LOCUS Exported 3223 bp ds-DNA circular SYN 12-MAY-2021 DEFINITION Expresses a S. pyogenes Cas9/dCas9 guide RNA in mammalian cells. ACCESSION . VERSION . KEYWORDS pSPgRNA SOURCE synthetic DNA construct ORGANISM synthetic DNA construct REFERENCE 1 (bases 1 to 3223) AUTHORS Perez-Pinera P, Kocak DD, Vockley CM, Adler AF, Kabadi AM, Polstein LR, Thakore PI, Glass KA, Ousterout DG, Leong KW, Guilak F, Crawford GE, Reddy TE, Gersbach CA TITLE RNA-guided gene activation by CRISPR-Cas9-based transcription factors. JOURNAL Nat Methods. 2013 Jul 25. doi: 10.1038/nmeth.2600. PUBMED 23892895 REFERENCE 2 (bases 1 to 3223) AUTHORS . TITLE Direct Submission JOURNAL Exported May 12, 2021 from SnapGene Server 1.1.58 http://www.snapgene.com FEATURES Location/Qualifiers source 1..3223 /organism="synthetic DNA construct" /mol_type="other DNA" promoter 1..241 /label=U6 promoter /note="RNA polymerase III promoter for human U6 snRNA" primer_bind 1..21 /label=hU6-F /note="Human U6 promoter, forward primer" misc_RNA 268..343 /label=gRNA scaffold /note="guide RNA scaffold for the Streptococcus pyogenes CRISPR/Cas9 system" primer_bind complement(377..397) /label=T3 /note="T3 promoter, forward primer" promoter complement(377..395) /label=T3 promoter /note="promoter for bacteriophage T3 RNA polymerase" primer_bind complement(416..432) /label=M13 rev /note="common sequencing primer, one of multiple similar variants" primer_bind complement(416..432) /label=M13 Reverse /note="In lacZ gene. Also called M13-rev" primer_bind complement(429..451) /label=M13/pUC Reverse /note="In lacZ gene" protein_bind 440..456 /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(464..494) /label=lac promoter /note="promoter for the E. coli lac operon" protein_bind 509..530 /label=CAP binding site /bound_moiety="E. coli catabolite activator protein" /note="CAP binding activates transcription in the presence of cAMP." primer_bind complement(647..664) /label=L4440 /note="L4440 vector, forward primer" rep_origin complement(818..1406) /direction=LEFT /label=ori /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of replication" primer_bind complement(898..917) /label=pBR322ori-F /note="pBR322 origin, forward primer" CDS complement(1577..2437) /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 2200..2219 /label=Amp-R /note="Ampicillin resistance gene, reverse primer" promoter complement(2438..2542) /gene="bla" /label=AmpR promoter rep_origin complement(2568..3023) /direction=LEFT /label=f1 ori /note="f1 bacteriophage origin of replication; arrow indicates direction of (+) strand synthesis" primer_bind complement(2705..2726) /label=F1ori-F /note="F1 origin, forward primer" primer_bind 2917..2936 /label=F1ori-R /note="F1 origin, reverse primer" primer_bind 3150..3172 /label=M13/pUC Forward /note="In lacZ gene" primer_bind 3164..3181 /label=M13 Forward /note="In lacZ gene. Also called M13-F20 or M13 (-21) Forward" primer_bind 3165..3181 /label=M13 fwd /note="common sequencing primer, one of multiple similar variants" primer_bind 3191..3210 /label=T7 /note="T7 promoter, forward primer" promoter 3191..3209 /label=T7 promoter /note="promoter for bacteriophage T7 RNA polymerase" ORIGIN 1 gagggcctat ttcccatgat tccttcatat ttgcatatac gatacaaggc tgttagagag 61 ataattggaa ttaatttgac tgtaaacaca aagatattag tacaaaatac gtgacgtaga 121 aagtaataat ttcttgggta gtttgcagtt ttaaaattat gttttaaaat ggactatcat 181 atgcttatcg taacttgaaa gtatttcgat ttcttggctt tatatatctt gtggaaagga 241 cgaaacaccg ggtcttcgag aagacctgtt ttagagctag aaatagcaag ttaaaataag 301 gctagtccgt tatcaacttg aaaaagtggc accgagtcgg tgcttttttt ccgcggtgga 361 gctccagctt ttgttccctt tagtgagggt taattgcgcg cttggcgtaa tcatggtcat 421 agctgtttcc tgtgtgaaat tgttatccgc tcacaattcc acacaacata cgagccggaa 481 gcataaagtg taaagcctgg ggtgcctaat gagtgagcta actcacatta attgcgttgc 541 gctcactgcc cgctttccag tcgggaaacc tgtcgtgcca gctgcattaa tgaatcggcc 601 aacgcgcggg gagaggcggt ttgcgtattg ggcgctcttc cgcttcctcg ctcactgact 661 cgctgcgctc ggtcgttcgg ctgcggcgag cggtatcagc tcactcaaag gcggtaatac 721 ggttatccac agaatcaggg gataacgcag gaaagaacat gtgagcaaaa ggccagcaaa 781 aggccaggaa ccgtaaaaag gccgcgttgc tggcgttttt ccataggctc cgcccccctg 841 acgagcatca caaaaatcga cgctcaagtc agaggtggcg aaacccgaca ggactataaa 901 gataccaggc gtttccccct ggaagctccc tcgtgcgctc tcctgttccg accctgccgc 961 ttaccggata cctgtccgcc tttctccctt cgggaagcgt ggcgctttct catagctcac 1021 gctgtaggta tctcagttcg gtgtaggtcg ttcgctccaa gctgggctgt gtgcacgaac 1081 cccccgttca gcccgaccgc tgcgccttat ccggtaacta tcgtcttgag tccaacccgg 1141 taagacacga cttatcgcca ctggcagcag ccactggtaa caggattagc agagcgaggt 1201 atgtaggcgg tgctacagag ttcttgaagt ggtggcctaa ctacggctac actagaagaa 1261 cagtatttgg tatctgcgct ctgctgaagc cagttacctt cggaaaaaga gttggtagct 1321 cttgatccgg caaacaaacc accgctggta gcggtggttt ttttgtttgc aagcagcaga 1381 ttacgcgcag aaaaaaagga tctcaagaag atcctttgat cttttctacg gggtctgacg 1441 ctcagtggaa cgaaaactca cgttaaggga ttttggtcat gagattatca aaaaggatct 1501 tcacctagat ccttttaaat taaaaatgaa gttttaaatc aatctaaagt atatatgagt 1561 aaacttggtc tgacagttac caatgcttaa tcagtgaggc acctatctca gcgatctgtc 1621 tatttcgttc atccatagtt gcctgactcc ccgtcgtgta gataactacg atacgggagg 1681 gcttaccatc tggccccagt gctgcaatga taccgcgaga cccacgctca ccggctccag 1741 atttatcagc aataaaccag ccagccggaa gggccgagcg cagaagtggt cctgcaactt 1801 tatccgcctc catccagtct attaattgtt gccgggaagc tagagtaagt agttcgccag 1861 ttaatagttt gcgcaacgtt gttgccattg ctacaggcat cgtggtgtca cgctcgtcgt 1921 ttggtatggc ttcattcagc tccggttccc aacgatcaag gcgagttaca tgatccccca 1981 tgttgtgcaa aaaagcggtt agctccttcg gtcctccgat cgttgtcaga agtaagttgg 2041 ccgcagtgtt atcactcatg gttatggcag cactgcataa ttctcttact gtcatgccat 2101 ccgtaagatg cttttctgtg actggtgagt actcaaccaa gtcattctga gaatagtgta 2161 tgcggcgacc gagttgctct tgcccggcgt caatacggga taataccgcg ccacatagca 2221 gaactttaaa agtgctcatc attggaaaac gttcttcggg gcgaaaactc tcaaggatct 2281 taccgctgtt gagatccagt tcgatgtaac ccactcgtgc acccaactga tcttcagcat 2341 cttttacttt caccagcgtt tctgggtgag caaaaacagg aaggcaaaat gccgcaaaaa 2401 agggaataag ggcgacacgg aaatgttgaa tactcatact cttccttttt caatattatt 2461 gaagcattta tcagggttat tgtctcatga gcggatacat atttgaatgt atttagaaaa 2521 ataaacaaat aggggttccg cgcacatttc cccgaaaagt gccacctaaa ttgtaagcgt 2581 taatattttg ttaaaattcg cgttaaattt ttgttaaatc agctcatttt ttaaccaata 2641 ggccgaaatc ggcaaaatcc cttataaatc aaaagaatag accgagatag ggttgagtgt 2701 tgttccagtt tggaacaaga gtccactatt aaagaacgtg gactccaacg tcaaagggcg 2761 aaaaaccgtc tatcagggcg atggcccact acgtgaacca tcaccctaat caagtttttt 2821 ggggtcgagg tgccgtaaag cactaaatcg gaaccctaaa gggagccccc gatttagagc 2881 ttgacgggga aagccggcga acgtggcgag aaaggaaggg aagaaagcga aaggagcggg 2941 cgctagggcg ctggcaagtg tagcggtcac gctgcgcgta accaccacac ccgccgcgct 3001 taatgcgccg ctacagggcg cgtcccattc gccattcagg ctgcgcaact gttgggaagg 3061 gcgatcggtg cgggcctctt cgctattacg ccagctggcg aaagggggat gtgctgcaag 3121 gcgattaagt tgggtaacgc cagggttttc ccagtcacga cgttgtaaaa cgacggccag 3181 tgagcgcgcg taatacgact cactataggg cgaattgggt acc //