 1 ctgcgcgctc gctcgctcac tgaggccgcc cgggcaaagc ccgggcgtcg ggcgaccttt 61 ggtcgcccgg cctcagtgag cgagcgagcg cgcagagagg gagtgtagcc atgctctagg 121 aagatcaatt cggtacaatt cacgcgtcaa aaaaataaac gatgcccctt aaagcagaag 181 ctttaagggg cagagcgttg cggcacatct tttcagacgg ccttattgta gcaacggttc 241 tcatttcgtt tccgaaatga gaaagggagc tacaacctcc caggcccagg gcggtcctcc 301 ggtgtttcgt cctttccaca agatatataa agccaagaaa tcgaaatact ttcaagttac 361 ggtaagcata tgatagtcca ttttaaaaca taattttaaa actgcaaact acccaagaaa 421 ttattacttt ctacgtcacg tattttgtac taatatcttt gtgtttacag tcaaattaat 481 tctaattatc tctctaacag ccttgtatcg tatatgcaaa tatgaaggaa tcatgggaaa 541 taggccctcc acgcgtcgac attgattatt gactagctct ggtcgttaca taacttacgg 601 taaatggccc gcctggctga ccgcccaacg acccccgccc attgacgtca ataatgacgt 661 atgttcccat agtaacgcca atagggactt tccattgacg tcaatgggtg gagtatttac 721 ggtaaactgc ccacttggca gtacatcaag tgtatcatat gccaagtacg ccccctattg 781 acgtcaatga cggtaaatgg cccgcctggc attatgccca gtacatgacc ttatgggact 841 ttcctacttg gcagtacatc tactcgaggc cacgttctgc ttcactctcc ccatctcccc 901 cccctcccca cccccaattt tgtatttatt tattttttaa ttattttgtg cagcgatggg 961 ggcggggggg gggggggggg gggcgcgcgc crggsggggc ggggcggggc gaggggcggg 1021 gcggggcgag gcggagaggt gcggcggcag ccaatcagag cggcgcgctc cgaaagtttc 1081 cttttatggc gaggcggcgg cggcggcggc cctataaaaa gcgaagcgcg cggcgggcgg 1141 gagcgggatc agccaccgcg gtggcggcct agagtcgacg aggaactgaa aaaccagaaa 1201 gttaactggt aagtttagtc tttttgtctt ttatttcagg tcccggatcc ggtggtggtg 1261 caaatcaaag aactgctcct cagtggatgt tgcctttact tctaggcctg tacggaagtg 1321 ttacttctgc tctaaaagct gcggaattgt acccgcggcc gatccaccgg tcgccaccat 1381 gttcaaacgg gccattattt tcaccagctt caacggcttc gagaaggtca gtcgcacgga 1441 aaaacggcgc cttgccaaga taattaacgc cagagttagt ataatcgacg agtatcttcg 1501 cgccaaagac accaacgcct ccttggacgg acagtataga gcattccttt tcaatgatga 1561 gtcaccagcg atgaccgaat tcctcgctaa gctcaaggcg ttcgcagaaa gctgtactgg 1621 tataagcatt gatgcgtggg agattgaaga gagcgagtat gttcgattgc ccgtcgaacg 1681 gagagatttc cttgcggcgg ccaatggcaa agaaattttc aaaattggcg gggattataa 1741 ggatgatgat gataagggct cccctaagaa gaagagaaag gtgtgataaa gcggccatca 1801 agcttatcga taccgtcgac tagagctcgc tgatcagcct cgactgtgcc ttctagttgc 1861 cagccatctg ttgtttgccc ctcccccgtg ccttccttga ccctggaagg tgccactccc 1921 actgtccttt cctaataaaa tgaggaaatt gcatcgcatt gtctgagtag gtgtcattct 1981 attctggggg gtggggtggg gcaggacagc aagggggagg attgggaaga caatagccta 2041 ggtagataag tagcatggcg ggttaatcat taactacaag gaacccctag tgatggagtt 2101 ggccactccc tctctgcgcg ctcgctcgct cactgaggcc gggcgaccaa aggtcgcccg 2161 acgcccgggc tttgcccggg cggcctcagt gagcgagcga gcgcgcagcc ttaattaacc 2221 taattcactg gccgtcgttt tacaacgtcg tgactgggaa aaccctggcg ttacccaact 2281 taatcgcctt gcagcacatc cccctttcgc cagctggcgt aatagcgaag aggcccgcac 2341 cgatcgccct tcccaacagt tgcgcagcct gaatggcgaa tgggacgcgc cctgtagcgg 2401 cgcattaagc gcggcgggtg tggtggttac gcgcagcgtg accgctacac ttgccagcgc 2461 cctagcgccc gctcctttcg ctttcttccc ttcctttctc gccacgttcg ccggctttcc 2521 ccgtcaagct ctaaatcggg ggctcccttt agggttccga tttagtgctt tacggcacct 2581 cgaccccaaa aaacttgatt agggtgatgg ttcacgtagt gggccatcgc cctgatagac 2641 ggtttttcgc cctttgacgt tggagtccac gttctttaat agtggactct tgttccaaac 2701 tggaacaaca ctcaacccta tctcggtcta ttcttttgat ttataaggga ttttgccgat 2761 ttcggcctat tggttaaaaa atgagctgat ttaacaaaaa tttaacgcga attttaacaa 2821 aatattaacg cttacaattt aggtggcact tttcggggaa atgtgcgcgg aacccctatt 2881 tgtttatttt tctaaataca ttcaaatatg tatccgctca tgagacaata accctgataa 2941 atgcttcaat aatattgaaa aaggaagagt atgagtattc aacatttccg tgtcgccctt 3001 attccctttt ttgcggcatt ttgccttcct gtttttgctc acccagaaac gctggtgaaa 3061 gtaaaagatg ctgaagatca gttgggtgca cgagtgggtt acatcgaact ggatctcaac 3121 agcggtaaga tccttgagag ttttcgcccc gaagaacgtt ttccaatgat gagcactttt 3181 aaagttctgc tatgtggcgc ggtattatcc cgtattgacg ccgggcaaga gcaactcggt 3241 cgccgcatac actattctca gaatgacttg gttgagtact caccagtcac agaaaagcat 3301 cttacggatg gcatgacagt aagagaatta tgcagtgctg ccataaccat gagtgataac 3361 actgcggcca acttacttct gacaacgatc ggaggaccga aggagctaac cgcttttttg 3421 cacaacatgg gggatcatgt aactcgcctt gatcgttggg aaccggagct gaatgaagcc 3481 ataccaaacg acgagcgtga caccacgatg cctgtagcaa tggcaacaac gttgcgcaaa 3541 ctattaactg gcgaactact tactctagct tcccggcaac aattaataga ctggatggag 3601 gcggataaag ttgcaggacc acttctgcgc tcggcccttc cggctggctg gtttattgct 3661 gataaatctg gagccggtga gcgtgggtct cgcggtatca ttgcagcact ggggccagat 3721 ggtaagccct cccgtatcgt agttatctac acgacgggga gtcaggcaac tatggatgaa 3781 cgaaatagac agatcgctga gataggtgcc tcactgatta agcattggta actgtcagac 3841 caagtttact catatatact ttagattgat ttaaaacttc atttttaatt taaaaggatc 3901 taggtgaaga tcctttttga taatctcatg accaaaatcc cttaacgtga gttttcgttc 3961 cactgagcgt cagaccccgt agaaaagatc aaaggatctt cttgagatcc tttttttctg 4021 cgcgtaatct gctgcttgca aacaaaaaaa ccaccgctac cagcggtggt ttgtttgccg 4081 gatcaagagc taccaactct ttttccgaag gtaactggct tcagcagagc gcagatacca 4141 aatactgttc ttctagtgta gccgtagtta ggccaccact tcaagaactc tgtagcaccg 4201 cctacatacc tcgctctgct aatcctgtta ccagtggctg ctgccagtgg cgataagtcg 4261 tgtcttaccg ggttggactc aagacgatag ttaccggata aggcgcagcg gtcgggctga 4321 acggggggtt cgtgcacaca gcccagcttg gagcgaacga cctacaccga actgagatac 4381 ctacagcgtg agctatgaga aagcgccacg cttcccgaag ggagaaaggc ggacaggtat 4441 ccggtaagcg gcagggtcgg aacaggagag cgcacgaggg agcttccagg gggaaacgcc 4501 tggtatcttt atagtcctgt cgggtttcgc cacctctgac ttgagcgtcg atttttgtga 4561 tgctcgtcag gggggcggag cctatggaaa aacgccagca acgcggcctt tttacggttc 4621 ctggcctttt gctggccttt tgctcacatg ttctttcctg cgttatcccc tgattctgtg 4681 gataaccgta ttaccgcctt tgagtgagct gataccgctc gccgcagccg aacgaccgag 4741 cgcagcgagt cagtgagcga ggaagcggaa gagcgcccaa tacgcaaacc gcctctcccc 4801 gcgcgttggc cgattcatta atgcagctgg cacgacaggt ttcccgactg gaaagcgggc 4861 agtgagcgca acgcaattaa tgtgagttag ctcactcatt aggcacccca ggctttacac 4921 tttatgcttc cggctcgtat gttgtgtgga attgtgagcg gataacaatt tcacacagga 4981 aacagctatg accatgatta cgccagattt aattaaggcc ttaattagg //