LOCUS       sequence-319298-        3217 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  tRNA and scaffold for the assembly of GBoligomers for position [2-3]
            of a polycistronic tRNA-gRNA.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 3217)
  AUTHORS   Vazquez-Vilar M, Garcia-Carpintero V, Selma S, Bernabe-Orts JM, 
            Sanchez-Vicente J, Salazar-Sarasua B, Ressa A, de Paola C, Ajenjo M,
            Quintela JC, Fernandez-Del-Carmen A, Granell A, Orzaez D
  TITLE     The GB4.0 Platform, an All-In-One Tool for CRISPR/Cas-Based 
            Multiplex Genome Engineering in Plants.
  JOURNAL   Front Plant Sci. 2021 Jul 1;12:689937. doi: 
            10.3389/fpls.2021.689937. eCollection 2021.
  PUBMED    34276739
REFERENCE   2  (bases 1 to 3217)
  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: "Front Plant
            Sci."; date: "2021-07-1"; volume: "12:689937. doi"; pages: " 
            10.3389/fpls.2021.689937. eCollection 2021"
COMMENT     Sequence Label: pVD1 tRNA-gRNA position E2 (GB2239)
FEATURES             Location/Qualifiers
     source          1..3217
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     rep_origin      complement(121..709)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(201..220)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(880..1671,1672..1740))
                     /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     1503..1522
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(1741..1845)
                     /gene="bla"
                     /label=AmpR promoter
     rep_origin      complement(1923..2378)
                     /direction=LEFT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(2060..2081)
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     2272..2291
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     2504..2526
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     primer_bind     2518..2535
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     2519..2535
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     2542..2561
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        2542..2560
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     regulatory      2591..2600
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     regulatory      2595..2604
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     misc_RNA        complement(2631..2705)
                     /label=gRNA scaffold
                     /note="guide RNA scaffold for the Streptococcus pyogenes 
                     CRISPR/Cas9 system"
     promoter        complement(2900..2918)
                     /label=SP6 promoter
                     /note="promoter for bacteriophage SP6 RNA polymerase"
     primer_bind     complement(2901..2918)
                     /label=SP6
                     /note="SP6 promoter, forward primer"
     primer_bind     complement(2936..2952)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(2936..2952)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(2949..2971)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    2960..2976
                     /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(2984..2990,2991..3008,3009..3014))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    3029..3050
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
ORIGIN
        1 agctcactca aaggcggtaa tacggttatc cacagaatca ggggataacg caggaaagaa
       61 catgtgagca aaaggccagc aaaaggccag gaaccgtaaa aaggccgcgt tgctggcgtt
      121 tttccatagg ctccgccccc ctgacgagca tcacaaaaat cgacgctcaa gtcagaggtg
      181 gcgaaacccg acaggactat aaagatacca ggcgtttccc cctggaagct ccctcgtgcg
      241 ctctcctgtt ccgaccctgc cgcttaccgg atacctgtcc gcctttctcc cttcgggaag
      301 cgtggcgctt tctcatagct cacgctgtag gtatctcagt tcggtgtagg tcgttcgctc
      361 caagctgggc tgtgtgcacg aaccccccgt tcagcccgac cgctgcgcct tatccggtaa
      421 ctatcgtctt gagtccaacc cggtaagaca cgacttatcg ccactggcag cagccactgg
      481 taacaggatt agcagagcga ggtatgtagg cggtgctaca gagttcttga agtggtggcc
      541 taactacggc tacactagaa gaacagtatt tggtatctgc gctctgctga agccagttac
      601 cttcggaaaa agagttggta gctcttgatc cggcaaacaa accaccgctg gtagcggtgg
      661 tttttttgtt tgcaagcagc agattacgcg cagaaaaaaa ggatctcaag aagatccttt
      721 gatcttttct acggggtctg acgctcagtg gaacgaaaac tcacgttaag ggattttggt
      781 catgagatta tcaaaaagga tcttcaccta gatcctttta aattaaaaat gaagttttaa
      841 atcaatctaa agtatatatg agtaaacttg gtctgacagt taccaatgct taatcagtga
      901 ggcacctatc tcagcgatct gtctatttcg ttcatccata gttgcctgac tccccgtcgt
      961 gtagataact acgatacggg agggcttacc atctggcccc agtgctgcaa tgataccgcg
     1021 agacccacgc tcaccggctc cagatttatc agcaataaac cagccagccg gaagggccga
     1081 gcgcagaagt ggtcctgcaa ctttatccgc ctccatccag tctattaatt gttgccggga
     1141 agctagagta agtagttcgc cagttaatag tttgcgcaac gttgttgcca ttgctacagg
     1201 catcgtggtg tcacgctcgt cgtttggtat ggcttcattc agctccggtt cccaacgatc
     1261 aaggcgagtt acatgatccc ccatgttgtg caaaaaagcg gttagctcct tcggtcctcc
     1321 gatcgttgtc agaagtaagt tggccgcagt gttatcactc atggttatgg cagcactgca
     1381 taattctctt actgtcatgc catccgtaag atgcttttct gtgactggtg agtactcaac
     1441 caagtcattc tgagaatagt gtatgcggcg accgagttgc tcttgcccgg cgtcaatacg
     1501 ggataatacc gcgccacata gcagaacttt aaaagtgctc atcattggaa aacgttcttc
     1561 ggggcgaaaa ctctcaagga tcttaccgct gttgagatcc agttcgatgt aacccactcg
     1621 tgcacccaac tgatcttcag catcttttac tttcaccagc gtttctgggt gagcaaaaac
     1681 aggaaggcaa aatgccgcaa aaaagggaat aagggcgaca cggaaatgtt gaatactcat
     1741 actcttcctt tttcaatatt attgaagcat ttatcagggt tattgtctca tgagcggata
     1801 catatttgaa tgtatttaga aaaataaaca aataggggtt ccgcgcacat ttccccgaaa
     1861 agtgccacct gatgcggtgt gaaataccgc acagatgcgt aaggagaaaa taccgcatca
     1921 ggaaattgta agcgttaata ttttgttaaa attcgcgtta aatttttgtt aaatcagctc
     1981 attttttaac caataggccg aaatcggcaa aatcccttat aaatcaaaag aatagaccga
     2041 gatagggttg agtgttgttc cagtttggaa caagagtcca ctattaaaga acgtggactc
     2101 caacgtcaaa gggcgaaaaa ccgtctatca gggcgatggc ccactacgtg aaccatcacc
     2161 ctaatcaagt tttttggggt cgaggtgccg taaagcacta aatcggaacc ctaaagggag
     2221 cccccgattt agagcttgac ggggaaagcc ggcgaacgtg gcgagaaagg aagggaagaa
     2281 agcgaaagga gcgggcgcta gggcgctggc aagtgtagcg gtcacgctgc gcgtaaccac
     2341 cacacccgcc gcgcttaatg cgccgctaca gggcgcgtcc attcgccatt caggctgcgc
     2401 aactgttggg aagggcgatc ggtgcgggcc tcttcgctat tacgccagct ggcgaaaggg
     2461 ggatgtgctg caaggcgatt aagttgggta acgccagggt tttcccagtc acgacgttgt
     2521 aaaacgacgg ccagtgaatt gtaatacgac tcactatagg gcgaattggg cccgacgtcg
     2581 catgctcccg gccgccatgg cggccgcggg aattcgattc ccgtctcgct caccgactcg
     2641 gtgccacttt ttcaagttga taacggacta gccttatttt aacttgctat ttctagctct
     2701 aaaactgaga cggcgcgcgc cgtctctgca ccagccggga atcgaacccg ggtctgtacc
     2761 gtggcagggt actattctac cactagacca ctggtgcttt gttgcaccga gcgagacggg
     2821 atcactagtg aattcgcggc cgcctgcagg tcgaccatat gggagagctc ccaacgcgtt
     2881 ggatgcatag cttgagtatt ctatagtgtc acctaaatag cttggcgtaa tcatggtcat
     2941 agctgtttcc tgtgtgaaat tgttatccgc tcacaattcc acacaacata cgagccggaa
     3001 gcataaagtg taaagcctgg ggtgcctaat gagtgagcta actcacatta attgcgttgc
     3061 gctcactgcc cgctttccag tcgggaaacc tgtcgtgcca gctgcattaa tgaatcggcc
     3121 aacgcgcggg gagaggcggt ttgcgtattg ggcgctcttc cgcttcctcg ctcactgact
     3181 cgctgcgctc ggtcgttcgg ctgcggcgag cggtatc
//
