LOCUS Exported 2144 bp ds-DNA circular SYN 25-MAY-2021 DEFINITION T7 promoter expression plasmid for in vitro transcription of SpCas9 sgRNA with spacer #2. ACCESSION . VERSION . KEYWORDS pT7-SpCas9_sgRNA-site2 (RTW448) SOURCE synthetic DNA construct ORGANISM synthetic DNA construct REFERENCE 1 (bases 1 to 2144) AUTHORS Walton RT, Hsu JY, Joung JK, Kleinstiver BP TITLE Scalable characterization of the PAM requirements of CRISPR-Cas enzymes using HT-PAMDA. JOURNAL Nat Protoc. 2021 Feb 5. pii: 10.1038/s41596-020-00465-2. doi: 10.1038/s41596-020-00465-2. PUBMED 33547443 REFERENCE 2 (bases 1 to 2144) AUTHORS . TITLE Direct Submission JOURNAL Exported May 25, 2021 from SnapGene Server 1.1.58 http://www.snapgene.com FEATURES Location/Qualifiers source 1..2144 /organism="synthetic DNA construct" /mol_type="other DNA" primer_bind 20..36 /label=M13 rev /note="common sequencing primer, one of multiple similar variants" primer_bind 20..36 /label=M13 Reverse /note="In lacZ gene. Also called M13-rev" promoter 165..182 /label=T7 promoter /note="promoter for bacteriophage T7 RNA polymerase" misc_RNA 202..277 /label=gRNA scaffold /note="guide RNA scaffold for the Streptococcus pyogenes CRISPR/Cas9 system" primer_bind complement(459..476) /label=M13 Forward /note="In lacZ gene. Also called M13-F20 or M13 (-21) Forward" primer_bind complement(459..475) /label=M13 fwd /note="common sequencing primer, one of multiple similar variants" rep_origin 516..1103 /direction=RIGHT /label=ori /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of replication" primer_bind 1004..1023 /label=pBR322ori-F /note="pBR322 origin, forward primer" terminator 1115..1142 /label=T7Te terminator /note="phage T7 early transcription terminator" CDS complement(1166..1981) /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="MSHIQRETSCLKPRLNSNMDADLYGYKWARDNVGQSGATIYRLYG KPDAPELFLKHGKGSVANDVTDEMVRLNWLTEFMPLPTIKHFIRTPDDAWLLTTAIPGK TAFQVLEEYPDSGENIVDALAVFLRRLHSIPVCNCPFNGDRVFRLAQAQSRMNNGLVGA SDFDDERNGWPVEQVWKEMHKLLPFSPDSVVTHGDFSLDNLIFDEGKLIGCIDVGRVGI ADRYQDLAILWNCLGEFSPSLQKRLFQKYGIDNPDMNKLQFHLMLDEFF" primer_bind 1889..1908 /label=Kan-R /note="Kanamycin resistance gene, reverse primer" promoter complement(1982..2084) /label=cat promoter /note="promoter of the E. coli cat gene encoding chloramphenicol acetyltransferase" terminator 2085..2116 /label=tonB terminator /note="bidirectional E. coli tonB-P14 transcription terminator" ORIGIN 1 aatggtcagt attgagcctc aggaaacagc tatgacatca agctgactag ataatctagc 61 tgatcgtgga ccgatcatac gtataatgcc gtaagatcac gggtcgcagc acagctcgcg 121 gtccagtagt gatcgacact gctcgatccg ctcgcaccgc tagctaatac gactcactat 181 agtcgccctc gaacttcacc tgttttagag ctagaaatag caagttaaaa taaggctagt 241 ccgttatcaa cttgaaaaag tggcaccgag tcggtgcttt taaaaagctt ggatcgacga 301 gagcagcgcg actggatctg tcgcccgtct caaacgcaac cctccggcgg tcgcatatca 361 ttcaggacga gcctcagact ccagcgtaac tggactgcaa tcaactcact ggctcacctt 421 ccggtccacg atcagctaga atcaagctga ctagataaac tggccgtcgt tttacacggg 481 tgggcctttc ttcggtagaa aatcaaagga tcttcttgag atcctttttt tctgcgcgta 541 atctgctgct tgcaaacaaa aaaaccaccg ctaccagcgg tggtttgttt gccggatcaa 601 gagctaccaa ctctttttcc gaggtaactg gcttcagcag agcgcagata ccaaatactg 661 ttcttctagt gtagccgtag ttaggccacc acttcaagaa ctctgtagca ccgcctacat 721 acctcgctct gctaatcctg ttaccagtgg ctgctgccag tggcgataag tcgtgtctta 781 ccgggttgga ctcaagacga tagttaccgg ataaggcgca gcggtcgggc tgaacggggg 841 gttcgtgcac acagcccagc ttggagcgaa cgacctacac cgaactgaga tacctacagc 901 gtgagctatg agaaagcgcc acgcttcccg aagggagaaa ggcggacagg tatccggtaa 961 gcggcagggt cggaacagga gagcgcacga gggagcttcc agggggaaac gcctggtatc 1021 tttatagtcc tgtcgggttt cgccacctct gacttgagca tcgatttttg tgatgctcgt 1081 caggggggcg gagcctatgg aaaaacgcca gcaacgcaga aaggcccacc cgaaggtgag 1141 ccaggtgatt acatttaggt cctcattaga aaaactcatc gagcatcaag tgaaactgca 1201 atttattcat atcaggatta tcaataccat atttttgaaa aagccgtttc tgtaatgaag 1261 gagaaaactc accgaggcag ttccatagga tggcaagatc ctggtatcgg tctgcgattc 1321 cgactcgtcc aacatcaata caacctatta atttcccctc gtcaaaaata aggttatcaa 1381 gtgagaaatc accatgagtg acgactgaat ccggtgagaa tggcaagagt ttatgcattt 1441 ctttccagac ttgttcaaca ggccagccat tacgctcgtc atcaaaatca ctcgcaccaa 1501 ccaaaccgtt attcattcgt gattgcgcct gagcgagacg aaatacgcga tcgccgttaa 1561 aaggacaatt acaaacagga atcgaatgca accggcgcag gaacactgcc agcgcatcaa 1621 caatattttc acctgaatca ggatattctt ctaatacctg gaatgctgtt ttccctggga 1681 tcgcagtggt gagtaaccat gcatcatcag gagtacggat aaaatgcttg atggtcggaa 1741 gaggcataaa ttccgtcagc cagtttagcc tgaccatctc atctgtaaca tcattggcaa 1801 cgctaccttt gccatgtttc agaaacaact ctggcgcatc gggcttccca tacaatcgat 1861 agattgtcgc acctgattgc ccgacattat cgcgagccca tttataccca tataaatcag 1921 catccatgtt ggaatttaat cgcggcttca agcaagacgt ttcccgttga atatggctca 1981 ttttagcttc cttagctcct gaaaatctcg ataactcaaa aaatacgccc ggtagtgatc 2041 ttatttcatt atggtgaaag ttggaacctc ttacgtgccg atcaagtcaa aagcctccgg 2101 tcggaggctt ttgactttct gctatggagg tcaggtatga ttta //