LOCUS Exported 949 bp ds-DNA linear SYN 17-MAY-2021 DEFINITION Cas9 from S. pyogenes with 2A-Puro, and cloning backbone for sgRNA (V2.0). ACCESSION . VERSION . KEYWORDS pSpCas9(BB)-2A-Puro (PX459) V2.0 SOURCE synthetic DNA construct ORGANISM synthetic DNA construct REFERENCE 1 (bases 1 to 949) AUTHORS Ran FA, Hsu PD, Wright J, Agarwala V, Scott DA, Zhang F TITLE Genome engineering using the CRISPR-Cas9 system. JOURNAL Nat Protoc. 2013 Nov;8(11):2281-308. doi: 10.1038/nprot.2013.143. Epub 2013 Oct 24. PUBMED 24157548 REFERENCE 2 (bases 1 to 949) AUTHORS . TITLE Direct Submission JOURNAL Exported May 17, 2021 from SnapGene Server 1.1.58 http://www.snapgene.com FEATURES Location/Qualifiers source 1..949 /organism="synthetic DNA construct" /mol_type="other DNA" CDS complement(7..606) /codon_start=1 /gene="pac from Streptomyces alboniger" /product="puromycin N-acetyltransferase" /label=PuroR /note="confers resistance to puromycin" /translation="MTEYKPTVRLATRDDVPRAVRTLAAAFADYPATRHTVDPDRHIER VTELQELFLTRVGLDIGKVWVADDGAAVAVWTTPESVEAGAVFAEIGPRMAELSGSRLA AQQQMEGLLAPHRPKEPAWFLATVGVSPDHQGKGLGSAVVLPGVEAAERAGVPAFLETS APRNLPFYERLGFTVTADVEVPEGPRTWCMTRKPGA" primer_bind complement(90..110) /label=Puro-F /note="Puromycin resistance gene, forward primer" primer_bind 587..606 /label=Puro-R /note="Puromycin resistance gene, reverse primer. Also called puro-variant-R" CDS complement(607..660) /codon_start=1 /product="2A peptide from Thosea asigna virus capsid protein" /label=T2A /note="Eukaryotic ribosomes fail to insert a peptide bond between the Gly and Pro residues, yielding separate polypeptides." /translation="EGRGSLLTCGDVEENPGP" CDS complement(676..723) /codon_start=1 /product="bipartite nuclear localization signal from nucleoplasmin" /label=nucleoplasmin NLS /translation="KRPAATKKAGQAKKKK" ORIGIN 1 gaattctcag gcaccgggct tgcgggtcat gcaccaggtg cgcggtcctt cgggcacctc 61 gacgtcggcg gtgacggtga agccgagccg ctcgtagaag gggaggttgc ggggcgcgga 121 ggtctccagg aaggcgggca ccccggcgcg ctcggccgcc tccactccgg ggagcacgac 181 ggcgctgccc agacccttgc cctggtggtc gggcgagact ccgacggtgg ccaggaacca 241 cgcgggctcc ttgggccggt gcggcgccag gaggccttcc atctgttgct gcgcggccag 301 ccgggaaccg ctcaactcgg ccatgcgcgg gccgatctcg gcgaacaccg cccccgcttc 361 gacgctctcc ggcgtggtcc agaccgccac cgcggcgccg tcgtccgcga cccacacctt 421 gccgatgtcg agcccgacgc gcgtgaggaa gagttcttgc agctcggtga cccgctcgat 481 gtggcggtcc ggatcgacgg tgtggcgcgt ggcggggtag tcggcgaacg cggcggcgag 541 ggtgcgtacg gccctgggga cgtcgtcgcg ggtggcgagg cgcaccgtgg gcttgtactc 601 ggtcattggg ccaggattct cctcgacgtc accgcatgtt agcagacttc ctctgccctc 661 tccactgccg aattcctttt tcttttttgc ctggccggcc tttttcgtgg ccgccggcct 721 tttgtcgcct cccagctgag acaggtcgat ccgtgtctcg tacaggccgg tgatgctctg 781 gtggatcagg gtggcgtcca gcacctcttt ggtgctggtg tacctcttcc ggtcgatggt 841 ggtgtcaaag tacttgaagg cggcaggggc tcccagattg gtcagggtaa acaggtggat 901 gatattctcg gcctgctctc tgatgggctt atcccggtgc ttgttgtag //