LOCUS       sequence-210673-        1122 bp    DNA     linear   UNA 15-DEC-2025
DEFINITION  Expresses sgRNA in barley.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      natural DNA sequence
  ORGANISM  unspecified
REFERENCE   1  (bases 1 to 1122)
  AUTHORS   Lawrenson T, Harwood WA
  TITLE     Creating Targeted Gene Knockouts in Barley Using CRISPR/Cas9.
  JOURNAL   Methods Mol Biol. 2019;1900:217-232. doi: 
            10.1007/978-1-4939-8944-7_14.
  PUBMED    30460568
REFERENCE   2  (bases 1 to 1122)
  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: "1900:217-232. doi"; pages: " 
            10.1007/978-1-4939-8944-7_14"
COMMENT     Sequence Label: pHvL1P3GA
FEATURES             Location/Qualifiers
     source          1..1122
                     /mol_type="genomic DNA"
                     /organism="unspecified"
     protein_bind    447..468
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     promoter        join(483..488,489..506,507..513)
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    521..537
                     /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     526..548
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     primer_bind     545..561
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     545..561
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     CDS             557..880
                     /codon_start=1
                     /gene="lacZ fragment"
                     /product="LacZ-alpha fragment of beta-galactosidase"
                     /label=lacZ-alpha
                     /translation="MTMITPSLHACRSTLEDPRVPSSNSLAVVLQRRDWENPGVTQLNR
                     LAAHPPFASWRNSEEARTDRPSQQLRSLNGEWRLMRYFLLTHLCGISHRIWCTLSTICS
                     DAA"
     misc_feature    574..630
                     /label=MCS
                     /note="pUC18/19 multiple cloning site"
     primer_bind     complement(631..648)
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     complement(631..647)
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(640..662)
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     primer_bind     complement(856..875)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     misc_RNA        977..1052
                     /label=gRNA scaffold
                     /note="guide RNA scaffold for the Streptococcus pyogenes 
                     CRISPR/Cas9 system"
     protein_bind    complement(1064..1088)
                     /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 agaccaagcc cgttattctg acagttctgg tgctcaacac
       61 atttatattt atcaaggagc acattgttac tcactgctag gagggaatcg aactaggaat
      121 attgatcaga ggaactacga gagagctgaa gataactgcc ctctagctct cactgatctg
      181 ggtcgcatag tgagatgcag cccacgtgag ttcagcaacg gtctagcgct gggcttttag
      241 gcccgcatga tcgggctttt gtcgggtggt cgacgtgttc acgattgggg agagcaacgc
      301 agcagttcct cttagtttag tcccacctcg cctgtccagc agagttctga ccggtttata
      361 aactcgcttg ctgcatcaga cttgtgagac cgcagctggc acgacaggtt tcccgactgg
      421 aaagcgggca gtgagcgcaa cgcaattaat gtgagttagc tcactcatta ggcaccccag
      481 gctttacact ttatgcttcc ggctcgtatg ttgtgtggaa ttgtgagcgg ataacaattt
      541 cacacaggaa acagctatga ccatgattac gccaagcttg catgcctgca ggtcgactct
      601 agaggatccc cgggtaccga gctcgaattc actggccgtc gttttacaac gtcgtgactg
      661 ggaaaaccct ggcgttaccc aacttaatcg ccttgcagca catccccctt tcgccagctg
      721 gcgtaatagc gaagaggccc gcaccgatcg cccttcccaa cagttgcgca gcctgaatgg
      781 cgaatggcgc ctgatgcggt attttctcct tacgcatctg tgcggtattt cacaccgcat
      841 atggtgcact ctcagtacaa tctgctctga tgccgcatag ttaagccagc cccgacaccc
      901 gccaacaccc gctgacgcgc cctgacgggc ttgtctgctc ccggcatccg cttacagaca
      961 agctgtgacg gtctcagttt tagagctaga aatagcaagt taaaataagg ctagtccgtt
     1021 atcaacttga aaaagtggca ccgagtcggt gctttttttc tagacccagc tttcttgtac
     1081 aaagttggca ttacgcttta cttacaagtc ttccacgaag tg
//
