LOCUS Exported 2789 bp ds-DNA circular SYN 12-MAY-2021 DEFINITION BsaI-based cloning of SpCas9 gRNA guide sequence, position 5/15/25 in the array. ACCESSION . VERSION . KEYWORDS pMA-SpCas9-g5 SOURCE synthetic DNA construct ORGANISM synthetic DNA construct REFERENCE 1 (bases 1 to 2789) AUTHORS Vad-Nielsen J, Lin L, Bolund L, Nielsen AL, Luo Y TITLE Golden Gate Assembly of CRISPR gRNA expression array for simultaneously targeting multiple genes. JOURNAL Cell Mol Life Sci. 2016 May 13. PUBMED 27178736 REFERENCE 2 (bases 1 to 2789) AUTHORS . TITLE Direct Submission JOURNAL Exported May 12, 2021 from SnapGene Server 1.1.58 http://www.snapgene.com FEATURES Location/Qualifiers source 1..2789 /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 403..643 /label=U6 promoter /note="RNA polymerase III promoter for human U6 snRNA" primer_bind 403..423 /label=hU6-F /note="Human U6 promoter, forward primer" primer_bind 574..593 /label=LKO.1 5' /note="Human U6 promoter, forward primer" misc_RNA 670..745 /label=gRNA scaffold /note="guide RNA scaffold for the Streptococcus pyogenes CRISPR/Cas9 system" primer_bind complement(932..949) /label=L4440 /note="L4440 vector, forward primer" rep_origin complement(1042..1630) /direction=LEFT /label=ori /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of replication" primer_bind complement(1122..1141) /label=pBR322ori-F /note="pBR322 origin, forward primer" CDS complement(1801..2661) /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 2424..2443 /label=Amp-R /note="Ampicillin resistance gene, reverse primer" promoter complement(2662..2766) /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 agctcggatc cggtctcctg ttgagggcct atttcccatg 421 attccttcat atttgcatat acgatacaag gctgttagag agataattgg aattaatttg 481 actgtaaaca caaagatatt agtacaaaat acgtgacgta gaaagtaata atttcttggg 541 tagtttgcag ttttaaaatt atgttttaaa atggactatc atatgcttac cgtaacttga 601 aagtatttcg atttcttggc tttatatatc ttgtggaaag gacgaaacac cgggtcttcg 661 agaagacctg ttttagagct agaaatagca agttaaaata aggctagtcc gttatcaact 721 tgaaaaagtg gcaccgagtc ggtgcttttt tgttttagag ctagaaatag caagttatgc 781 aggagacctg tacaacgcgt ggtacctgga gcacaagact ggcctcacgg gccttccgct 841 cactgcccgc tttccagtcg ggaaacctgt cgtgccagct gcattaacat ggtcatagct 901 gtttccttgc gtattgggcg ctctccgctt cctcgctcac tgactcgctg cgctcggtcg 961 ttcgggtaaa gcctggggtg cctaatgagc aaaaggccag caaaaggcca ggaaccgtaa 1021 aaaggccgcg ttgctggcgt ttttccatag gctccgcccc cctgacgagc atcacaaaaa 1081 tcgacgctca agtcagaggt ggcgaaaccc gacaggacta taaagatacc aggcgtttcc 1141 ccctggaagc tccctcgtgc gctctcctgt tccgaccctg ccgcttaccg gatacctgtc 1201 cgcctttctc ccttcgggaa gcgtggcgct ttctcatagc tcacgctgta ggtatctcag 1261 ttcggtgtag gtcgttcgct ccaagctggg ctgtgtgcac gaaccccccg ttcagcccga 1321 ccgctgcgcc ttatccggta actatcgtct tgagtccaac ccggtaagac acgacttatc 1381 gccactggca gcagccactg gtaacaggat tagcagagcg aggtatgtag gcggtgctac 1441 agagttcttg aagtggtggc ctaactacgg ctacactaga agaacagtat ttggtatctg 1501 cgctctgctg aagccagtta ccttcggaaa aagagttggt agctcttgat ccggcaaaca 1561 aaccaccgct ggtagcggtg gtttttttgt ttgcaagcag cagattacgc gcagaaaaaa 1621 aggatctcaa gaagatcctt tgatcttttc tacggggtct gacgctcagt ggaacgaaaa 1681 ctcacgttaa gggattttgg tcatgagatt atcaaaaagg atcttcacct agatcctttt 1741 aaattaaaaa tgaagtttta aatcaatcta aagtatatat gagtaaactt ggtctgacag 1801 ttaccaatgc ttaatcagtg aggcacctat ctcagcgatc tgtctatttc gttcatccat 1861 agttgcctga ctccccgtcg tgtagataac tacgatacgg gagggcttac catctggccc 1921 cagtgctgca atgataccgc gagaaccacg ctcaccggct ccagatttat cagcaataaa 1981 ccagccagcc ggaagggccg agcgcagaag tggtcctgca actttatccg cctccatcca 2041 gtctattaat tgttgccggg aagctagagt aagtagttcg ccagttaata gtttgcgcaa 2101 cgttgttgcc attgctacag gcatcgtggt gtcacgctcg tcgtttggta tggcttcatt 2161 cagctccggt tcccaacgat caaggcgagt tacatgatcc cccatgttgt gcaaaaaagc 2221 ggttagctcc ttcggtcctc cgatcgttgt cagaagtaag ttggccgcag tgttatcact 2281 catggttatg gcagcactgc ataattctct tactgtcatg ccatccgtaa gatgcttttc 2341 tgtgactggt gagtactcaa ccaagtcatt ctgagaatag tgtatgcggc gaccgagttg 2401 ctcttgcccg gcgtcaatac gggataatac cgcgccacat agcagaactt taaaagtgct 2461 catcattgga aaacgttctt cggggcgaaa actctcaagg atcttaccgc tgttgagatc 2521 cagttcgatg taacccactc gtgcacccaa ctgatcttca gcatctttta ctttcaccag 2581 cgtttctggg tgagcaaaaa caggaaggca aaatgccgca aaaaagggaa taagggcgac 2641 acggaaatgt tgaatactca tactcttcct ttttcaatat tattgaagca tttatcaggg 2701 ttattgtctc atgagcggat acatatttga atgtatttag aaaaataaac aaataggggt 2761 tccgcgcaca tttccccgaa aagtgccac //