LOCUS       sequence-151818-        1009 bp    DNA     linear   UNA 15-DEC-2025
DEFINITION  tRNA and scaffold for the assembly of GBoligomers for the 
            intermediate position (positon [2_n-1]) of a polycistronic tRNA-gRNA
            (3-part multiplexing).
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      natural DNA sequence
  ORGANISM  unspecified
REFERENCE   1  (bases 1 to 1009)
  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 1009)
  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 [2_n-1] (GB1206)
FEATURES             Location/Qualifiers
     source          1..1009
                     /mol_type="genomic DNA"
                     /organism="unspecified"
     regulatory      2..11
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     regulatory      6..15
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     misc_RNA        146..221
                     /label=gRNA scaffold
                     /note="guide RNA scaffold for the Streptococcus pyogenes 
                     CRISPR/Cas9 system"
     promoter        complement(316..334)
                     /label=SP6 promoter
                     /note="promoter for bacteriophage SP6 RNA polymerase"
     primer_bind     complement(317..334)
                     /label=SP6
                     /note="SP6 promoter, forward primer"
     primer_bind     complement(352..368)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(352..368)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(365..387)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    376..392
                     /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(400..406,407..424,425..430))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    445..466
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(583..600)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     primer_bind     complement(834..853)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
ORIGIN
        1 ggccgccatg gcggccgcgg gaattcgatt cccgtctcgc tcggtgcaac aaagcaccag
       61 tggtctagtg gtagaatagt accctgccac ggtacagacc cgggttcgat tcccggctgg
      121 tgcagagacg gcgcgcgccg tctcagtttt agagctagaa atagcaagtt aaaataaggc
      181 tagtccgtta tcaacttgaa aaagtggcac cgagtcggtg caatgagcga gacgggatca
      241 ctagtgaatt cgcggccgcc tgcaggtcga ccatatggga gagctcccaa cgcgttggat
      301 gcatagcttg agtattctat agtgtcacct aaatagcttg gcgtaatcat ggtcatagct
      361 gtttcctgtg tgaaattgtt atccgctcac aattccacac aacatacgag ccggaagcat
      421 aaagtgtaaa gcctggggtg cctaatgagt gagctaactc acattaattg cgttgcgctc
      481 actgcccgct ttccagtcgg gaaacctgtc gtgccagctg cattaatgaa tcggccaacg
      541 cgcggggaga ggcggtttgc gtattgggcg ctcttccgct tcctcgctca ctgactcgct
      601 gcgctcggtc gttcggctgc ggcgagcggt atcagctcac tcaaaggcgg taatacggtt
      661 atccacagaa tcaggggata acgcaggaaa gaacatgtga gcaaaaggcc agcaaaaggc
      721 caggaaccgt aaaaaggccg cgttgctggc gtttttccat aggctccgcc cccctgacga
      781 gcatcacaaa aatcgacgct caagtcagag gtggcgaaac ccgacaggac tataaagata
      841 ccaggcgttt ccccctggaa gctccctcgt gcgctctcct gttccgaccc tgccgcttac
      901 cggatacctg tccgcctttc tcccttcggg aagcgtggcg ctttctcata gctcacgctg
      961 taggtatctc agttcggtgt aggtcgttcg ctccaagctg ggctgtgtg
//
