LOCUS Exported 4481 bp ds-DNA circular SYN 12-MAY-2021 DEFINITION pAL374 plasmid carrying the pyk (NT) sgRNA targeting the non-template strand of pyk, SpecR. ACCESSION . VERSION . KEYWORDS pAL-pyk_NT SOURCE synthetic DNA construct ORGANISM synthetic DNA construct REFERENCE 1 (bases 1 to 4481) AUTHORS Cleto S, Jensen JV, Wendisch VF, Lu TK TITLE Corynebacterium glutamicum Metabolic Engineering with CRISPR Interference (CRISPRi). JOURNAL ACS Synth Biol. 2016 Feb 16. PUBMED 26829286 REFERENCE 2 (bases 1 to 4481) AUTHORS . TITLE Direct Submission JOURNAL Exported May 12, 2021 from SnapGene Server 1.1.58 http://www.snapgene.com FEATURES Location/Qualifiers source 1..4481 /organism="synthetic DNA construct" /mol_type="other DNA" misc_RNA 1..76 /label=gRNA scaffold /note="guide RNA scaffold for the Streptococcus pyogenes CRISPR/Cas9 system" oriT 2341..2450 /note="incP origin of transfer" protein_bind 3182..3196 /label=boxB /bound_moiety="lambda-N peptide" /note="lambda N peptide binding site consisting of an RNA hairpin within the N-utliization (nut) site of the lambda bacteriophage transcript (Tan and Frankel, 1995)" CDS 3316..4107 /codon_start=1 /gene="aadA" /product="aminoglycoside adenylyltransferase (Murphy, 1985)" /label=SmR /note="confers resistance to spectinomycin and streptomycin" /translation="MREAVIAEVSTQLSEVVGVIERHLEPTLLAVHLYGSAVDGGLKPH SDIDLLVTVTVRLDETTRQALINDLLETSASPGESEILRAVEVTIVVHDDIIPWRYPAK RELQFGEWQRNDILAGIFEPATIDIDLAILLTKAREHSVALVGPAAEELFDPVPEQDLF EALNETLTLWNSPPDWAGDERNVVLTLSRIWYSAVTGKIAPKDVAADWAMERLPAQYQP VILEARQAYLGQEEDRLASRADQLEEFVHYVKGEITKVVGK" ORIGIN 1 gttttagagc tagaaatagc aagttaaaat aaggctagtc cgttatcaac ttgaaaaagt 61 ggcaccgagt cggtgctttt ttatggaatt ggatccgtgg aagacatctg tcgaagacac 121 cttgtggtcg aagacacctt gtctcttccc agaggcgtgg tcgcatacta atttgtaaac 181 ggccatgacg aaatagtcgt acggaacaag gagccactag tctagaattc ggtgaggtta 241 tggcggaggg ttgcgaggtc taggagaaca gaggaagtca tgctttgaag catataagct 301 gccctgcccc tcaaggtttt cttcaagtga ggttttatct aactgcctaa cggcagggga 361 accgtatatt gcttacggta tgagacccct taaacgtccg gatagtcacc gctcttcttt 421 agctccgcga catgcctagc aaccgtggcg cgagagactc ctacctctgc ccctatttca 481 gcccacgtgg gaactgtccc tgtctggaaa tactgatcgt tcaccatttg gctaatacgg 541 gacttcgtag atcgtccttg agcctttttc ttacggtgcg tcttttcaag cttcgacctt 601 tgtgcttgcg catatttgcc ctcggggtct gttttccagc gttgtgcggc tttttgtccg 661 cctctgcgtc ccatcgtggc caaggctttc cgctcgctgc tggtggcttt acctggtgcg 721 ttagagccgc tgtaggtctc gctcttggat tgggcgacat acccgcgcac gcgccttgcc 781 atggtttggc ggtcgcgcat gggtggcatc tcgttgtcgc ggcctgcacc gccgtgggtg 841 tgtgcgacgt tgtaggcgtg ctcataggcg tcgatgattg ctgcgtctgt caggcgttgg 901 ccttgctggc gcaagcggtg gccagtctta agcgcatgtc taaaggctgt ttcgtcgcgt 961 gctgcggttc cttggacaat ccagagcaca cgcacaccgt cgataagttc cgggtcatac 1021 tggtcgagac caccggcgat ttccgcgtct acgtcctggg cgagtgcttt gaatgcttgg 1081 gcttcttcac ggcgggtctt gaccgcgttg ataagttcgc ggccagagct gaattgctgg 1141 cgtggggtgg ggttgaactg gtcgtgtcct gccatatccc ttacctgctt tatcaagtct 1201 ccaaggcgca tcacccggtt gtgctgccta taccaacgat aagcggtagg ggctttgcct 1261 gtgtagaacg ggttgcggct aaagcggtgg gaaaagtgcg ggtcatggtc taaaagctca 1321 cccagcacac gcgtggttgc tgcaagaagc ttcatctgcg cagatttacc gttacggtca 1381 gcgtagacag ggtcaataag ccatatgaac tgggctttgc cgttagttgg gttaataccc 1441 acccaggctg gcccgacgct atgagtaatc agtgagcgca ccacgtcgcg gacgtacggg 1501 tttaagtctg cggggtcacc gcctgcggta cctacttggt caacgtctac gaccaggacg 1561 gcggcgtact gcttggtggt gagcatggcg tactcgcacc gtcctaaagc atcagtctcg 1621 aagcgataca tacgcggcga gttcgtgccg tcagcgttgc gtcgataggc ctttttaaag 1681 tctcgtgtga ctgaaccgtg gagtacatcg cggcctagat gatcgcgtaa aaggtcgcgg 1741 tcactggcag atgctggggt gttgtccagt ccaccacggt cgcgctcgac gcgggtaggt 1801 gttttagtgt gcgcattctg cgcatgagtc tgtaaactca tgaccgtgat ttctcccagg 1861 tgtgtgctgg gtgataagcg aaagtcatcg ggttgccgcc cggtggcttt cttcgttttt 1921 cattgtcttt ccctgactct aaatgacacc ggtgttattt actagccatg acacgcgaaa 1981 aatatgcctt ttacctgcgg ttacaattcg cccttgctct agagatcgtg gcatcaccga 2041 accgcgccgt gcgcgggtcg tcggtgagcc agagtttcag caggccgccc aggcggccca 2101 ggtcgccatt gatgcgggcc agctcgcgga cgtgctcata gtccacgacg cccgtgattt 2161 tgtagccctg gccgacggcc agcaggtagg ccgacaggct catgccggcc gccgccgcct 2221 tttcctcaat cgctcttcgt tcgtctggaa ggcagtacac cttgataggt gggctgccct 2281 tcctggttgg cttggtttca tcagccatcc gcttgccctc atctgttacg ccggcggtag 2341 ccggccagcc tcgcagagca ggattcccgt tgagcaccgc caggtgcgaa taagggacag 2401 tgaagaagga acacccgctc gcgggtgggc ctacttcacc tatcctgccc ggctgacgcc 2461 gttggataca ccaaggaaag tctacacgaa ccctttggca aaatcctgta tatcgtgcga 2521 aaaaggatgg atataccgaa aaaatcgcta taatgacccc gaagcagggt tatgcagcgg 2581 aaaagcgctg cttccctgct gttttgtgga atatctaccg actggaaaca ggcaaatgca 2641 ggaaattact gaactgaggg gacaggcgag agacgatgcc aaagagctac accgacgagc 2701 tggccgagtg ggttgaatcc cgcgcggcca agaagcgccg gcgtgatgag gctgcggttg 2761 cgttcctggc ggtgagggcg gatgtcgagg cggcgttagc gtccggctat gcgctcgtca 2821 ccatttggga gcacatgcgg gaaacgggga aggtcaagtt ctcctacgag acgttccgct 2881 cgcacgccag gcggcacatc aaggccaagc ccgccgatgt gcccgcaccg caggccaagg 2941 ctgcggaacc cgcgccggca cccaagacgc cggagccacg gcggccgaag caggggggca 3001 aggctgaaaa gccggccccc gctgcggccc cgaccggctt caccttcaac ccaacaccgg 3061 acaaaaagga tcaattcgcc cttgcaacgc gtgctgagcg acataaatac cactggcggt 3121 gatactgagc acatcagcag gacgcactga ccaccatgaa ggtgacgctc ttaaaaatta 3181 agccctgaag aagggcagca ttcaaagcag aaggctttgg ggtgtgtgat acgaaacgaa 3241 gcattggccg taagtgcgat tccggattag ctgccaatga agtactagat ccacggttag 3301 cggaggaggc cccccatgag ggaagcggtg atcgccgaag tatcgactca actatcagag 3361 gtagttggcg tcatcgagcg ccatctcgaa ccgacgttgc tggccgtaca tttgtacggc 3421 tccgcagtgg atggcggcct gaagccacac agtgatattg atttgctggt tacggtgacc 3481 gtaaggcttg atgaaacaac gcggcaagct ttgatcaacg accttttgga aacttcggct 3541 tcccctggag agagcgagat tctccgcgct gtagaagtca ccattgttgt gcacgacgac 3601 atcattccgt ggcgttatcc agctaagcgc gaactgcaat ttggagaatg gcagcgcaat 3661 gacattcttg caggtatctt cgagccagcc acgatcgaca ttgatctggc tatcttgctg 3721 acaaaagcaa gagaacatag cgttgccttg gtaggtccag cggcggagga actctttgat 3781 ccggttcctg aacaggatct atttgaggcg ctaaatgaaa ccttaacgct atggaactcg 3841 ccgcccgact gggctggcga tgagcgaaat gtagtgctta cgttgtcccg catttggtac 3901 agcgcagtaa ccggcaaaat cgcgccgaag gatgtcgctg ccgactgggc aatggagcgc 3961 ctgccggccc agtatcagcc cgtcatactt gaagctagac aggcttatct tggacaagaa 4021 gaagatcgct tggcctcgcg cgcagatcag ttggaagaat ttgtccacta cgtgaaaggc 4081 gagatcacca aggtagtcgg caaataattc gaaacgcgtt gcaagggcga attggaaggg 4141 cgaattgtaa ccgcgtttca gctggcacga caggtttccc gactggaaag cgggcagtga 4201 gcgcaacgca attaatgtaa gttagcgcga attgatctgg tttgacagct tatcatcgac 4261 tgcacggtgc accaatgctt ctggcgtcag gcagccatcg gaagctgtgg tatggctgtg 4321 caggtcgtaa atcactgcat aattcgtgtc gctcaaggcg cactcccgtt ctggataatg 4381 ttttttgcgc cgacatcata acggttctgg caaatattct gaatgagctg tacaattaat 4441 catcggctcg tataatgatt ccatctgcac tagccaccgc t //