LOCUS       sequence-212866-         965 bp    DNA     linear   UNA 15-DEC-2025
DEFINITION  Express a cloned sgRNA from AtU626 promoter.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      natural DNA sequence
  ORGANISM  unspecified
REFERENCE   1  (bases 1 to 965)
  AUTHORS   Lawrenson T, Hundleby P, Harwood W
  TITLE     Creating Targeted Gene Knockouts in Brassica oleracea Using 
            CRISPR/Cas9.
  JOURNAL   Methods Mol Biol. 2019;1917:155-170. doi: 
            10.1007/978-1-4939-8991-1_12.
  PUBMED    30610635
REFERENCE   2  (bases 1 to 965)
  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: "Methods Mol
            Biol."; date: "2019"; volume: "1917:155-170. doi"; pages: " 
            10.1007/978-1-4939-8991-1_12"
COMMENT     Sequence Label: pBoL1P3GA
FEATURES             Location/Qualifiers
     source          1..965
                     /mol_type="genomic DNA"
                     /organism="unspecified"
     protein_bind    290..311
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     promoter        join(326..331,332..349,350..356)
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    364..380
                     /label=lac operator
                     /bound_moiety="lac repressor encoded by lacI"
                     /note="The lac repressor binds to the lac operator to 
                     inhibit transcription in E. coli. This inhibition can be 
                     relieved by adding lactose or 
                     isopropyl-beta-D-thiogalactopyranoside (IPTG)."
     primer_bind     369..391
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     primer_bind     388..404
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     388..404
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     CDS             400..723
                     /codon_start=1
                     /gene="lacZ fragment"
                     /product="LacZ-alpha fragment of beta-galactosidase"
                     /label=lacZ-alpha
                     /translation="MTMITPSLHACRSTLEDPRVPSSNSLAVVLQRRDWENPGVTQLNR
                     LAAHPPFASWRNSEEARTDRPSQQLRSLNGEWRLMRYFLLTHLCGISHRIWCTLSTICS
                     DAA"
     misc_feature    417..473
                     /label=MCS
                     /note="pUC18/19 multiple cloning site"
     primer_bind     complement(474..491)
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     complement(474..490)
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(483..505)
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     primer_bind     complement(699..718)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     misc_RNA        820..895
                     /label=gRNA scaffold
                     /note="guide RNA scaffold for the Streptococcus pyogenes 
                     CRISPR/Cas9 system"
     protein_bind    complement(907..931)
                     /gene="mutant version of attB"
                     /label=attB2
                     /bound_moiety="BP Clonase(TM)"
                     /note="recombination site for the Gateway(R) BP reaction"
ORIGIN
        1 cactctgtgg aagacaaact acatcttcat tcttaagata tgaagataat cttcaaaagg
       61 cccctgggaa tctgaaagaa gagaagcagg cccatttata tgggaaagaa caatagtatt
      121 tcttatatag gcccatttaa gttgaaaaca atcttcaaaa gtcccacatc gcttagataa
      181 gaaaacgaag ctgagtttat atacagctag agtcgaagta gtgattgtga gaccgcagct
      241 ggcacgacag gtttcccgac tggaaagcgg gcagtgagcg caacgcaatt aatgtgagtt
      301 agctcactca ttaggcaccc caggctttac actttatgct tccggctcgt atgttgtgtg
      361 gaattgtgag cggataacaa tttcacacag gaaacagcta tgaccatgat tacgccaagc
      421 ttgcatgcct gcaggtcgac tctagaggat ccccgggtac cgagctcgaa ttcactggcc
      481 gtcgttttac aacgtcgtga ctgggaaaac cctggcgtta cccaacttaa tcgccttgca
      541 gcacatcccc ctttcgccag ctggcgtaat agcgaagagg cccgcaccga tcgcccttcc
      601 caacagttgc gcagcctgaa tggcgaatgg cgcctgatgc ggtattttct ccttacgcat
      661 ctgtgcggta tttcacaccg catatggtgc actctcagta caatctgctc tgatgccgca
      721 tagttaagcc agccccgaca cccgccaaca cccgctgacg cgccctgacg ggcttgtctg
      781 ctcccggcat ccgcttacag acaagctgtg acggtctcag ttttagagct agaaatagca
      841 agttaaaata aggctagtcc gttatcaact tgaaaaagtg gcaccgagtc ggtgcttttt
      901 ttctagaccc agctttcttg tacaaagttg gcattacgct ttacttacaa gtcttccacg
      961 aagtg
//
