LOCUS       sequence-151821-        1030 bp    DNA     linear   UNA 15-DEC-2025
DEFINITION  tRNA and scaffold for the assembly of GBoligomers for the last 
            position (positon [n]) of a polycistronic tRNA-gRNA (2- and 3-part 
            multiplexing).
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      natural DNA sequence
  ORGANISM  unspecified
REFERENCE   1  (bases 1 to 1030)
  AUTHORS   Vazquez-Vilar M, Bernabe-Orts JM, Fernandez-Del-Carmen A, Ziarsolo 
            P, Blanca J, Granell A, Orzaez D
  TITLE     A modular toolbox for gRNA-Cas9 genome engineering in plants based 
            on the GoldenBraid standard.
  JOURNAL   Plant Methods. 2016 Feb 1;12:10. doi: 10.1186/s13007-016-0101-2. 
            eCollection 2016.
  PUBMED    26839579
REFERENCE   2  (bases 1 to 1030)
  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: "Plant 
            Methods."; date: "2016-02-1"; volume: "12:10. doi"; pages: " 
            10.1186/s13007-016-0101-2. eCollection 2016"
COMMENT     Sequence Label: tRNA-gRNA position [n] (GB1207)
FEATURES             Location/Qualifiers
     source          1..1030
                     /mol_type="genomic DNA"
                     /organism="unspecified"
     regulatory      6..15
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     regulatory      10..19
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     misc_RNA        148..223
                     /label=gRNA scaffold
                     /note="guide RNA scaffold for the Streptococcus pyogenes 
                     CRISPR/Cas9 system"
     protein_bind    complement(235..259)
                     /gene="mutant version of attB"
                     /label=attB2
                     /bound_moiety="BP Clonase(TM)"
                     /note="recombination site for the Gateway(R) BP reaction"
     promoter        complement(361..379)
                     /label=SP6 promoter
                     /note="promoter for bacteriophage SP6 RNA polymerase"
     primer_bind     complement(362..379)
                     /label=SP6
                     /note="SP6 promoter, forward primer"
     primer_bind     complement(397..413)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(397..413)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(410..432)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    421..437
                     /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)."
     promoter        complement(join(445..451,452..469,470..475))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    490..511
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(628..645)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     primer_bind     complement(879..898)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
ORIGIN
        1 tcccggccgc catggcggcc gcgggaattc gattcccgtc tcgctcggca acaaagcacc
       61 agtggtctag tggtagaata gtaccctgcc acggtacaga cccgggttcg attcccggct
      121 ggtgcagaga cggcgcgcgc cgtctcagtt ttagagctag aaatagcaag ttaaaataag
      181 gctagtccgt tatcaacttg aaaaagtggc accgagtcgg tgcttttttt ctagacccag
      241 ctttcttgta caaagttggc attacgcttg agcgagacgg gatcactagt gaattcgcgg
      301 ccgcctgcag gtcgaccata tgggagagct cccaacgcgt tggatgcata gcttgagtat
      361 tctatagtgt cacctaaata gcttggcgta atcatggtca tagctgtttc ctgtgtgaaa
      421 ttgttatccg ctcacaattc cacacaacat acgagccgga agcataaagt gtaaagcctg
      481 gggtgcctaa tgagtgagct aactcacatt aattgcgttg cgctcactgc ccgctttcca
      541 gtcgggaaac ctgtcgtgcc agctgcatta atgaatcggc caacgcgcgg ggagaggcgg
      601 tttgcgtatt gggcgctctt ccgcttcctc gctcactgac tcgctgcgct cggtcgttcg
      661 gctgcggcga gcggtatcag ctcactcaaa ggcggtaata cggttatcca cagaatcagg
      721 ggataacgca ggaaagaaca tgtgagcaaa aggccagcaa aaggccagga accgtaaaaa
      781 ggccgcgttg ctggcgtttt tccataggct ccgcccccct gacgagcatc acaaaaatcg
      841 acgctcaagt cagaggtggc gaaacccgac aggactataa agataccagg cgtttccccc
      901 tggaagctcc ctcgtgcgct ctcctgttcc gaccctgccg cttaccggat acctgtccgc
      961 ctttctccct tcgggaagcg tggcgctttc tcatagctca cgctgtaggt atctcagttc
     1021 ggtgtaggtc
//
