LOCUS       sequence-319296-        3277 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 [M1_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 3277)
  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 3277)
  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 M1_3-pTRNA-scf 2.1 (GB2073)
FEATURES             Location/Qualifiers
     source          1..3277
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     misc_RNA        complement(14..89)
                     /label=gRNA scaffold
                     /note="guide RNA scaffold for the Streptococcus pyogenes 
                     CRISPR/Cas9 system"
     promoter        complement(284..302)
                     /label=SP6 promoter
                     /note="promoter for bacteriophage SP6 RNA polymerase"
     primer_bind     complement(285..302)
                     /label=SP6
                     /note="SP6 promoter, forward primer"
     primer_bind     complement(320..336)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(320..336)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(333..355)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    344..360
                     /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(368..374,375..392,393..398))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    413..434
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(551..568)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(722..1310)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(802..821)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(1481..2272,2273..2341))
                     /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     2104..2123
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(2342..2446)
                     /gene="bla"
                     /label=AmpR promoter
     rep_origin      complement(2524..2979)
                     /direction=LEFT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(2661..2682)
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     2873..2892
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     3105..3127
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     primer_bind     3119..3136
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     3120..3136
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     3143..3162
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        3143..3161
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     regulatory      3192..3201
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     regulatory      3196..3205
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
ORIGIN
        1 ctcatgtgct cccgcaccga ctcggtgcca ctttttcaag ttgataacgg actagcctta
       61 ttttaacttg ctatttctag ctctaaaact gagacggcgc gcgccgtctc tgcaccagcc
      121 gggaatcgaa cccgggtctg taccgtggca gggtactatt ctaccactag accactggtg
      181 ctttgttcaa tcgagcgaga cgggatcact agtgaattcg cggccgcctg caggtcgacc
      241 atatgggaga gctcccaacg cgttggatgc atagcttgag tattctatag tgtcacctaa
      301 atagcttggc gtaatcatgg tcatagctgt ttcctgtgtg aaattgttat ccgctcacaa
      361 ttccacacaa catacgagcc ggaagcataa agtgtaaagc ctggggtgcc taatgagtga
      421 gctaactcac attaattgcg ttgcgctcac tgcccgcttt ccagtcggga aacctgtcgt
      481 gccagctgca ttaatgaatc ggccaacgcg cggggagagg cggtttgcgt attgggcgct
      541 cttccgcttc ctcgctcact gactcgctgc gctcggtcgt tcggctgcgg cgagcggtat
      601 cagctcactc aaaggcggta atacggttat ccacagaatc aggggataac gcaggaaaga
      661 acatgtgagc aaaaggccag caaaaggcca ggaaccgtaa aaaggccgcg ttgctggcgt
      721 ttttccatag gctccgcccc cctgacgagc atcacaaaaa tcgacgctca agtcagaggt
      781 ggcgaaaccc gacaggacta taaagatacc aggcgtttcc ccctggaagc tccctcgtgc
      841 gctctcctgt tccgaccctg ccgcttaccg gatacctgtc cgcctttctc ccttcgggaa
      901 gcgtggcgct ttctcatagc tcacgctgta ggtatctcag ttcggtgtag gtcgttcgct
      961 ccaagctggg ctgtgtgcac gaaccccccg ttcagcccga ccgctgcgcc ttatccggta
     1021 actatcgtct tgagtccaac ccggtaagac acgacttatc gccactggca gcagccactg
     1081 gtaacaggat tagcagagcg aggtatgtag gcggtgctac agagttcttg aagtggtggc
     1141 ctaactacgg ctacactaga agaacagtat ttggtatctg cgctctgctg aagccagtta
     1201 ccttcggaaa aagagttggt agctcttgat ccggcaaaca aaccaccgct ggtagcggtg
     1261 gtttttttgt ttgcaagcag cagattacgc gcagaaaaaa aggatctcaa gaagatcctt
     1321 tgatcttttc tacggggtct gacgctcagt ggaacgaaaa ctcacgttaa gggattttgg
     1381 tcatgagatt atcaaaaagg atcttcacct agatcctttt aaattaaaaa tgaagtttta
     1441 aatcaatcta aagtatatat gagtaaactt ggtctgacag ttaccaatgc ttaatcagtg
     1501 aggcacctat ctcagcgatc tgtctatttc gttcatccat agttgcctga ctccccgtcg
     1561 tgtagataac tacgatacgg gagggcttac catctggccc cagtgctgca atgataccgc
     1621 gagacccacg ctcaccggct ccagatttat cagcaataaa ccagccagcc ggaagggccg
     1681 agcgcagaag tggtcctgca actttatccg cctccatcca gtctattaat tgttgccggg
     1741 aagctagagt aagtagttcg ccagttaata gtttgcgcaa cgttgttgcc attgctacag
     1801 gcatcgtggt gtcacgctcg tcgtttggta tggcttcatt cagctccggt tcccaacgat
     1861 caaggcgagt tacatgatcc cccatgttgt gcaaaaaagc ggttagctcc ttcggtcctc
     1921 cgatcgttgt cagaagtaag ttggccgcag tgttatcact catggttatg gcagcactgc
     1981 ataattctct tactgtcatg ccatccgtaa gatgcttttc tgtgactggt gagtactcaa
     2041 ccaagtcatt ctgagaatag tgtatgcggc gaccgagttg ctcttgcccg gcgtcaatac
     2101 gggataatac cgcgccacat agcagaactt taaaagtgct catcattgga aaacgttctt
     2161 cggggcgaaa actctcaagg atcttaccgc tgttgagatc cagttcgatg taacccactc
     2221 gtgcacccaa ctgatcttca gcatctttta ctttcaccag cgtttctggg tgagcaaaaa
     2281 caggaaggca aaatgccgca aaaaagggaa taagggcgac acggaaatgt tgaatactca
     2341 tactcttcct ttttcaatat tattgaagca tttatcaggg ttattgtctc atgagcggat
     2401 acatatttga atgtatttag aaaaataaac aaataggggt tccgcgcaca tttccccgaa
     2461 aagtgccacc tgatgcggtg tgaaataccg cacagatgcg taaggagaaa ataccgcatc
     2521 aggaaattgt aagcgttaat attttgttaa aattcgcgtt aaatttttgt taaatcagct
     2581 cattttttaa ccaataggcc gaaatcggca aaatccctta taaatcaaaa gaatagaccg
     2641 agatagggtt gagtgttgtt ccagtttgga acaagagtcc actattaaag aacgtggact
     2701 ccaacgtcaa agggcgaaaa accgtctatc agggcgatgg cccactacgt gaaccatcac
     2761 cctaatcaag ttttttgggg tcgaggtgcc gtaaagcact aaatcggaac cctaaaggga
     2821 gcccccgatt tagagcttga cggggaaagc cggcgaacgt ggcgagaaag gaagggaaga
     2881 aagcgaaagg agcgggcgct agggcgctgg caagtgtagc ggtcacgctg cgcgtaacca
     2941 ccacacccgc cgcgcttaat gcgccgctac agggcgcgtc cattcgccat tcaggctgcg
     3001 caactgttgg gaagggcgat cggtgcgggc ctcttcgcta ttacgccagc tggcgaaagg
     3061 gggatgtgct gcaaggcgat taagttgggt aacgccaggg ttttcccagt cacgacgttg
     3121 taaaacgacg gccagtgaat tgtaatacga ctcactatag ggcgaattgg gcccgacgtc
     3181 gcatgctccc ggccgccatg gcggccgcgg gaattcgatt cccgtctcgc tcagggaaga
     3241 ctccccagtg actgtgggag tcgcacatgg gtgattc
//
