LOCUS       sequence-146005-        3222 bp    DNA     circular SYN 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      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 [2_n-1] (GB1206)
FEATURES             Location/Qualifiers
     source          1..3222
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     misc_RNA        111..186
                     /label=gRNA scaffold
                     /note="guide RNA scaffold for the Streptococcus pyogenes 
                     CRISPR/Cas9 system"
     promoter        complement(281..299)
                     /label=SP6 promoter
                     /note="promoter for bacteriophage SP6 RNA polymerase"
     primer_bind     complement(282..299)
                     /label=SP6
                     /note="SP6 promoter, forward primer"
     primer_bind     complement(317..333)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(317..333)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(330..352)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    341..357
                     /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(365..371,372..389,390..395))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    410..431
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(548..565)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(719..1307)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(799..818)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(1478..2269,2270..2338))
                     /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     2101..2120
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(2339..2443)
                     /gene="bla"
                     /label=AmpR promoter
     rep_origin      complement(2521..2976)
                     /direction=LEFT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(2658..2679)
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     2870..2889
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     3102..3124
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     primer_bind     3116..3133
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     3117..3133
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     3140..3159
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        3140..3158
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     regulatory      3189..3198
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     regulatory      3193..3202
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
ORIGIN
        1 ctcgctcggt gcaacaaagc accagtggtc tagtggtaga atagtaccct gccacggtac
       61 agacccgggt tcgattcccg gctggtgcag agacggcgcg cgccgtctca gttttagagc
      121 tagaaatagc aagttaaaat aaggctagtc cgttatcaac ttgaaaaagt ggcaccgagt
      181 cggtgcaatg agcgagacgg gatcactagt gaattcgcgg ccgcctgcag gtcgaccata
      241 tgggagagct cccaacgcgt tggatgcata gcttgagtat tctatagtgt cacctaaata
      301 gcttggcgta atcatggtca tagctgtttc ctgtgtgaaa ttgttatccg ctcacaattc
      361 cacacaacat acgagccgga agcataaagt gtaaagcctg gggtgcctaa tgagtgagct
      421 aactcacatt aattgcgttg cgctcactgc ccgctttcca gtcgggaaac ctgtcgtgcc
      481 agctgcatta atgaatcggc caacgcgcgg ggagaggcgg tttgcgtatt gggcgctctt
      541 ccgcttcctc gctcactgac tcgctgcgct cggtcgttcg gctgcggcga gcggtatcag
      601 ctcactcaaa ggcggtaata cggttatcca cagaatcagg ggataacgca ggaaagaaca
      661 tgtgagcaaa aggccagcaa aaggccagga accgtaaaaa ggccgcgttg ctggcgtttt
      721 tccataggct ccgcccccct gacgagcatc acaaaaatcg acgctcaagt cagaggtggc
      781 gaaacccgac aggactataa agataccagg cgtttccccc tggaagctcc ctcgtgcgct
      841 ctcctgttcc gaccctgccg cttaccggat acctgtccgc ctttctccct tcgggaagcg
      901 tggcgctttc tcatagctca cgctgtaggt atctcagttc ggtgtaggtc gttcgctcca
      961 agctgggctg tgtgcacgaa ccccccgttc agcccgaccg ctgcgcctta tccggtaact
     1021 atcgtcttga gtccaacccg gtaagacacg acttatcgcc actggcagca gccactggta
     1081 acaggattag cagagcgagg tatgtaggcg gtgctacaga gttcttgaag tggtggccta
     1141 actacggcta cactagaaga acagtatttg gtatctgcgc tctgctgaag ccagttacct
     1201 tcggaaaaag agttggtagc tcttgatccg gcaaacaaac caccgctggt agcggtggtt
     1261 tttttgtttg caagcagcag attacgcgca gaaaaaaagg atctcaagaa gatcctttga
     1321 tcttttctac ggggtctgac gctcagtgga acgaaaactc acgttaaggg attttggtca
     1381 tgagattatc aaaaaggatc ttcacctaga tccttttaaa ttaaaaatga agttttaaat
     1441 caatctaaag tatatatgag taaacttggt ctgacagtta ccaatgctta atcagtgagg
     1501 cacctatctc agcgatctgt ctatttcgtt catccatagt tgcctgactc cccgtcgtgt
     1561 agataactac gatacgggag ggcttaccat ctggccccag tgctgcaatg ataccgcgag
     1621 acccacgctc accggctcca gatttatcag caataaacca gccagccgga agggccgagc
     1681 gcagaagtgg tcctgcaact ttatccgcct ccatccagtc tattaattgt tgccgggaag
     1741 ctagagtaag tagttcgcca gttaatagtt tgcgcaacgt tgttgccatt gctacaggca
     1801 tcgtggtgtc acgctcgtcg tttggtatgg cttcattcag ctccggttcc caacgatcaa
     1861 ggcgagttac atgatccccc atgttgtgca aaaaagcggt tagctccttc ggtcctccga
     1921 tcgttgtcag aagtaagttg gccgcagtgt tatcactcat ggttatggca gcactgcata
     1981 attctcttac tgtcatgcca tccgtaagat gcttttctgt gactggtgag tactcaacca
     2041 agtcattctg agaatagtgt atgcggcgac cgagttgctc ttgcccggcg tcaatacggg
     2101 ataataccgc gccacatagc agaactttaa aagtgctcat cattggaaaa cgttcttcgg
     2161 ggcgaaaact ctcaaggatc ttaccgctgt tgagatccag ttcgatgtaa cccactcgtg
     2221 cacccaactg atcttcagca tcttttactt tcaccagcgt ttctgggtga gcaaaaacag
     2281 gaaggcaaaa tgccgcaaaa aagggaataa gggcgacacg gaaatgttga atactcatac
     2341 tcttcctttt tcaatattat tgaagcattt atcagggtta ttgtctcatg agcggataca
     2401 tatttgaatg tatttagaaa aataaacaaa taggggttcc gcgcacattt ccccgaaaag
     2461 tgccacctga tgcggtgtga aataccgcac agatgcgtaa ggagaaaata ccgcatcagg
     2521 aaattgtaag cgttaatatt ttgttaaaat tcgcgttaaa tttttgttaa atcagctcat
     2581 tttttaacca ataggccgaa atcggcaaaa tcccttataa atcaaaagaa tagaccgaga
     2641 tagggttgag tgttgttcca gtttggaaca agagtccact attaaagaac gtggactcca
     2701 acgtcaaagg gcgaaaaacc gtctatcagg gcgatggccc actacgtgaa ccatcaccct
     2761 aatcaagttt tttggggtcg aggtgccgta aagcactaaa tcggaaccct aaagggagcc
     2821 cccgatttag agcttgacgg ggaaagccgg cgaacgtggc gagaaaggaa gggaagaaag
     2881 cgaaaggagc gggcgctagg gcgctggcaa gtgtagcggt cacgctgcgc gtaaccacca
     2941 cacccgccgc gcttaatgcg ccgctacagg gcgcgtccat tcgccattca ggctgcgcaa
     3001 ctgttgggaa gggcgatcgg tgcgggcctc ttcgctatta cgccagctgg cgaaaggggg
     3061 atgtgctgca aggcgattaa gttgggtaac gccagggttt tcccagtcac gacgttgtaa
     3121 aacgacggcc agtgaattgt aatacgactc actatagggc gaattgggcc cgacgtcgca
     3181 tgctcccggc cgccatggcg gccgcgggaa ttcgattccc gt
//
