LOCUS Exported 2866 bp ds-DNA circular SYN 12-MAY-2021 DEFINITION An empty sgRNA expression vector. sgRNA sequence can be cloned into its Bbs I site. Its gRNA scaffold has efficient ver. 2 structure.. ACCESSION . VERSION . KEYWORDS gRNA Cloning Vector Bbs I ver. 2 SOURCE synthetic DNA construct ORGANISM synthetic DNA construct REFERENCE 1 (bases 1 to 2866) AUTHORS Fujita T, Yuno M, Fujii H TITLE Allele-specific locus binding and genome editing by CRISPR at the p16INK4a locus. JOURNAL Sci Rep. 2016 Jul 28;6:30485. doi: 10.1038/srep30485. PUBMED 27465215 REFERENCE 2 (bases 1 to 2866) AUTHORS . TITLE Direct Submission JOURNAL Exported May 12, 2021 from SnapGene Server 1.1.58 http://www.snapgene.com FEATURES Location/Qualifiers source 1..2866 /organism="synthetic DNA construct" /mol_type="other DNA" primer_bind 278..300 /label=M13/pUC Forward /note="In lacZ gene" primer_bind 292..309 /label=M13 Forward /note="In lacZ gene. Also called M13-F20 or M13 (-21) Forward" primer_bind 293..309 /label=M13 fwd /note="common sequencing primer, one of multiple similar variants" primer_bind 320..339 /label=T7 /note="T7 promoter, forward primer" promoter 320..338 /label=T7 promoter /note="promoter for bacteriophage T7 RNA polymerase" promoter 469..709 /label=U6 promoter /note="RNA polymerase III promoter for human U6 snRNA" primer_bind 469..489 /label=hU6-F /note="Human U6 promoter, forward primer" primer_bind 640..659 /label=LKO.1 5' /note="Human U6 promoter, forward primer" protein_bind complement(833..857) /gene="mutant version of attB" /label=attB2 /bound_moiety="BP Clonase(TM)" /note="recombination site for the Gateway(R) BP reaction" primer_bind complement(1009..1026) /label=L4440 /note="L4440 vector, forward primer" rep_origin complement(1119..1707) /direction=LEFT /label=ori /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of replication" primer_bind complement(1199..1218) /label=pBR322ori-F /note="pBR322 origin, forward primer" CDS complement(1878..2738) /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 2501..2520 /label=Amp-R /note="Ampicillin resistance gene, reverse primer" promoter complement(2739..2843) /gene="bla" /label=AmpR promoter ORIGIN 1 ctaaattgta agcgttaata ttttgttaaa attcgcgtta aatttttgtt aaatcagctc 61 attttttaac caataggccg aaatcggcaa aatcccttat aaatcaaaag aatagaccga 121 gatagggttg agtggccgct acagggcgct cccattcgcc attcaggctg cgcaactgtt 181 gggaagggcg tttcggtgcg ggcctcttcg ctattacgcc agctggcgaa agggggatgt 241 gctgcaaggc gattaagttg ggtaacgcca gggttttccc agtcacgacg ttgtaaaacg 301 acggccagtg agcgcgacgt aatacgactc actatagggc gaattggcgg aaggccgtca 361 aggccacgtg tcttgtccag gcgcgccact cgagaattct gtacaaaaaa gcaggcttta 421 aaggaaccaa ttcagtcgac tggatccggt accaaggtcg ggcaggaaga gggcctattt 481 cccatgattc cttcatattt gcatatacga tacaaggctg ttagagagat aattggaatt 541 aatttgactg taaacacaaa gatattagta caaaatacgt gacgtagaaa gtaataattt 601 cttgggtagt ttgcagtttt aaaattatgt tttaaaatgg actatcatat gcttaccgta 661 acttgaaagt atttcgattt cttggcttta tatatcttgt ggaaaggacg aaacaccggg 721 tcttcgagaa gacctgttta agagctatgc tggaaacagc atagcaagtt taaataaggc 781 tagtccgtta tcaacttgaa aaagtggcac cgagtcggtg ctttttttct agacccagct 841 ttcttgtaca aagttggcat tagaattcaa gcttattaat taatggagca caagactggc 901 ctcatgggcc ttccgctcac tgcccgcttt ccagtcggga aacctgtcgt gccagctgca 961 ttaacatggt catagctgtt tccttgcgta ttgggcgctc tccgcttcct cgctcactga 1021 ctcgctgcgc tcggtcgttc gggtaaagcc tggggtgcct aatgagcaaa aggccagcaa 1081 aaggccagga accgtaaaaa ggccgcgttg ctggcgtttt tccataggct ccgcccccct 1141 gacgagcatc acaaaaatcg acgctcaagt cagaggtggc gaaacccgac aggactataa 1201 agataccagg cgtttccccc tggaagctcc ctcgtgcgct ctcctgttcc gaccctgccg 1261 cttaccggat acctgtccgc ctttctccct tcgggaagcg tggcgctttc tcatagctca 1321 cgctgtaggt atctcagttc ggtgtaggtc gttcgctcca agctgggctg tgtgcacgaa 1381 ccccccgttc agcccgaccg ctgcgcctta tccggtaact atcgtcttga gtccaacccg 1441 gtaagacacg acttatcgcc actggcagca gccactggta acaggattag cagagcgagg 1501 tatgtaggcg gtgctacaga gttcttgaag tggtggccta actacggcta cactagaaga 1561 acagtatttg gtatctgcgc tctgctgaag ccagttacct tcggaaaaag agttggtagc 1621 tcttgatccg gcaaacaaac caccgctggt agcggtggtt tttttgtttg caagcagcag 1681 attacgcgca gaaaaaaagg atctcaagaa gatcctttga tcttttctac ggggtctgac 1741 gctcagtgga acgaaaactc acgttaaggg attttggtca tgagattatc aaaaaggatc 1801 ttcacctaga tccttttaaa ttaaaaatga agttttaaat caatctaaag tatatatgag 1861 taaacttggt ctgacagtta ccaatgctta atcagtgagg cacctatctc agcgatctgt 1921 ctatttcgtt catccatagt tgcctgactc cccgtcgtgt agataactac gatacgggag 1981 ggcttaccat ctggccccag tgctgcaatg ataccgcgag aaccacgctc accggctcca 2041 gatttatcag caataaacca gccagccgga agggccgagc gcagaagtgg tcctgcaact 2101 ttatccgcct ccatccagtc tattaattgt tgccgggaag ctagagtaag tagttcgcca 2161 gttaatagtt tgcgcaacgt tgttgccatt gctacaggca tcgtggtgtc acgctcgtcg 2221 tttggtatgg cttcattcag ctccggttcc caacgatcaa ggcgagttac atgatccccc 2281 atgttgtgca aaaaagcggt tagctccttc ggtcctccga tcgttgtcag aagtaagttg 2341 gccgcagtgt tatcactcat ggttatggca gcactgcata attctcttac tgtcatgcca 2401 tccgtaagat gcttttctgt gactggtgag tactcaacca agtcattctg agaatagtgt 2461 atgcggcgac cgagttgctc ttgcccggcg tcaatacggg ataataccgc gccacatagc 2521 agaactttaa aagtgctcat cattggaaaa cgttcttcgg ggcgaaaact ctcaaggatc 2581 ttaccgctgt tgagatccag ttcgatgtaa cccactcgtg cacccaactg atcttcagca 2641 tcttttactt tcaccagcgt ttctgggtga gcaaaaacag gaaggcaaaa tgccgcaaaa 2701 aagggaataa gggcgacacg gaaatgttga atactcatac tcttcctttt tcaatattat 2761 tgaagcattt atcagggtta ttgtctcatg agcggataca tatttgaatg tatttagaaa 2821 aataaacaaa taggggttcc gcgcacattt ccccgaaaag tgccac //