LOCUS       sequence-119201-        4718 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Level 1 Golden Gate Cassette: sgRNA cassette targeting BolC.GA4a-2 
            in Brassica oleracea.
ACCESSION   .
VERSION     .
KEYWORDS    .
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 Dec 15, 2025 from SnapGene Server 8.0.1
            https://www.snapgene.com
COMMENT     SGRef: number: 1; type: "Journal Article"; journalName: "Genome 
            Biol."; date: "2015-11-30"; volume: "16(1):258. doi"; pages: " 
            10.1186/s13059-015-0826-7"
COMMENT     Sequence Label: pICSL11062
FEATURES             Location/Qualifiers
     source          1..4718
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     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(join(2815..3606,3607..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
//
