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 #1. ACCESSION . VERSION . KEYWORDS pT7-SpCas9_sgRNA-site1 (RTW443) 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 26..42 /label=M13 rev /note="common sequencing primer, one of multiple similar variants" primer_bind 26..42 /label=M13 Reverse /note="In lacZ gene. Also called M13-rev" primer_bind 171..190 /label=T7 /note="T7 promoter, forward primer" promoter 171..189 /label=T7 promoter /note="promoter for bacteriophage T7 RNA polymerase" misc_RNA 208..283 /label=gRNA scaffold /note="guide RNA scaffold for the Streptococcus pyogenes CRISPR/Cas9 system" primer_bind complement(465..482) /label=M13 Forward /note="In lacZ gene. Also called M13-F20 or M13 (-21) Forward" primer_bind complement(465..481) /label=M13 fwd /note="common sequencing primer, one of multiple similar variants" rep_origin 522..1109 /direction=RIGHT /label=ori /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of replication" primer_bind 1010..1029 /label=pBR322ori-F /note="pBR322 origin, forward primer" terminator 1121..1148 /label=T7Te terminator /note="phage T7 early transcription terminator" CDS complement(1172..1987) /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 1895..1914 /label=Kan-R /note="Kanamycin resistance gene, reverse primer" promoter complement(1988..2090) /label=cat promoter /note="promoter of the E. coli cat gene encoding chloramphenicol acetyltransferase" terminator 2091..2122 /label=tonB terminator /note="bidirectional E. coli tonB-P14 transcription terminator" ORIGIN 1 gatttaaatg gtcagtattg agcctcagga aacagctatg acatcaagct gactagataa 61 tctagctgat cgtggaccga tcatacgtat aatgccgtaa gatcacgggt cgcagcacag 121 ctcgcggtcc agtagtgatc gacactgctc gatccgctcg caccgctagc taatacgact 181 cactataggg cacgggcagc ttgccgggtt ttagagctag aaatagcaag ttaaaataag 241 gctagtccgt tatcaacttg aaaaagtggc accgagtcgg tgcttttaaa aagcttggat 301 cgacgagagc agcgcgactg gatctgtcgc ccgtctcaaa cgcaaccctc cggcggtcgc 361 atatcattca ggacgagcct cagactccag cgtaactgga ctgcaatcaa ctcactggct 421 caccttccgg tccacgatca gctagaatca agctgactag ataaactggc cgtcgtttta 481 cacgggtggg cctttcttcg gtagaaaatc aaaggatctt cttgagatcc tttttttctg 541 cgcgtaatct gctgcttgca aacaaaaaaa ccaccgctac cagcggtggt ttgtttgccg 601 gatcaagagc taccaactct ttttccgagg taactggctt cagcagagcg cagataccaa 661 atactgttct tctagtgtag ccgtagttag gccaccactt caagaactct gtagcaccgc 721 ctacatacct cgctctgcta atcctgttac cagtggctgc tgccagtggc gataagtcgt 781 gtcttaccgg gttggactca agacgatagt taccggataa ggcgcagcgg tcgggctgaa 841 cggggggttc gtgcacacag cccagcttgg agcgaacgac ctacaccgaa ctgagatacc 901 tacagcgtga gctatgagaa agcgccacgc ttcccgaagg gagaaaggcg gacaggtatc 961 cggtaagcgg cagggtcgga acaggagagc gcacgaggga gcttccaggg ggaaacgcct 1021 ggtatcttta tagtcctgtc gggtttcgcc acctctgact tgagcatcga tttttgtgat 1081 gctcgtcagg ggggcggagc ctatggaaaa acgccagcaa cgcagaaagg cccacccgaa 1141 ggtgagccag gtgattacat ttaggtcctc attagaaaaa ctcatcgagc atcaagtgaa 1201 actgcaattt attcatatca ggattatcaa taccatattt ttgaaaaagc cgtttctgta 1261 atgaaggaga aaactcaccg aggcagttcc ataggatggc aagatcctgg tatcggtctg 1321 cgattccgac tcgtccaaca tcaatacaac ctattaattt cccctcgtca aaaataaggt 1381 tatcaagtga gaaatcacca tgagtgacga ctgaatccgg tgagaatggc aagagtttat 1441 gcatttcttt ccagacttgt tcaacaggcc agccattacg ctcgtcatca aaatcactcg 1501 caccaaccaa accgttattc attcgtgatt gcgcctgagc gagacgaaat acgcgatcgc 1561 cgttaaaagg acaattacaa acaggaatcg aatgcaaccg gcgcaggaac actgccagcg 1621 catcaacaat attttcacct gaatcaggat attcttctaa tacctggaat gctgttttcc 1681 ctgggatcgc agtggtgagt aaccatgcat catcaggagt acggataaaa tgcttgatgg 1741 tcggaagagg cataaattcc gtcagccagt ttagcctgac catctcatct gtaacatcat 1801 tggcaacgct acctttgcca tgtttcagaa acaactctgg cgcatcgggc ttcccataca 1861 atcgatagat tgtcgcacct gattgcccga cattatcgcg agcccattta tacccatata 1921 aatcagcatc catgttggaa tttaatcgcg gcttcaagca agacgtttcc cgttgaatat 1981 ggctcatttt agcttcctta gctcctgaaa atctcgataa ctcaaaaaat acgcccggta 2041 gtgatcttat ttcattatgg tgaaagttgg aacctcttac gtgccgatca agtcaaaagc 2101 ctccggtcgg aggcttttga ctttctgcta tggaggtcag gtat //