LOCUS Exported 3120 bp ds-DNA circular SYN 12-MAY-2021 DEFINITION Mammalian U6-driven sgRNA (SPm) targeting GTCCCCTCCACCCCACAGTG. ACCESSION . VERSION . KEYWORDS M-SP-sgRNA SOURCE synthetic DNA construct ORGANISM synthetic DNA construct REFERENCE 1 (bases 1 to 3120) AUTHORS Esvelt KM, Mali P, Braff JL, Moosburner M, Yaung SJ, Church GM TITLE Orthogonal Cas9 proteins for RNA-guided gene regulation and editing. JOURNAL Nat Methods. 2013 Sep 29. doi: 10.1038/nmeth.2681. PUBMED 24076762 REFERENCE 2 (bases 1 to 3120) AUTHORS . TITLE Direct Submission JOURNAL Exported May 12, 2021 from SnapGene Server 1.1.58 http://www.snapgene.com FEATURES Location/Qualifiers source 1..3120 /organism="synthetic DNA construct" /mol_type="other DNA" primer_bind complement(29..51) /label=pGEX 3' /note="pGEX vectors, reverse primer" primer_bind 151..170 /label=pRS-marker /note="pRS vectors, use to sequence yeast selectable marker" primer_bind 364..386 /label=M13/pUC Forward /note="In lacZ gene" primer_bind 378..395 /label=M13 Forward /note="In lacZ gene. Also called M13-F20 or M13 (-21) Forward" primer_bind 379..395 /label=M13 fwd /note="common sequencing primer, one of multiple similar variants" promoter 501..741 /label=U6 promoter /note="RNA polymerase III promoter for human U6 snRNA" primer_bind 501..521 /label=hU6-F /note="Human U6 promoter, forward primer" primer_bind 672..691 /label=LKO.1 5' /note="Human U6 promoter, forward primer" misc_RNA 770..845 /label=gRNA scaffold /note="guide RNA scaffold for the Streptococcus pyogenes CRISPR/Cas9 system" protein_bind complement(857..881) /gene="mutant version of attB" /label=attB2 /bound_moiety="BP Clonase(TM)" /note="recombination site for the Gateway(R) BP reaction" primer_bind complement(944..960) /label=M13 rev /note="common sequencing primer, one of multiple similar variants" primer_bind complement(944..960) /label=M13 Reverse /note="In lacZ gene. Also called M13-rev" primer_bind complement(957..979) /label=M13/pUC Reverse /note="In lacZ gene" protein_bind 968..984 /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(992..1022) /label=lac promoter /note="promoter for the E. coli lac operon" protein_bind 1037..1058 /label=CAP binding site /bound_moiety="E. coli catabolite activator protein" /note="CAP binding activates transcription in the presence of cAMP." primer_bind complement(1175..1192) /label=L4440 /note="L4440 vector, forward primer" rep_origin complement(1346..1934) /direction=LEFT /label=ori /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of replication" primer_bind complement(1426..1445) /label=pBR322ori-F /note="pBR322 origin, forward primer" CDS complement(2105..2920) /codon_start=1 /gene="aph(3')-Ia" /product="aminoglycoside phosphotransferase" /label=KanR /note="confers resistance to kanamycin in bacteria or G418 (Geneticin(R)) in eukaryotes" /translation="MSHIQRETSCSRPRLNSNMDADLYGYKWARDNVGQSGATIYRLYG KPDAPELFLKHGKGSVANDVTDEMVRLNWLTEFMPLPTIKHFIRTPDDAWLLTTAIPGK TAFQVLEEYPDSGENIVDALAVFLRRLHSIPVCNCPFNSDRVFRLAQAQSRMNNGLVDA SDFDDERNGWPVEQVWKEMHKLLPFSPDSVVTHGDFSLDNLIFDEGKLIGCIDVGRVGI ADRYQDLAILWNCLGEFSPSLQKRLFQKYGIDNPDMNKLQFHLMLDEFF" primer_bind 2828..2847 /label=Kan-R /note="Kanamycin resistance gene, reverse primer" promoter complement(2921..3025) /gene="bla" /label=AmpR promoter primer_bind 3093..3111 /label=pBRforEco /note="pBR322 vectors, upsteam of EcoRI site, forward primer" ORIGIN 1 tcgcgcgttt cggtgatgac ggtgaaaacc tctgacacat gcagctcccg gagacggtca 61 cagcttgtct gtaagcggat gccgggagca gacaagcccg tcagggcgcg tcagcgggtg 121 ttggcgggtg tcggggctgg cttaactatg cggcatcaga gcagattgta ctgagagtgc 181 accatatgcg gtgtgaaata ccgcacagat gcgtaaggag aaaataccgc atcaggcgcc 241 attcgccatt caggctgcgc aactgttggg aagggcgatc ggtgcgggcc tcttcgctat 301 tacgccagct ggcgaaaggg ggatgtgctg caaggcgatt aagttgggta acgccagggt 361 tttcccagtc acgacgttgt aaaacgacgg ccagtgaatt cgagctcggt acctcgcgaa 421 tgcatctaga ttgtacaaaa aagcaggctt taaaggaacc aattcagtcg actggatccg 481 gtaccaaggt cgggcaggaa gagggcctat ttcccatgat tccttcatat ttgcatatac 541 gatacaaggc tgttagagag ataattagaa ttaatttgac tgtaaacaca aagatattag 601 tacaaaatac gtgacgtaga aagtaataat ttcttgggta gtttgcagtt ttaaaattat 661 gttttaaaat ggactatcat atgcttaccg taacttgaaa gtatttcgat ttcttggctt 721 tatatatctt gtggaaagga cgaaacaccg tcccctccac cccacagtgg ttttagagct 781 agaaatagca agttaaaata aggctagtcc gttatcaact tgaaaaagtg gcaccgagtc 841 ggtgcttttt ttctagaccc agctttcttg tacaaagttg gcattaatcg gatcccgggc 901 ccgtcgactg cagaggcctg catgcaagct tggcgtaatc atggtcatag ctgtttcctg 961 tgtgaaattg ttatccgctc acaattccac acaacatacg agccggaagc ataaagtgta 1021 aagcctgggg tgcctaatga gtgagctaac tcacattaat tgcgttgcgc tcactgcccg 1081 ctttccagtc gggaaacctg tcgtgccagc tgcattaatg aatcggccaa cgcgcgggga 1141 gaggcggttt gcgtattggg cgctcttccg cttcctcgct cactgactcg ctgcgctcgg 1201 tcgttcggct gcggcgagcg gtatcagctc actcaaaggc ggtaatacgg ttatccacag 1261 aatcagggga taacgcagga aagaacatgt gagcaaaagg ccagcaaaag gccaggaacc 1321 gtaaaaaggc cgcgttgctg gcgtttttcc ataggctccg cccccctgac gagcatcaca 1381 aaaatcgacg ctcaagtcag aggtggcgaa acccgacagg actataaaga taccaggcgt 1441 ttccccctgg aagctccctc gtgcgctctc ctgttccgac cctgccgctt accggatacc 1501 tgtccgcctt tctcccttcg ggaagcgtgg cgctttctca tagctcacgc tgtaggtatc 1561 tcagttcggt gtaggtcgtt cgctccaagc tgggctgtgt gcacgaaccc cccgttcagc 1621 ccgaccgctg cgccttatcc ggtaactatc gtcttgagtc caacccggta agacacgact 1681 tatcgccact ggcagcagcc actggtaaca ggattagcag agcgaggtat gtaggcggtg 1741 ctacagagtt cttgaagtgg tggcctaact acggctacac tagaagaaca gtatttggta 1801 tctgcgctct gctgaagcca gttaccttcg gaaaaagagt tggtagctct tgatccggca 1861 aacaaaccac cgctggtagc ggtggttttt ttgtttgcaa gcagcagatt acgcgcagaa 1921 aaaaaggatc tcaagaagat cctttgatct tttctacggg gtctgacgct cagtggaacg 1981 aaaactcacg ttaagggatt ttggtcatga gattatcaaa aaggatcttc acctagatcc 2041 ttttaaatta aaaatgaagt tttaaatcaa tctaaagtat atatgagtaa acttggtctg 2101 acagttagaa aaactcatcg agcatcaaat gaaactgcaa tttattcata tcaggattat 2161 caataccata tttttgaaaa agccgtttct gtaatgaagg agaaaactca ccgaggcagt 2221 tccataggat ggcaagatcc tggtatcggt ctgcgattcc gactcgtcca acatcaatac 2281 aacctattaa tttcccctcg tcaaaaataa ggttatcaag tgagaaatca ccatgagtga 2341 cgactgaatc cggtgagaat ggcaaaagtt tatgcatttc tttccagact tgttcaacag 2401 gccagccatt acgctcgtca tcaaaatcac tcgcatcaac caaaccgtta ttcattcgtg 2461 attgcgcctg agcgagacga aatacgcgat cgctgttaaa aggacaatta caaacaggaa 2521 tcgaatgcaa ccggcgcagg aacactgcca gcgcatcaac aatattttca cctgaatcag 2581 gatattcttc taatacctgg aatgctgttt tcccagggat cgcagtggtg agtaaccatg 2641 catcatcagg agtacggata aaatgcttga tggtcggaag aggcataaat tccgtcagcc 2701 agtttagtct gaccatctca tctgtaacat cattggcaac gctacctttg ccatgtttca 2761 gaaacaactc tggcgcatcg ggcttcccat acaatcgata gattgtcgca cctgattgcc 2821 cgacattatc gcgagcccat ttatacccat ataaatcagc atccatgttg gaatttaatc 2881 gcggcctaga gcaagacgtt tcccgttgaa tatggctcat actcttcctt tttcaatatt 2941 attgaagcat ttatcagggt tattgtctca tgagcggata catatttgaa tgtatttaga 3001 aaaataaaca aataggggtt ccgcgcacat ttccccgaaa agtgccacct gacgtctaag 3061 aaaccattat tatcatgaca ttaacctata aaaataggcg tatcacgagg ccctttcgtc //