LOCUS       sequence-210676-        1605 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 1605)
  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 1605)
  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: pHvL1P4GA
FEATURES             Location/Qualifiers
     source          1..1605
                     /mol_type="genomic DNA"
                     /organism="unspecified"
     protein_bind    488..509
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     promoter        join(524..529,530..547,548..554)
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    562..578
                     /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)."
     RBS             596..607
                     /note="strong bacterial ribosome binding site (Elowitz and 
                     Leibler, 2000)"
     CDS             614..1291
                     /codon_start=1
                     /product="monomeric derivative of DsRed (Campbell et al., 
                     2002)"
                     /label=mRFP1
                     /translation="MASSEDVIKEFMRFKVRMEGSVNGHEFEIEGEGEGRPYEGTQTAK
                     LKVTKGGPLPFAWDILSPQFQYGSKAYVKHPADIPDYLKLSFPEGFKWERVMNFEDGGV
                     VTVTQDSSLQDGEFIYKVKLRGTNFPSDGPVMQKKTMGWEASTERMYPEDGALKGEIKM
                     RLKLKDGGHYDAEVKTTYMAKKPVQLPGAYKTDIKLDITSHNEDYTIVEQYERAEGRHS
                     TGA"
     terminator      1336..1407
                     /label=rrnB T1 terminator
                     /note="transcription terminator T1 from the E. coli rrnB 
                     gene"
     terminator      1423..1450
                     /label=T7Te terminator
                     /note="phage T7 early transcription terminator"
     protein_bind    complement(1547..1571)
                     /gene="mutant version of attB"
                     /label=attB2
                     /bound_moiety="BP Clonase(TM)"
                     /note="recombination site for the Gateway(R) BP reaction"
ORIGIN
        1 cactctgtgg aagacaatta cgaccaagcc 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 cgagacgcaa tacgcaaacc gcctctcccc gcgcgttggc
      421 cgattcatta atgcagctgg cacgacaggt ttcccgactg gaaagcgggc agtgagcgca
      481 acgcaattaa tgtgagttag ctcactcatt aggcacccca ggctttacac tttatgcttc
      541 cggctcgtat gttgtgtgga attgtgagcg gataacaatt tcacacatac tagagaaaga
      601 ggagaaatac tagatggctt cctccgagga cgttatcaaa gagttcatgc gtttcaaagt
      661 tcgtatggaa ggttccgtta acggtcacga gttcgaaatc gaaggtgaag gtgaaggtcg
      721 tccgtacgaa ggtacccaga ccgctaaact gaaagttacc aaaggtggtc cgctgccgtt
      781 cgcttgggac atcctgtccc cgcagttcca gtacggttcc aaagcttacg ttaaacaccc
      841 ggctgacatc ccggactacc tgaaactgtc cttcccggaa ggtttcaaat gggaacgtgt
      901 tatgaacttc gaggacggtg gtgttgttac cgttacccag gactcctccc tgcaagacgg
      961 tgagttcatc tacaaagtta aactgcgtgg taccaacttc ccgtccgacg gtccggttat
     1021 gcagaaaaaa accatgggtt gggaagcttc caccgaacgt atgtacccgg aggacggtgc
     1081 tctgaaaggt gaaatcaaaa tgcgtctgaa actgaaagac ggtggtcact acgacgctga
     1141 agttaaaacc acctacatgg ctaaaaaacc ggttcagctg ccgggtgctt acaaaaccga
     1201 catcaaactg gacatcacct cccacaacga ggactacacc atcgttgaac agtacgaacg
     1261 tgctgaaggt cgtcactcca ccggtgctta ataacgctga tagtgctagt gtagatcgct
     1321 actagagcca ggcatcaaat aaaacgaaag gctcagtcga aagactgggc ctttcgtttt
     1381 atctgttgtt tgtcggtgaa cgctctctac tagagtcaca ctggctcacc ttcgggtggg
     1441 cctttctgcg tttatacgtc tcttagagct agaaatagca agttaaaata aggctagtcc
     1501 gttatcaact tgaaaaagtg gcaccgagtc ggtgcttttt ttctagaccc agctttcttg
     1561 tacaaagttg gcattacgct ttacgggaaa gtcttccacg aagtg
//
