LOCUS       sequence-146009-        3263 bp    DNA     circular SYN 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      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 3263)
  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 3263)
  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..3263
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     misc_RNA        109..184
                     /label=gRNA scaffold
                     /note="guide RNA scaffold for the Streptococcus pyogenes 
                     CRISPR/Cas9 system"
     protein_bind    complement(196..220)
                     /gene="mutant version of attB"
                     /label=attB2
                     /bound_moiety="BP Clonase(TM)"
                     /note="recombination site for the Gateway(R) BP reaction"
     promoter        complement(322..340)
                     /label=SP6 promoter
                     /note="promoter for bacteriophage SP6 RNA polymerase"
     primer_bind     complement(323..340)
                     /label=SP6
                     /note="SP6 promoter, forward primer"
     primer_bind     complement(358..374)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(358..374)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(371..393)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    382..398
                     /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(406..412,413..430,431..436))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    451..472
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(589..606)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(760..1348)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(840..859)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(1519..2310,2311..2379))
                     /codon_start=1
                     /gene="bla"
                     /product="beta-lactamase"
                     /label=AmpR
                     /note="confers resistance to ampicillin, carbenicillin, and
                     related antibiotics"
                     /translation="MSIQHFRVALIPFFAAFCLPVFAHPETLVKVKDAEDQLGARVGYI
                     ELDLNSGKILESFRPEERFPMMSTFKVLLCGAVLSRIDAGQEQLGRRIHYSQNDLVEYS
                     PVTEKHLTDGMTVRELCSAAITMSDNTAANLLLTTIGGPKELTAFLHNMGDHVTRLDRW
                     EPELNEAIPNDERDTTMPVAMATTLRKLLTGELLTLASRQQLIDWMEADKVAGPLLRSA
                     LPAGWFIADKSGAGERGSRGIIAALGPDGKPSRIVVIYTTGSQATMDERNRQIAEIGAS
                     LIKHW"
     primer_bind     2142..2161
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(2380..2484)
                     /gene="bla"
                     /label=AmpR promoter
     rep_origin      complement(2562..3017)
                     /direction=LEFT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(2699..2720)
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     2911..2930
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     3143..3165
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     primer_bind     3157..3174
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     3158..3174
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     3181..3200
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        3181..3199
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     regulatory      3230..3239
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     regulatory      3234..3243
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
ORIGIN
        1 ctcgctcggc aacaaagcac cagtggtcta gtggtagaat agtaccctgc cacggtacag
       61 acccgggttc gattcccggc tggtgcagag acggcgcgcg ccgtctcagt tttagagcta
      121 gaaatagcaa gttaaaataa ggctagtccg ttatcaactt gaaaaagtgg caccgagtcg
      181 gtgctttttt tctagaccca gctttcttgt acaaagttgg cattacgctt gagcgagacg
      241 ggatcactag tgaattcgcg gccgcctgca ggtcgaccat atgggagagc tcccaacgcg
      301 ttggatgcat agcttgagta ttctatagtg tcacctaaat agcttggcgt aatcatggtc
      361 atagctgttt cctgtgtgaa attgttatcc gctcacaatt ccacacaaca tacgagccgg
      421 aagcataaag tgtaaagcct ggggtgccta atgagtgagc taactcacat taattgcgtt
      481 gcgctcactg cccgctttcc agtcgggaaa cctgtcgtgc cagctgcatt aatgaatcgg
      541 ccaacgcgcg gggagaggcg gtttgcgtat tgggcgctct tccgcttcct cgctcactga
      601 ctcgctgcgc tcggtcgttc ggctgcggcg agcggtatca gctcactcaa aggcggtaat
      661 acggttatcc acagaatcag gggataacgc aggaaagaac atgtgagcaa aaggccagca
      721 aaaggccagg aaccgtaaaa aggccgcgtt gctggcgttt ttccataggc tccgcccccc
      781 tgacgagcat cacaaaaatc gacgctcaag tcagaggtgg cgaaacccga caggactata
      841 aagataccag gcgtttcccc ctggaagctc cctcgtgcgc tctcctgttc cgaccctgcc
      901 gcttaccgga tacctgtccg cctttctccc ttcgggaagc gtggcgcttt ctcatagctc
      961 acgctgtagg tatctcagtt cggtgtaggt cgttcgctcc aagctgggct gtgtgcacga
     1021 accccccgtt cagcccgacc gctgcgcctt atccggtaac tatcgtcttg agtccaaccc
     1081 ggtaagacac gacttatcgc cactggcagc agccactggt aacaggatta gcagagcgag
     1141 gtatgtaggc ggtgctacag agttcttgaa gtggtggcct aactacggct acactagaag
     1201 aacagtattt ggtatctgcg ctctgctgaa gccagttacc ttcggaaaaa gagttggtag
     1261 ctcttgatcc ggcaaacaaa ccaccgctgg tagcggtggt ttttttgttt gcaagcagca
     1321 gattacgcgc agaaaaaaag gatctcaaga agatcctttg atcttttcta cggggtctga
     1381 cgctcagtgg aacgaaaact cacgttaagg gattttggtc atgagattat caaaaaggat
     1441 cttcacctag atccttttaa attaaaaatg aagttttaaa tcaatctaaa gtatatatga
     1501 gtaaacttgg tctgacagtt accaatgctt aatcagtgag gcacctatct cagcgatctg
     1561 tctatttcgt tcatccatag ttgcctgact ccccgtcgtg tagataacta cgatacggga
     1621 gggcttacca tctggcccca gtgctgcaat gataccgcga gacccacgct caccggctcc
     1681 agatttatca gcaataaacc agccagccgg aagggccgag cgcagaagtg gtcctgcaac
     1741 tttatccgcc tccatccagt ctattaattg ttgccgggaa gctagagtaa gtagttcgcc
     1801 agttaatagt ttgcgcaacg ttgttgccat tgctacaggc atcgtggtgt cacgctcgtc
     1861 gtttggtatg gcttcattca gctccggttc ccaacgatca aggcgagtta catgatcccc
     1921 catgttgtgc aaaaaagcgg ttagctcctt cggtcctccg atcgttgtca gaagtaagtt
     1981 ggccgcagtg ttatcactca tggttatggc agcactgcat aattctctta ctgtcatgcc
     2041 atccgtaaga tgcttttctg tgactggtga gtactcaacc aagtcattct gagaatagtg
     2101 tatgcggcga ccgagttgct cttgcccggc gtcaatacgg gataataccg cgccacatag
     2161 cagaacttta aaagtgctca tcattggaaa acgttcttcg gggcgaaaac tctcaaggat
     2221 cttaccgctg ttgagatcca gttcgatgta acccactcgt gcacccaact gatcttcagc
     2281 atcttttact ttcaccagcg tttctgggtg agcaaaaaca ggaaggcaaa atgccgcaaa
     2341 aaagggaata agggcgacac ggaaatgttg aatactcata ctcttccttt ttcaatatta
     2401 ttgaagcatt tatcagggtt attgtctcat gagcggatac atatttgaat gtatttagaa
     2461 aaataaacaa ataggggttc cgcgcacatt tccccgaaaa gtgccacctg atgcggtgtg
     2521 aaataccgca cagatgcgta aggagaaaat accgcatcag gaaattgtaa gcgttaatat
     2581 tttgttaaaa ttcgcgttaa atttttgtta aatcagctca ttttttaacc aataggccga
     2641 aatcggcaaa atcccttata aatcaaaaga atagaccgag atagggttga gtgttgttcc
     2701 agtttggaac aagagtccac tattaaagaa cgtggactcc aacgtcaaag ggcgaaaaac
     2761 cgtctatcag ggcgatggcc cactacgtga accatcaccc taatcaagtt ttttggggtc
     2821 gaggtgccgt aaagcactaa atcggaaccc taaagggagc ccccgattta gagcttgacg
     2881 gggaaagccg gcgaacgtgg cgagaaagga agggaagaaa gcgaaaggag cgggcgctag
     2941 ggcgctggca agtgtagcgg tcacgctgcg cgtaaccacc acacccgccg cgcttaatgc
     3001 gccgctacag ggcgcgtcca ttcgccattc aggctgcgca actgttggga agggcgatcg
     3061 gtgcgggcct cttcgctatt acgccagctg gcgaaagggg gatgtgctgc aaggcgatta
     3121 agttgggtaa cgccagggtt ttcccagtca cgacgttgta aaacgacggc cagtgaattg
     3181 taatacgact cactataggg cgaattgggc ccgacgtcgc atgctcccgg ccgccatggc
     3241 ggccgcggga attcgattcc cgt
//
