LOCUS Exported 3455 bp ds-DNA circular SYN 12-MAY-2021 DEFINITION gRNA targeting human HPRT gene. ACCESSION . VERSION . KEYWORDS HPRT2b-pGEM SOURCE synthetic DNA construct ORGANISM synthetic DNA construct REFERENCE 1 (bases 1 to 3455) AUTHORS Liao S, Tammaro M, Yan H TITLE Enriching CRISPR-Cas9 targeted cells by co-targeting the HPRT gene. JOURNAL Nucleic Acids Res. 2015 Jun 29. pii: gkv675. PUBMED 26130722 REFERENCE 2 (bases 1 to 3455) AUTHORS . TITLE Direct Submission JOURNAL Exported May 12, 2021 from SnapGene Server 1.1.58 http://www.snapgene.com FEATURES Location/Qualifiers source 1..3455 /organism="synthetic DNA construct" /mol_type="other DNA" regulatory 33..42 /regulatory_class="other" /note="vertebrate consensus sequence for strong initiation of translation (Kozak, 1987)" promoter 121..361 /label=U6 promoter /note="RNA polymerase III promoter for human U6 snRNA" primer_bind 121..141 /label=hU6-F /note="Human U6 promoter, forward primer" primer_bind 292..311 /label=LKO.1 5' /note="Human U6 promoter, forward primer" misc_RNA 390..465 /label=gRNA scaffold /note="guide RNA scaffold for the Streptococcus pyogenes CRISPR/Cas9 system" protein_bind complement(477..501) /gene="mutant version of attB" /label=attB2 /bound_moiety="BP Clonase(TM)" /note="recombination site for the Gateway(R) BP reaction" promoter complement(580..598) /label=SP6 promoter /note="promoter for bacteriophage SP6 RNA polymerase" primer_bind complement(581..598) /label=SP6 /note="SP6 promoter, forward primer" primer_bind complement(616..632) /label=M13 rev /note="common sequencing primer, one of multiple similar variants" primer_bind complement(616..632) /label=M13 Reverse /note="In lacZ gene. Also called M13-rev" primer_bind complement(629..651) /label=M13/pUC Reverse /note="In lacZ gene" protein_bind 640..656 /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(664..694) /label=lac promoter /note="promoter for the E. coli lac operon" protein_bind 709..730 /label=CAP binding site /bound_moiety="E. coli catabolite activator protein" /note="CAP binding activates transcription in the presence of cAMP." primer_bind complement(847..864) /label=L4440 /note="L4440 vector, forward primer" rep_origin complement(1018..1606) /direction=LEFT /label=ori /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of replication" primer_bind complement(1098..1117) /label=pBR322ori-F /note="pBR322 origin, forward primer" CDS complement(1777..2637) /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 2400..2419 /label=Amp-R /note="Ampicillin resistance gene, reverse primer" promoter complement(2638..2742) /gene="bla" /label=AmpR promoter rep_origin complement(2820..3275) /direction=LEFT /label=f1 ori /note="f1 bacteriophage origin of replication; arrow indicates direction of (+) strand synthesis" primer_bind complement(2957..2978) /label=F1ori-F /note="F1 origin, forward primer" primer_bind 3169..3188 /label=F1ori-R /note="F1 origin, reverse primer" primer_bind 3401..3423 /label=M13/pUC Forward /note="In lacZ gene" primer_bind 3415..3432 /label=M13 Forward /note="In lacZ gene. Also called M13-F20 or M13 (-21) Forward" primer_bind 3416..3432 /label=M13 fwd /note="common sequencing primer, one of multiple similar variants" primer_bind 3439..3 /label=T7 /note="T7 promoter, forward primer" promoter 3439..2 /label=T7 promoter /note="promoter for bacteriophage T7 RNA polymerase" ORIGIN 1 gggcgaattg ggcccgacgt cgcatgctcc cggccgccat ggccgcggga ttgtacaaaa 61 aagcaggctt taaaggaacc aattcagtcg actggatccg gtaccaaggt cgggcaggaa 121 gagggcctat ttcccatgat tccttcatat ttgcatatac gatacaaggc tgttagagag 181 ataattagaa ttaatttgac tgtaaacaca aagatattag tacaaaatac gtgacgtaga 241 aagtaataat ttcttgggta gtttgcagtt ttaaaattat gttttaaaat ggactatcat 301 atgcttaccg taacttgaaa gtatttcgat ttcttggctt tatatatctt gtggaaagga 361 cgaaacaccg aagtaattca cttacagtcg ttttagagct agaaatagca agttaaaata 421 aggctagtcc gttatcaact tgaaaaagtg gcaccgagtc ggtgcttttt ttctagaccc 481 agctttcttg tacaaagttg gcattaatca ctagtgcggc cgcctgcagg tcgaccatat 541 gggagagctc ccaacgcgtt ggatgcatag cttgagtatt ctatagtgtc acctaaatag 601 cttggcgtaa tcatggtcat agctgtttcc tgtgtgaaat tgttatccgc tcacaattcc 661 acacaacata cgagccggaa gcataaagtg taaagcctgg ggtgcctaat gagtgagcta 721 actcacatta attgcgttgc gctcactgcc cgctttccag tcgggaaacc tgtcgtgcca 781 gctgcattaa tgaatcggcc aacgcgcggg gagaggcggt ttgcgtattg ggcgctcttc 841 cgcttcctcg ctcactgact cgctgcgctc ggtcgttcgg ctgcggcgag cggtatcagc 901 tcactcaaag gcggtaatac ggttatccac agaatcaggg gataacgcag gaaagaacat 961 gtgagcaaaa ggccagcaaa aggccaggaa ccgtaaaaag gccgcgttgc tggcgttttt 1021 ccataggctc cgcccccctg acgagcatca caaaaatcga cgctcaagtc agaggtggcg 1081 aaacccgaca ggactataaa gataccaggc gtttccccct ggaagctccc tcgtgcgctc 1141 tcctgttccg accctgccgc ttaccggata cctgtccgcc tttctccctt cgggaagcgt 1201 ggcgctttct catagctcac gctgtaggta tctcagttcg gtgtaggtcg ttcgctccaa 1261 gctgggctgt gtgcacgaac cccccgttca gcccgaccgc tgcgccttat ccggtaacta 1321 tcgtcttgag tccaacccgg taagacacga cttatcgcca ctggcagcag ccactggtaa 1381 caggattagc agagcgaggt atgtaggcgg tgctacagag ttcttgaagt ggtggcctaa 1441 ctacggctac actagaagaa cagtatttgg tatctgcgct ctgctgaagc cagttacctt 1501 cggaaaaaga gttggtagct cttgatccgg caaacaaacc accgctggta gcggtggttt 1561 ttttgtttgc aagcagcaga ttacgcgcag aaaaaaagga tctcaagaag atcctttgat 1621 cttttctacg gggtctgacg ctcagtggaa cgaaaactca cgttaaggga ttttggtcat 1681 gagattatca aaaaggatct tcacctagat ccttttaaat taaaaatgaa gttttaaatc 1741 aatctaaagt atatatgagt aaacttggtc tgacagttac caatgcttaa tcagtgaggc 1801 acctatctca gcgatctgtc tatttcgttc atccatagtt gcctgactcc ccgtcgtgta 1861 gataactacg atacgggagg gcttaccatc tggccccagt gctgcaatga taccgcgaga 1921 cccacgctca ccggctccag atttatcagc aataaaccag ccagccggaa gggccgagcg 1981 cagaagtggt cctgcaactt tatccgcctc catccagtct attaattgtt gccgggaagc 2041 tagagtaagt agttcgccag ttaatagttt gcgcaacgtt gttgccattg ctacaggcat 2101 cgtggtgtca cgctcgtcgt ttggtatggc ttcattcagc tccggttccc aacgatcaag 2161 gcgagttaca tgatccccca tgttgtgcaa aaaagcggtt agctccttcg gtcctccgat 2221 cgttgtcaga agtaagttgg ccgcagtgtt atcactcatg gttatggcag cactgcataa 2281 ttctcttact gtcatgccat ccgtaagatg cttttctgtg actggtgagt actcaaccaa 2341 gtcattctga gaatagtgta tgcggcgacc gagttgctct tgcccggcgt caatacggga 2401 taataccgcg ccacatagca gaactttaaa agtgctcatc attggaaaac gttcttcggg 2461 gcgaaaactc tcaaggatct taccgctgtt gagatccagt tcgatgtaac ccactcgtgc 2521 acccaactga tcttcagcat cttttacttt caccagcgtt tctgggtgag caaaaacagg 2581 aaggcaaaat gccgcaaaaa agggaataag ggcgacacgg aaatgttgaa tactcatact 2641 cttccttttt caatattatt gaagcattta tcagggttat tgtctcatga gcggatacat 2701 atttgaatgt atttagaaaa ataaacaaat aggggttccg cgcacatttc cccgaaaagt 2761 gccacctgat gcggtgtgaa ataccgcaca gatgcgtaag gagaaaatac cgcatcagga 2821 aattgtaagc gttaatattt tgttaaaatt cgcgttaaat ttttgttaaa tcagctcatt 2881 ttttaaccaa taggccgaaa tcggcaaaat cccttataaa tcaaaagaat agaccgagat 2941 agggttgagt gttgttccag tttggaacaa gagtccacta ttaaagaacg tggactccaa 3001 cgtcaaaggg cgaaaaaccg tctatcaggg cgatggccca ctacgtgaac catcacccta 3061 atcaagtttt ttggggtcga ggtgccgtaa agcactaaat cggaacccta aagggagccc 3121 ccgatttaga gcttgacggg gaaagccggc gaacgtggcg agaaaggaag ggaagaaagc 3181 gaaaggagcg ggcgctaggg cgctggcaag tgtagcggtc acgctgcgcg taaccaccac 3241 acccgccgcg cttaatgcgc cgctacaggg cgcgtccatt cgccattcag gctgcgcaac 3301 tgttgggaag ggcgatcggt gcgggcctct tcgctattac gccagctggc gaaaggggga 3361 tgtgctgcaa ggcgattaag ttgggtaacg ccagggtttt cccagtcacg acgttgtaaa 3421 acgacggcca gtgaattgta atacgactca ctata //