LOCUS Exported 4718 bp ds-DNA circular SYN 13-MAY-2021 DEFINITION Level 1 Golden Gate Cassette: sgRNA cassette targeting BolC.GA4a-2 in Brassica oleracea. ACCESSION . VERSION . KEYWORDS pICSL11062 SOURCE synthetic DNA construct ORGANISM synthetic DNA construct REFERENCE 1 (bases 1 to 4718) AUTHORS Lawrenson T, Shorinola O, Stacey N, Li C, Ostergaard L, Patron N, Uauy C, Harwood W TITLE Induction of targeted, heritable mutations in barley and Brassica oleracea using RNA-guided Cas9 nuclease. JOURNAL Genome Biol. 2015 Nov 30;16(1):258. doi: 10.1186/s13059-015-0826-7. PUBMED 26616834 REFERENCE 2 (bases 1 to 4718) AUTHORS . TITLE Direct Submission JOURNAL Exported May 13, 2021 from SnapGene Server 1.1.58 http://www.snapgene.com FEATURES Location/Qualifiers source 1..4718 /organism="synthetic DNA construct" /mol_type="other DNA" misc_RNA 230..305 /label=gRNA scaffold /note="guide RNA scaffold for the Streptococcus pyogenes CRISPR/Cas9 system" protein_bind complement(317..341) /gene="mutant version of attB" /label=attB2 /bound_moiety="BP Clonase(TM)" /note="recombination site for the Gateway(R) BP reaction" misc_feature 392..416 /label=RB T-DNA repeat /note="right border repeat from nopaline C58 T-DNA" CDS complement(572..1720) /codon_start=1 /product="trans-acting replication protein that binds to and activates oriV" /label=trfA /translation="MNRTFDRKAYRQELIDAGFSAEDAETIASRTVMRAPRETFQSVGS MVQQATAKIERDSVQLAPPALPAPSAAVERSRRLEQEAAGLAKSMTIDTRGTMTTKKRK TAGEDLAKQVSEAKQAALLKHTKQQIKEMQLSLFDIAPWPDTMRAMPNDTARSALFTTR NKKIPREALQNKVIFHVNKDVKITYTGVELRADDDELVWQQVLEYAKRTPIGEPITFTF YELCQDLGWSINGRYYTKAEECLSRLQATAMGFTSDRVGHLESVSLLHRFRVLDRGKKT SRCQVLIDEEIVVLFAGDHYTKFIWEKYRKLSPTARRMFDYFSSHREPYPLKLETFRLM CGSDSTRVKKWREQVGEACEELRGSGLVEHAWVNDDLVHCKR" rep_origin complement(2056..2644) /direction=LEFT /label=ori /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of replication" primer_bind complement(2136..2155) /label=pBR322ori-F /note="pBR322 origin, forward primer" CDS complement(2815..3675) /codon_start=1 /gene="bla" /product="beta-lactamase" /label=AmpR /note="confers resistance to ampicillin, carbenicillin, and related antibiotics" /translation="MSIQHFRVALIPFFAAFCLPVFAHPETLVKVKDAEDQLGARVGYI ELDLNSGKILESFRPEERFPMMSTFKVLLCGAVLSRIDAGQEQLGRRIHYSQNDLVEYS PVTEKHLTDGMTVRELCSAAITMSDNTAANLLLTTIGGPKELTAFLHNMGDHVTRLDRW EPELNEAIPNDERDTTMPVAMATTLRKLLTGELLTLASRQQLIDWMEADKVAGPLLRSA LPAGWFIADKSGAGERGSRGIIAALGPDGKPSRIVVIYTTGSQATMDERNRQIAEIGAS LIKHW" primer_bind 3438..3457 /label=Amp-R /note="Ampicillin resistance gene, reverse primer" promoter complement(3676..3780) /gene="bla" /label=AmpR promoter rep_origin 3867..4498 /label=oriV /note="incP origin of replication" misc_feature 4590..4614 /label=LB T-DNA repeat /note="left border repeat from nopaline C58 T-DNA" ORIGIN 1 ggagcatctt cattcttaag atatgaagat aatcttcaaa aggcccctgg gaatctgaaa 61 gaagagaagc aggcccattt atatgggaaa gaacaatagt atttcttata taggcccatt 121 taagttgaaa acaatcttca aaagtcccac atcgcttaga taagaaaacg aagctgagtt 181 tatatacagc tagagtcgaa gtagtgattg ttgagagggg agccggtgag ttttagagct 241 agaaatagca agttaaaata aggctagtcc gttatcaact tgaaaaagtg gcaccgagtc 301 ggtgcttttt ttctagaccc agctttcttg tacaaagttg gcattacgct cagattgtct 361 tctgcacgaa gtggtttaaa ctatcagtgt ttgacaggat atattggcgg gtaaacctaa 421 gagaaaagag cgtttattag aataatcgga tatttaaaag ggcgtgaaaa ggtttatccg 481 ttcgtccatt tgtatgtgca tgccaaccac agggttcccc agatcaggcg ctggctgctg 541 aacccccagc cggaactgac cccacaaggc cctagcgttt gcaatgcacc aggtcatcat 601 tgacccaggc gtgttccacc aggccgctgc ctcgcaactc ttcgcaggct tcgccgacct 661 gctcgcgcca cttcttcacg cgggtggaat ccgatccgca catgaggcgg aaggtttcca 721 gcttgagcgg gtacggctcc cggtgcgagc tgaaatagtc gaacatccgt cgggccgtcg 781 gcgacagctt gcggtacttc tcccatatga atttcgtgta gtggtcgcca gcaaacagca 841 cgacgatttc ctcgtcgatc aggacctggc aacgggacgt tttcttgcca cggtccagga 901 cgcggaagcg gtgcagcagc gacaccgatt ccaggtgccc aacgcggtcg gacgtgaagc 961 ccatcgccgt cgcctgtagg cgcgacaggc attcctcggc cttcgtgtaa taccggccat 1021 tgatcgacca gcccaggtcc tggcaaagct cgtagaacgt gaaggtgatc ggctcgccga 1081 taggggtgcg cttcgcgtac tccaacacct gctgccacac cagttcgtca tcgtcggccc 1141 gcagctcgac gccggtgtag gtgatcttca cgtccttgtt gacgtggaaa atgaccttgt 1201 tttgcagcgc ctcgcgcggg attttcttgt tgcgcgtggt gaacagggca gagcgggccg 1261 tgtcgtttgg catcgctcgc atcgtgtccg gccacggcgc aatatcgaac aaggaaagct 1321 gcatttcctt gatctgctgc ttcgtgtgtt tcagcaacgc ggcctgcttg gcctcgctga 1381 cctgttttgc caggtcctcg ccggcggttt ttcgcttctt ggtcgtcata gttcctcgcg 1441 tgtcgatggt catcgacttc gccaaacctg ccgcctcctg ttcaagacga cgcgaacgct 1501 ccacggcggc cgatggcgcg ggcagggcag ggggagccag ttgcacgctg tcgcgctcga 1561 tcttggccgt agcttgctgg accatcgagc cgacggactg gaaggtttcg cggggcgcac 1621 gcatgacggt gcggcttgcg atggtttcgg catcctcggc ggaaaacccc gcgtcgatca 1681 gttcttgcct gtatgccttc cggtcaaacg tccgattcat tcaccctcct tgcgggattg 1741 ccccgactca cgccggggca atgtgccctt attcctgatt tgacccgcct ggtgccttgg 1801 tgtccagata atccacctta tcggcaatga agtcggtccc gtagaccgtc tggccgtcct 1861 tctcgtactt ggtattccga atcttgccct gcacgaatac cagcgacccc ttgcccaaat 1921 acttgccgtg ggcctcggcc tgagagccaa aacacttgat gcggaagaag tcggtgcgct 1981 cctgcttgtc gccggcatcg ttgcgccaca tctaggatct gccaggaacc gtaaaaaggc 2041 cgcgttgctg gcgtttttcc ataggctccg cccccctgac gagcatcaca aaaatcgacg 2101 ctcaagtcag aggtggcgaa acccgacagg actataaaga taccaggcgt ttccccctgg 2161 aagctccctc gtgcgctctc ctgttccgac cctgccgctt accggatacc tgtccgcctt 2221 tctcccttcg ggaagcgtgg cgctttctca tagctcacgc tgtaggtatc tcagttcggt 2281 gtaggtcgtt cgctccaagc tgggctgtgt gcacgaaccc cccgttcagc ccgaccgctg 2341 cgccttatcc ggtaactatc gtcttgagtc caacccggta agacacgact tatcgccact 2401 ggcagcagcc actggtaaca ggattagcag agcgaggtat gtaggcggtg ctacagagtt 2461 cttgaagtgg tggcctaact acggctacac tagaagaaca gtatttggta tctgcgctct 2521 gctgaagcca gttaccttcg gaaaaagagt tggtagctct tgatccggca aacaaaccac 2581 cgctggtagc ggtggttttt ttgtttgcaa gcagcagatt acgcgcagaa aaaaaggatc 2641 tcaagaagat cctttgatct tttctacggg gtctgacgct cagtggaacg aaaactcacg 2701 ttaagggatt ttggtcatga gattatcaaa aaggatcttc acctagatcc ttttaaatta 2761 aaaatgaagt tttaaatcaa tctaaagtat atatgagtaa acttggtctg acagttacca 2821 atgcttaatc agtgaggcac ctatctcagc gatctgtcta tttcgttcat ccatagttgc 2881 ctgactcccc gtcgtgtaga taactacgat acgggagggc ttaccatctg gccccagtgc 2941 tgcaatgata ccgcgagaac cacgctcacc ggctccagat ttatcagcaa taaaccagcc 3001 agccggaagg gccgagcgca gaagtggtcc tgcaacttta tccgcctcca tccagtctat 3061 taattgttgc cgggaagcta gagtaagtag ttcgccagtt aatagtttgc gcaacgttgt 3121 tgccattgct acaggcatcg tggtgtcacg ctcgtcgttt ggtatggctt cattcagctc 3181 cggttcccaa cgatcaaggc gagttacatg atcccccatg ttgtgcaaaa aagcggttag 3241 ctccttcggt cctccgatcg ttgtcagaag taagttggcc gcagtgttat cactcatggt 3301 tatggcagca ctgcataatt ctcttactgt catgccatcc gtaagatgct tttctgtgac 3361 tggtgagtac tcaaccaagt cattctgaga atagtgtatg cggcgaccga gttgctcttg 3421 cccggcgtca atacgggata ataccgcgcc acatagcaga actttaaaag tgctcatcat 3481 tggaaaacgt tcttcggggc gaaaactctc aaggatctta ccgctgttga gatccagttc 3541 gatgtaaccc actcgtgcac ccaactgatc ttcagcatct tttactttca ccagcgtttc 3601 tgggtgagca aaaacaggaa ggcaaaatgc cgcaaaaaag ggaataaggg cgacacggaa 3661 atgttgaata ctcatactct tcctttttca atattattga agcatttatc aaggttattg 3721 tctcatgagc ggatacatat ttgaatgtat ttagaaaaat aaacaaatag gggttccgcg 3781 cacgaattgg ccagcgctgc catttttggg gtgaggccgt tcgcggccga ggggcgcagc 3841 ccctgggggg atgggaggcc cgcgttagcg ggccgggagg gttcgagaag ggggggcacc 3901 ccccttcggc gtgcgcggtc acgcgcacag ggcgcagccc tggttaaaaa caaggtttat 3961 aaatattggt ttaaaagcag gttaaaagac aggttagcgg tggccgaaaa acgggcggaa 4021 acccttgcaa atgctggatt ttctgcctgt ggacagcccc tcaaatgtca ataggtgcgc 4081 ccctcatctg tcagcactct gcccctcaag tgtcaaggat cgcgcccctc atctgtcagt 4141 agtcgcgccc ctcaagtgtc aataccgcag ggcacttatc cccaggcttg tccacatcat 4201 ctgtgggaaa ctcgcgtaaa atcaggcgtt ttcgccgatt tgcgaggctg gccagctcca 4261 cgtcgccggc cgaaatcgag cctgcccctc atctgtcaac gccgcgccgg gtgagtcggc 4321 ccctcaagtg tcaacgtccg cccctcatct gtcagtgagg gccaagtttt ccgcgaggta 4381 tccacaacgc cggcggccgc ggtgtctcgc acacggcttc gacggcgttt ctggcgcgtt 4441 tgcagggcca tagacggccg ccagcccagc ggcgagggca accagcccgg tgagcgtcgc 4501 aaaggagatc ctgatctgac tgatgggctg cctgtatcga gtggtgattt tgtgccgagc 4561 tgccggtcgg ggagctgttg gctggctggt ggcaggatat attgtggtgt aaacaaattg 4621 acgcttagac aacttaataa cacattgcgg acgtttttaa tgtactgggg tggatgcagt 4681 gggccccact ctgtgaagac aattacgaat tcccatgg //