LOCUS       sequence-152039-         966 bp    DNA     linear   UNA 15-DEC-2025
DEFINITION  tRNA and scaffold for the assembly of GBoligomers for the first 
            position (positon [M1_2]) of a polycistronic tRNA-gRNA regulated by 
            the (monocot) U3 promoter (3-part multiplexing).
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      natural DNA sequence
  ORGANISM  unspecified
REFERENCE   1  (bases 1 to 966)
  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 966)
  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 [M1_2] (GB1209)
FEATURES             Location/Qualifiers
     source          1..966
                     /mol_type="genomic DNA"
                     /organism="unspecified"
     regulatory      7..16
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     regulatory      11..20
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     misc_RNA        complement(49..124)
                     /label=gRNA scaffold
                     /note="guide RNA scaffold for the Streptococcus pyogenes 
                     CRISPR/Cas9 system"
     promoter        complement(319..337)
                     /label=SP6 promoter
                     /note="promoter for bacteriophage SP6 RNA polymerase"
     primer_bind     complement(320..337)
                     /label=SP6
                     /note="SP6 promoter, forward primer"
     primer_bind     complement(355..371)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(355..371)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(368..390)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    379..395
                     /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(403..409,410..427,428..433))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    448..469
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(586..603)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     primer_bind     complement(837..856)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
ORIGIN
        1 ctcccggccg ccatggcggc cgcgggaatt cgattcccgt ctcgctcagc accgactcgg
       61 tgccactttt tcaagttgat aacggactag ccttatttta acttgctatt tctagctcta
      121 aaactgagac ggcgcgcgcc gtctctgcac cagccgggaa tcgaacccgg gtctgtaccg
      181 tggcagggta ctattctacc actagaccac tggtgctttg tttgcccgag cgagacggga
      241 tcactagtga attcgcggcc gcctgcaggt cgaccatatg ggagagctcc caacgcgttg
      301 gatgcatagc ttgagtattc tatagtgtca cctaaatagc ttggcgtaat catggtcata
      361 gctgtttcct gtgtgaaatt gttatccgct cacaattcca cacaacatac gagccggaag
      421 cataaagtgt aaagcctggg gtgcctaatg agtgagctaa ctcacattaa ttgcgttgcg
      481 ctcactgccc gctttccagt cgggaaacct gtcgtgccag ctgcattaat gaatcggcca
      541 acgcgcgggg agaggcggtt tgcgtattgg gcgctcttcc gcttcctcgc tcactgactc
      601 gctgcgctcg gtcgttcggc tgcggcgagc ggtatcagct cactcaaagg cggtaatacg
      661 gttatccaca gaatcagggg ataacgcagg aaagaacatg tgagcaaaag gccagcaaaa
      721 ggccaggaac cgtaaaaagg ccgcgttgct ggcgtttttc cataggctcc gcccccctga
      781 cgagcatcac aaaaatcgac gctcaagtca gaggtggcga aacccgacag gactataaag
      841 ataccaggcg tttccccctg gaagctccct cgtgcgctct cctgttccga ccctgccgct
      901 taccggatac ctgtccgcct ttctcccttc gggaagcgtg gcgctttctc atagctcacg
      961 ctgtag
//
