LOCUS       sequence-319297-        3320 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 [M3_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 3320)
  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 3320)
  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 M3_3-pTRNA-scf 2.1 (GB2075)
FEATURES             Location/Qualifiers
     source          1..3320
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     147..166
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     400..417
                     /label=L4440
                     /note="L4440 vector, forward primer"
     protein_bind    534..555
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     promoter        join(570..575,576..593,594..600)
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    608..624
                     /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)."
     primer_bind     613..635
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     primer_bind     632..648
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     632..648
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     promoter        666..684
                     /label=SP6 promoter
                     /note="promoter for bacteriophage SP6 RNA polymerase"
     primer_bind     666..683
                     /label=SP6
                     /note="SP6 promoter, forward primer"
     misc_RNA        879..954
                     /label=gRNA scaffold
                     /note="guide RNA scaffold for the Streptococcus pyogenes 
                     CRISPR/Cas9 system"
     protein_bind    complement(1021..1045)
                     /gene="mutant version of attB"
                     /label=attB2
                     /bound_moiety="BP Clonase(TM)"
                     /note="recombination site for the Gateway(R) BP reaction"
     regulatory      1083..1092
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     regulatory      1087..1096
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     primer_bind     complement(1126..1145)
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        complement(1127..1145)
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     primer_bind     complement(1152..1169)
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     complement(1152..1168)
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(1161..1183)
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     rep_origin      1309..1764
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(1396..1415)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     1606..1627
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     promoter        1842..1946
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(1947..2015,2016..2807)
                     /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     complement(2165..2184)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      join(2978..3320,1..246)
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
ORIGIN
        1 agcttggagc gaacgaccta caccgaactg agatacctac agcgtgagct atgagaaagc
       61 gccacgcttc ccgaagggag aaaggcggac aggtatccgg taagcggcag ggtcggaaca
      121 ggagagcgca cgagggagct tccaggggga aacgcctggt atctttatag tcctgtcggg
      181 tttcgccacc tctgacttga gcgtcgattt ttgtgatgct cgtcaggggg gcggagccta
      241 tggaaaaacg ccagcaacgc ggccttttta cggttcctgg ccttttgctg gccttttgct
      301 cacatgttct ttcctgcgtt atcccctgat tctgtggata accgtattac cgcctttgag
      361 tgagctgata ccgctcgccg cagccgaacg accgagcgca gcgagtcagt gagcgaggaa
      421 gcggaagagc gcccaatacg caaaccgcct ctccccgcgc gttggccgat tcattaatgc
      481 agctggcacg acaggtttcc cgactggaaa gcgggcagtg agcgcaacgc aattaatgtg
      541 agttagctca ctcattaggc accccaggct ttacacttta tgcttccggc tcgtatgttg
      601 tgtggaattg tgagcggata acaatttcac acaggaaaca gctatgacca tgattacgcc
      661 aagctattta ggtgacacta tagaatactc aagctatgca tccaacgcgt tgggagctct
      721 cccatatggt cgacctgcag gcggccgcga attcactagt gatcccgtct cgctcgccaa
      781 aacaaagcac cagtggtcta gtggtagaat agtaccctgc cacggtacag acccgggttc
      841 gattcccggc tggtgcagag acggcgcgcg ccgtctcagt tttagagcta gaaatagcaa
      901 gttaaaataa ggctagtccg ttatcaactt gaaaaagtgg caccgagtcg gtgcgggagc
      961 acatgaggaa tcacccatgt gcgactccca cagtcactgg ggagtcttcc cttttttctg
     1021 acccagcttt cttgtacaaa gttggcatta cgcttgagcg agacgggaat cgaattcccg
     1081 cggccgccat ggcggccggg agcatgcgac gtcgggccca attcgcccta tagtgagtcg
     1141 tattacaatt cactggccgt cgttttacaa cgtcgtgact gggaaaaccc tggcgttacc
     1201 caacttaatc gccttgcagc acatccccct ttcgccagct ggcgtaatag cgaagaggcc
     1261 cgcaccgatc gcccttccca acagttgcgc agcctgaatg gcgaatggac gcgccctgta
     1321 gcggcgcatt aagcgcggcg ggtgtggtgg ttacgcgcag cgtgaccgct acacttgcca
     1381 gcgccctagc gcccgctcct ttcgctttct tcccttcctt tctcgccacg ttcgccggct
     1441 ttccccgtca agctctaaat cgggggctcc ctttagggtt ccgatttagt gctttacggc
     1501 acctcgaccc caaaaaactt gattagggtg atggttcacg tagtgggcca tcgccctgat
     1561 agacggtttt tcgccctttg acgttggagt ccacgttctt taatagtgga ctcttgttcc
     1621 aaactggaac aacactcaac cctatctcgg tctattcttt tgatttataa gggattttgc
     1681 cgatttcggc ctattggtta aaaaatgagc tgatttaaca aaaatttaac gcgaatttta
     1741 acaaaatatt aacgcttaca atttcctgat gcggtatttt ctccttacgc atctgtgcgg
     1801 tatttcacac cgcatcaggt ggcacttttc ggggaaatgt gcgcggaacc cctatttgtt
     1861 tatttttcta aatacattca aatatgtatc cgctcatgag acaataaccc tgataaatgc
     1921 ttcaataata ttgaaaaagg aagagtatga gtattcaaca tttccgtgtc gcccttattc
     1981 ccttttttgc ggcattttgc cttcctgttt ttgctcaccc agaaacgctg gtgaaagtaa
     2041 aagatgctga agatcagttg ggtgcacgag tgggttacat cgaactggat ctcaacagcg
     2101 gtaagatcct tgagagtttt cgccccgaag aacgttttcc aatgatgagc acttttaaag
     2161 ttctgctatg tggcgcggta ttatcccgta ttgacgccgg gcaagagcaa ctcggtcgcc
     2221 gcatacacta ttctcagaat gacttggttg agtactcacc agtcacagaa aagcatctta
     2281 cggatggcat gacagtaaga gaattatgca gtgctgccat aaccatgagt gataacactg
     2341 cggccaactt acttctgaca acgatcggag gaccgaagga gctaaccgct tttttgcaca
     2401 acatggggga tcatgtaact cgccttgatc gttgggaacc ggagctgaat gaagccatac
     2461 caaacgacga gcgtgacacc acgatgcctg tagcaatggc aacaacgttg cgcaaactat
     2521 taactggcga actacttact ctagcttccc ggcaacaatt aatagactgg atggaggcgg
     2581 ataaagttgc aggaccactt ctgcgctcgg cccttccggc tggctggttt attgctgata
     2641 aatctggagc cggtgagcgt gggtctcgcg gtatcattgc agcactgggg ccagatggta
     2701 agccctcccg tatcgtagtt atctacacga cggggagtca ggcaactatg gatgaacgaa
     2761 atagacagat cgctgagata ggtgcctcac tgattaagca ttggtaactg tcagaccaag
     2821 tttactcata tatactttag attgatttaa aacttcattt ttaatttaaa aggatctagg
     2881 tgaagatcct ttttgataat ctcatgacca aaatccctta acgtgagttt tcgttccact
     2941 gagcgtcaga ccccgtagaa aagatcaaag gatcttcttg agatcctttt tttctgcgcg
     3001 taatctgctg cttgcaaaca aaaaaaccac cgctaccagc ggtggtttgt ttgccggatc
     3061 aagagctacc aactcttttt ccgaaggtaa ctggcttcag cagagcgcag ataccaaata
     3121 ctgttcttct agtgtagccg tagttaggcc accacttcaa gaactctgta gcaccgccta
     3181 catacctcgc tctgctaatc ctgttaccag tggctgctgc cagtggcgat aagtcgtgtc
     3241 ttaccgggtt ggactcaaga cgatagttac cggataaggc gcagcggtcg ggctgaacgg
     3301 ggggttcgtg cacacagccc
//
