LOCUS       sequence-319683-        3320 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  tRNA and scaffold 2.1 scRNA aptamer Ms2 for the assembly of 
            GBoligomers for a single position 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 M1_1-pTRNA-scf 2.1 (GB2603)
FEATURES             Location/Qualifiers
     source          1..3320
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     388..407
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(626..730)
                     /gene="bla"
                     /label=AmpR promoter
     rep_origin      complement(808..1263)
                     /direction=LEFT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(945..966)
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     1157..1176
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     1389..1411
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     primer_bind     1403..1420
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     1404..1420
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     1427..1446
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        1427..1445
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     regulatory      1476..1485
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     regulatory      1480..1489
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     misc_RNA        1620..1695
                     /label=gRNA scaffold
                     /note="guide RNA scaffold for the Streptococcus pyogenes 
                     CRISPR/Cas9 system"
     protein_bind    complement(1762..1786)
                     /gene="mutant version of attB"
                     /label=attB2
                     /bound_moiety="BP Clonase(TM)"
                     /note="recombination site for the Gateway(R) BP reaction"
     promoter        complement(1888..1906)
                     /label=SP6 promoter
                     /note="promoter for bacteriophage SP6 RNA polymerase"
     primer_bind     complement(1889..1906)
                     /label=SP6
                     /note="SP6 promoter, forward primer"
     primer_bind     complement(1924..1940)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(1924..1940)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(1937..1959)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    1948..1964
                     /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(1972..1978,1979..1996,1997..2002))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    2017..2038
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(2155..2172)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(2326..2914)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(2406..2425)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(3085..3320,1..556,557..625))
                     /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"
ORIGIN
        1 tccagtctat taattgttgc cgggaagcta gagtaagtag ttcgccagtt aatagtttgc
       61 gcaacgttgt tgccattgct acaggcatcg tggtgtcacg ctcgtcgttt ggtatggctt
      121 cattcagctc cggttcccaa cgatcaaggc gagttacatg atcccccatg ttgtgcaaaa
      181 aagcggttag ctccttcggt cctccgatcg ttgtcagaag taagttggcc gcagtgttat
      241 cactcatggt tatggcagca ctgcataatt ctcttactgt catgccatcc gtaagatgct
      301 tttctgtgac tggtgagtac tcaaccaagt cattctgaga atagtgtatg cggcgaccga
      361 gttgctcttg cccggcgtca atacgggata ataccgcgcc acatagcaga actttaaaag
      421 tgctcatcat tggaaaacgt tcttcggggc gaaaactctc aaggatctta ccgctgttga
      481 gatccagttc gatgtaaccc actcgtgcac ccaactgatc ttcagcatct tttactttca
      541 ccagcgtttc tgggtgagca aaaacaggaa ggcaaaatgc cgcaaaaaag ggaataaggg
      601 cgacacggaa atgttgaata ctcatactct tcctttttca atattattga agcatttatc
      661 agggttattg tctcatgagc ggatacatat ttgaatgtat ttagaaaaat aaacaaatag
      721 gggttccgcg cacatttccc cgaaaagtgc cacctgatgc ggtgtgaaat accgcacaga
      781 tgcgtaagga gaaaataccg catcaggaaa ttgtaagcgt taatattttg ttaaaattcg
      841 cgttaaattt ttgttaaatc agctcatttt ttaaccaata ggccgaaatc ggcaaaatcc
      901 cttataaatc aaaagaatag accgagatag ggttgagtgt tgttccagtt tggaacaaga
      961 gtccactatt aaagaacgtg gactccaacg tcaaagggcg aaaaaccgtc tatcagggcg
     1021 atggcccact acgtgaacca tcaccctaat caagtttttt ggggtcgagg tgccgtaaag
     1081 cactaaatcg gaaccctaaa gggagccccc gatttagagc ttgacgggga aagccggcga
     1141 acgtggcgag aaaggaaggg aagaaagcga aaggagcggg cgctagggcg ctggcaagtg
     1201 tagcggtcac gctgcgcgta accaccacac ccgccgcgct taatgcgccg ctacagggcg
     1261 cgtccattcg ccattcaggc tgcgcaactg ttgggaaggg cgatcggtgc gggcctcttc
     1321 gctattacgc cagctggcga aagggggatg tgctgcaagg cgattaagtt gggtaacgcc
     1381 agggttttcc cagtcacgac gttgtaaaac gacggccagt gaattgtaat acgactcact
     1441 atagggcgaa ttgggcccga cgtcgcatgc tcccggccgc catggcggcc gcgggaattc
     1501 gattcccgtc tcgctcgatt gaacaaagca ccagtggtct agtggtagaa tagtaccctg
     1561 ccacggtaca gacccgggtt cgattcccgg ctggtgcaga gacggcgcgc gccgtctcag
     1621 ttttagagct agaaatagca agttaaaata aggctagtcc gttatcaact tgaaaaagtg
     1681 gcaccgagtc ggtgcgggag cacatgagga atcacccatg tgcgactccc acagtcactg
     1741 gggagtcttc ccttttttct gacccagctt tcttgtacaa agttggcatt acgcttgagc
     1801 gagacgggat cactagtgaa ttcgcggccg cctgcaggtc gaccatatgg gagagctccc
     1861 aacgcgttgg atgcatagct tgagtattct atagtgtcac ctaaatagct tggcgtaatc
     1921 atggtcatag ctgtttcctg tgtgaaattg ttatccgctc acaattccac acaacatacg
     1981 agccggaagc ataaagtgta aagcctgggg tgcctaatga gtgagctaac tcacattaat
     2041 tgcgttgcgc tcactgcccg ctttccagtc gggaaacctg tcgtgccagc tgcattaatg
     2101 aatcggccaa cgcgcgggga gaggcggttt gcgtattggg cgctcttccg cttcctcgct
     2161 cactgactcg ctgcgctcgg tcgttcggct gcggcgagcg gtatcagctc actcaaaggc
     2221 ggtaatacgg ttatccacag aatcagggga taacgcagga aagaacatgt gagcaaaagg
     2281 ccagcaaaag gccaggaacc gtaaaaaggc cgcgttgctg gcgtttttcc ataggctccg
     2341 cccccctgac gagcatcaca aaaatcgacg ctcaagtcag aggtggcgaa acccgacagg
     2401 actataaaga taccaggcgt ttccccctgg aagctccctc gtgcgctctc ctgttccgac
     2461 cctgccgctt accggatacc tgtccgcctt tctcccttcg ggaagcgtgg cgctttctca
     2521 tagctcacgc tgtaggtatc tcagttcggt gtaggtcgtt cgctccaagc tgggctgtgt
     2581 gcacgaaccc cccgttcagc ccgaccgctg cgccttatcc ggtaactatc gtcttgagtc
     2641 caacccggta agacacgact tatcgccact ggcagcagcc actggtaaca ggattagcag
     2701 agcgaggtat gtaggcggtg ctacagagtt cttgaagtgg tggcctaact acggctacac
     2761 tagaagaaca gtatttggta tctgcgctct gctgaagcca gttaccttcg gaaaaagagt
     2821 tggtagctct tgatccggca aacaaaccac cgctggtagc ggtggttttt ttgtttgcaa
     2881 gcagcagatt acgcgcagaa aaaaaggatc tcaagaagat cctttgatct tttctacggg
     2941 gtctgacgct cagtggaacg aaaactcacg ttaagggatt ttggtcatga gattatcaaa
     3001 aaggatcttc acctagatcc ttttaaatta aaaatgaagt tttaaatcaa tctaaagtat
     3061 atatgagtaa acttggtctg acagttacca atgcttaatc agtgaggcac ctatctcagc
     3121 gatctgtcta tttcgttcat ccatagttgc ctgactcccc gtcgtgtaga taactacgat
     3181 acgggagggc ttaccatctg gccccagtgc tgcaatgata ccgcgagacc cacgctcacc
     3241 ggctccagat ttatcagcaa taaaccagcc agccggaagg gccgagcgca gaagtggtcc
     3301 tgcaacttta tccgcctcca
//
