LOCUS       sequence-211947-        3222 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  tRNA and scaffold for the assembly of GBoligomers for the first 
            position (positon [M1_n-1]) of a polycistronic tRNA-gRNA regulated 
            by the (monocot) U3 promoter (2-part multiplexing).
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 3222)
  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 3222)
  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_n-1] (GB1210)
FEATURES             Location/Qualifiers
     source          1..3222
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     1..17
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     24..43
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        24..42
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     regulatory      73..82
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     regulatory      77..86
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     misc_RNA        217..292
                     /label=gRNA scaffold
                     /note="guide RNA scaffold for the Streptococcus pyogenes 
                     CRISPR/Cas9 system"
     promoter        complement(387..405)
                     /label=SP6 promoter
                     /note="promoter for bacteriophage SP6 RNA polymerase"
     primer_bind     complement(388..405)
                     /label=SP6
                     /note="SP6 promoter, forward primer"
     primer_bind     complement(423..439)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(423..439)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(436..458)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    447..463
                     /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(471..477,478..495,496..501))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    516..537
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(654..671)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(825..1413)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(905..924)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(1584..2375,2376..2444))
                     /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     2207..2226
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(2445..2549)
                     /gene="bla"
                     /label=AmpR promoter
     rep_origin      complement(2627..3082)
                     /direction=LEFT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(2764..2785)
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     2976..2995
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     join(3208..3222,1..8)
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     primer_bind     join(3222,1..17)
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
ORIGIN
        1 gtaaaacgac ggccagtgaa ttgtaatacg actcactata gggcgaattg ggcccgacgt
       61 cgcatgctcc cggccgccat ggcggccgcg ggaattcgat tcccgtctcg ctcgggcaaa
      121 caaagcacca gtggtctagt ggtagaatag taccctgcca cggtacagac ccgggttcga
      181 ttcccggctg gtgcagagac ggcgcgcgcc gtctcagttt tagagctaga aatagcaagt
      241 taaaataagg ctagtccgtt atcaacttga aaaagtggca ccgagtcggt gcaatgagcg
      301 agacgggatc actagtgaat tcgcggccgc ctgcaggtcg accatatggg agagctccca
      361 acgcgttgga tgcatagctt gagtattcta tagtgtcacc taaatagctt ggcgtaatca
      421 tggtcatagc tgtttcctgt gtgaaattgt tatccgctca caattccaca caacatacga
      481 gccggaagca taaagtgtaa agcctggggt gcctaatgag tgagctaact cacattaatt
      541 gcgttgcgct cactgcccgc tttccagtcg ggaaacctgt cgtgccagct gcattaatga
      601 atcggccaac gcgcggggag aggcggtttg cgtattgggc gctcttccgc ttcctcgctc
      661 actgactcgc tgcgctcggt cgttcggctg cggcgagcgg tatcagctca ctcaaaggcg
      721 gtaatacggt tatccacaga atcaggggat aacgcaggaa agaacatgtg agcaaaaggc
      781 cagcaaaagg ccaggaaccg taaaaaggcc gcgttgctgg cgtttttcca taggctccgc
      841 ccccctgacg agcatcacaa aaatcgacgc tcaagtcaga ggtggcgaaa cccgacagga
      901 ctataaagat accaggcgtt tccccctgga agctccctcg tgcgctctcc tgttccgacc
      961 ctgccgctta ccggatacct gtccgccttt ctcccttcgg gaagcgtggc gctttctcat
     1021 agctcacgct gtaggtatct cagttcggtg taggtcgttc gctccaagct gggctgtgtg
     1081 cacgaacccc ccgttcagcc cgaccgctgc gccttatccg gtaactatcg tcttgagtcc
     1141 aacccggtaa gacacgactt atcgccactg gcagcagcca ctggtaacag gattagcaga
     1201 gcgaggtatg taggcggtgc tacagagttc ttgaagtggt ggcctaacta cggctacact
     1261 agaagaacag tatttggtat ctgcgctctg ctgaagccag ttaccttcgg aaaaagagtt
     1321 ggtagctctt gatccggcaa acaaaccacc gctggtagcg gtggtttttt tgtttgcaag
     1381 cagcagatta cgcgcagaaa aaaaggatct caagaagatc ctttgatctt ttctacgggg
     1441 tctgacgctc agtggaacga aaactcacgt taagggattt tggtcatgag attatcaaaa
     1501 aggatcttca cctagatcct tttaaattaa aaatgaagtt ttaaatcaat ctaaagtata
     1561 tatgagtaaa cttggtctga cagttaccaa tgcttaatca gtgaggcacc tatctcagcg
     1621 atctgtctat ttcgttcatc catagttgcc tgactccccg tcgtgtagat aactacgata
     1681 cgggagggct taccatctgg ccccagtgct gcaatgatac cgcgagaccc acgctcaccg
     1741 gctccagatt tatcagcaat aaaccagcca gccggaaggg ccgagcgcag aagtggtcct
     1801 gcaactttat ccgcctccat ccagtctatt aattgttgcc gggaagctag agtaagtagt
     1861 tcgccagtta atagtttgcg caacgttgtt gccattgcta caggcatcgt ggtgtcacgc
     1921 tcgtcgtttg gtatggcttc attcagctcc ggttcccaac gatcaaggcg agttacatga
     1981 tcccccatgt tgtgcaaaaa agcggttagc tccttcggtc ctccgatcgt tgtcagaagt
     2041 aagttggccg cagtgttatc actcatggtt atggcagcac tgcataattc tcttactgtc
     2101 atgccatccg taagatgctt ttctgtgact ggtgagtact caaccaagtc attctgagaa
     2161 tagtgtatgc ggcgaccgag ttgctcttgc ccggcgtcaa tacgggataa taccgcgcca
     2221 catagcagaa ctttaaaagt gctcatcatt ggaaaacgtt cttcggggcg aaaactctca
     2281 aggatcttac cgctgttgag atccagttcg atgtaaccca ctcgtgcacc caactgatct
     2341 tcagcatctt ttactttcac cagcgtttct gggtgagcaa aaacaggaag gcaaaatgcc
     2401 gcaaaaaagg gaataagggc gacacggaaa tgttgaatac tcatactctt cctttttcaa
     2461 tattattgaa gcatttatca gggttattgt ctcatgagcg gatacatatt tgaatgtatt
     2521 tagaaaaata aacaaatagg ggttccgcgc acatttcccc gaaaagtgcc acctgatgcg
     2581 gtgtgaaata ccgcacagat gcgtaaggag aaaataccgc atcaggaaat tgtaagcgtt
     2641 aatattttgt taaaattcgc gttaaatttt tgttaaatca gctcattttt taaccaatag
     2701 gccgaaatcg gcaaaatccc ttataaatca aaagaataga ccgagatagg gttgagtgtt
     2761 gttccagttt ggaacaagag tccactatta aagaacgtgg actccaacgt caaagggcga
     2821 aaaaccgtct atcagggcga tggcccacta cgtgaaccat caccctaatc aagttttttg
     2881 gggtcgaggt gccgtaaagc actaaatcgg aaccctaaag ggagcccccg atttagagct
     2941 tgacggggaa agccggcgaa cgtggcgaga aaggaaggga agaaagcgaa aggagcgggc
     3001 gctagggcgc tggcaagtgt agcggtcacg ctgcgcgtaa ccaccacacc cgccgcgctt
     3061 aatgcgccgc tacagggcgc gtccattcgc cattcaggct gcgcaactgt tgggaagggc
     3121 gatcggtgcg ggcctcttcg ctattacgcc agctggcgaa agggggatgt gctgcaaggc
     3181 gattaagttg ggtaacgcca gggttttccc agtcacgacg tt
//
