LOCUS       sequence-310922-        3278 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  tRNA and scaffold 2.1scRNA aptamer Ms2 for the assembly of 
            GBoligomers for the first position (positon [M2_3]) of a 
            polycistronic tRNA-gRNA regulated by the U6-26 or U6-1 promoter.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 3278)
  AUTHORS   Selma S, Bernabe-Orts JM, Vazquez-Vilar M, Diego-Martin B, Ajenjo M,
            Garcia-Carpintero V, Granell A, Orzaez D
  TITLE     Strong gene activation in plants with genome-wide specificity using 
            a new orthogonal CRISPR/Cas9-based programmable transcriptional 
            activator.
  JOURNAL   Plant Biotechnol J. 2019 Sep;17(9):1703-1705. doi: 
            10.1111/pbi.13138. Epub 2019 May 23.
  PUBMED    31034138
REFERENCE   2  (bases 1 to 3278)
  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"; doi: "10.1111/pbi.13138";
            journalName: "Plant Biotechnol J"; date: "2019-09"; volume: "17"; 
            issue: "9"; pages: "1703-1705"
COMMENT     Sequence Label: pVD1 M2_3-pTRNA-scf 2.1 (GB2074)
FEATURES             Location/Qualifiers
     source          1..3278
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     misc_RNA        116..191
                     /label=gRNA scaffold
                     /note="guide RNA scaffold for the Streptococcus pyogenes 
                     CRISPR/Cas9 system"
     promoter        complement(343..361)
                     /label=SP6 promoter
                     /note="promoter for bacteriophage SP6 RNA polymerase"
     primer_bind     complement(344..361)
                     /label=SP6
                     /note="SP6 promoter, forward primer"
     primer_bind     complement(379..395)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(379..395)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(392..414)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    403..419
                     /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(427..433,434..451,452..457))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    472..493
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(610..627)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(781..1369)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(861..880)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(1540..2331,2332..2400))
                     /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     2163..2182
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(2401..2505)
                     /gene="bla"
                     /label=AmpR promoter
     rep_origin      complement(2583..3038)
                     /direction=LEFT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(2720..2741)
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     2932..2951
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     3164..3186
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     primer_bind     3178..3195
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     3179..3195
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     3202..3221
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        3202..3220
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     regulatory      3251..3260
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     regulatory      3255..3264
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
ORIGIN
        1 cccgtctcgc tcgtcccaac aaagcaccag tggtctagtg gtagaatagt accctgccac
       61 ggtacagacc cgggttcgat tcccggctgg tgcagagacg gcgcgcgccg tctcagtttt
      121 agagctagaa atagcaagtt aaaataaggc tagtccgtta tcaacttgaa aaagtggcac
      181 cgagtcggtg cgggagcaca tgaggaatca cccatgtgcg actcccacag tcactgggga
      241 gtcttcccaa tgagcgagac gggatcacta gtgaattcgc ggccgcctgc aggtcgacca
      301 tatgggagag ctcccaacgc gttggatgca tagcttgagt attctatagt gtcacctaaa
      361 tagcttggcg taatcatggt catagctgtt tcctgtgtga aattgttatc cgctcacaat
      421 tccacacaac atacgagccg gaagcataaa gtgtaaagcc tggggtgcct aatgagtgag
      481 ctaactcaca ttaattgcgt tgcgctcact gcccgctttc cagtcgggaa acctgtcgtg
      541 ccagctgcat taatgaatcg gccaacgcgc ggggagaggc ggtttgcgta ttgggcgctc
      601 ttccgcttcc tcgctcactg actcgctgcg ctcggtcgtt cggctgcggc gagcggtatc
      661 agctcactca aaggcggtaa tacggttatc cacagaatca ggggataacg caggaaagaa
      721 catgtgagca aaaggccagc aaaaggccag gaaccgtaaa aaggccgcgt tgctggcgtt
      781 tttccatagg ctccgccccc ctgacgagca tcacaaaaat cgacgctcaa gtcagaggtg
      841 gcgaaacccg acaggactat aaagatacca ggcgtttccc cctggaagct ccctcgtgcg
      901 ctctcctgtt ccgaccctgc cgcttaccgg atacctgtcc gcctttctcc cttcgggaag
      961 cgtggcgctt tctcatagct cacgctgtag gtatctcagt tcggtgtagg tcgttcgctc
     1021 caagctgggc tgtgtgcacg aaccccccgt tcagcccgac cgctgcgcct tatccggtaa
     1081 ctatcgtctt gagtccaacc cggtaagaca cgacttatcg ccactggcag cagccactgg
     1141 taacaggatt agcagagcga ggtatgtagg cggtgctaca gagttcttga agtggtggcc
     1201 taactacggc tacactagaa gaacagtatt tggtatctgc gctctgctga agccagttac
     1261 cttcggaaaa agagttggta gctcttgatc cggcaaacaa accaccgctg gtagcggtgg
     1321 tttttttgtt tgcaagcagc agattacgcg cagaaaaaaa ggatctcaag aagatccttt
     1381 gatcttttct acggggtctg acgctcagtg gaacgaaaac tcacgttaag ggattttggt
     1441 catgagatta tcaaaaagga tcttcaccta gatcctttta aattaaaaat gaagttttaa
     1501 atcaatctaa agtatatatg agtaaacttg gtctgacagt taccaatgct taatcagtga
     1561 ggcacctatc tcagcgatct gtctatttcg ttcatccata gttgcctgac tccccgtcgt
     1621 gtagataact acgatacggg agggcttacc atctggcccc agtgctgcaa tgataccgcg
     1681 agacccacgc tcaccggctc cagatttatc agcaataaac cagccagccg gaagggccga
     1741 gcgcagaagt ggtcctgcaa ctttatccgc ctccatccag tctattaatt gttgccggga
     1801 agctagagta agtagttcgc cagttaatag tttgcgcaac gttgttgcca ttgctacagg
     1861 catcgtggtg tcacgctcgt cgtttggtat ggcttcattc agctccggtt cccaacgatc
     1921 aaggcgagtt acatgatccc ccatgttgtg caaaaaagcg gttagctcct tcggtcctcc
     1981 gatcgttgtc agaagtaagt tggccgcagt gttatcactc atggttatgg cagcactgca
     2041 taattctctt actgtcatgc catccgtaag atgcttttct gtgactggtg agtactcaac
     2101 caagtcattc tgagaatagt gtatgcggcg accgagttgc tcttgcccgg cgtcaatacg
     2161 ggataatacc gcgccacata gcagaacttt aaaagtgctc atcattggaa aacgttcttc
     2221 ggggcgaaaa ctctcaagga tcttaccgct gttgagatcc agttcgatgt aacccactcg
     2281 tgcacccaac tgatcttcag catcttttac tttcaccagc gtttctgggt gagcaaaaac
     2341 aggaaggcaa aatgccgcaa aaaagggaat aagggcgaca cggaaatgtt gaatactcat
     2401 actcttcctt tttcaatatt attgaagcat ttatcagggt tattgtctca tgagcggata
     2461 catatttgaa tgtatttaga aaaataaaca aataggggtt ccgcgcacat ttccccgaaa
     2521 agtgccacct gatgcggtgt gaaataccgc acagatgcgt aaggagaaaa taccgcatca
     2581 ggaaattgta agcgttaata ttttgttaaa attcgcgtta aatttttgtt aaatcagctc
     2641 attttttaac caataggccg aaatcggcaa aatcccttat aaatcaaaag aatagaccga
     2701 gatagggttg agtgttgttc cagtttggaa caagagtcca ctattaaaga acgtggactc
     2761 caacgtcaaa gggcgaaaaa ccgtctatca gggcgatggc ccactacgtg aaccatcacc
     2821 ctaatcaagt tttttggggt cgaggtgccg taaagcacta aatcggaacc ctaaagggag
     2881 cccccgattt agagcttgac ggggaaagcc ggcgaacgtg gcgagaaagg aagggaagaa
     2941 agcgaaagga gcgggcgcta gggcgctggc aagtgtagcg gtcacgctgc gcgtaaccac
     3001 cacacccgcc gcgcttaatg cgccgctaca gggcgcgtcc attcgccatt caggctgcgc
     3061 aactgttggg aagggcgatc ggtgcgggcc tcttcgctat tacgccagct ggcgaaaggg
     3121 ggatgtgctg caaggcgatt aagttgggta acgccagggt tttcccagtc acgacgttgt
     3181 aaaacgacgg ccagtgaatt gtaatacgac tcactatagg gcgaattggg cccgacgtcg
     3241 catgctcccg gccgccatgg cggccgcggg aattcgat
//
