LOCUS       sequence-319301-        3218 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  tRNA and scaffold for the assembly of GBoligomers for the third 
            position (positon [D3_4]) 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 3218)
  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 3218)
  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 E3 (GB2240)
FEATURES             Location/Qualifiers
     source          1..3218
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     promoter        complement(181..285)
                     /gene="bla"
                     /label=AmpR promoter
     rep_origin      complement(363..818)
                     /direction=LEFT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(500..521)
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     712..731
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     944..966
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     primer_bind     958..975
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     959..975
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     982..1001
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        982..1000
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     regulatory      1031..1040
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     regulatory      1035..1044
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     misc_RNA        1178..1253
                     /label=gRNA scaffold
                     /note="guide RNA scaffold for the Streptococcus pyogenes 
                     CRISPR/Cas9 system"
     promoter        complement(1341..1359)
                     /label=SP6 promoter
                     /note="promoter for bacteriophage SP6 RNA polymerase"
     primer_bind     complement(1342..1359)
                     /label=SP6
                     /note="SP6 promoter, forward primer"
     primer_bind     complement(1377..1393)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(1377..1393)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(1390..1412)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    1401..1417
                     /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(1425..1431,1432..1449,1450..1455))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    1470..1491
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(1608..1625)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(1779..2367)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(1859..1878)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(2538..3218,1..111,112..180))
                     /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     3161..3180
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
ORIGIN
        1 ggggcgaaaa ctctcaagga tcttaccgct gttgagatcc agttcgatgt aacccactcg
       61 tgcacccaac tgatcttcag catcttttac tttcaccagc gtttctgggt gagcaaaaac
      121 aggaaggcaa aatgccgcaa aaaagggaat aagggcgaca cggaaatgtt gaatactcat
      181 actcttcctt tttcaatatt attgaagcat ttatcagggt tattgtctca tgagcggata
      241 catatttgaa tgtatttaga aaaataaaca aataggggtt ccgcgcacat ttccccgaaa
      301 agtgccacct gatgcggtgt gaaataccgc acagatgcgt aaggagaaaa taccgcatca
      361 ggaaattgta agcgttaata ttttgttaaa attcgcgtta aatttttgtt aaatcagctc
      421 attttttaac caataggccg aaatcggcaa aatcccttat aaatcaaaag aatagaccga
      481 gatagggttg agtgttgttc cagtttggaa caagagtcca ctattaaaga acgtggactc
      541 caacgtcaaa gggcgaaaaa ccgtctatca gggcgatggc ccactacgtg aaccatcacc
      601 ctaatcaagt tttttggggt cgaggtgccg taaagcacta aatcggaacc ctaaagggag
      661 cccccgattt agagcttgac ggggaaagcc ggcgaacgtg gcgagaaagg aagggaagaa
      721 agcgaaagga gcgggcgcta gggcgctggc aagtgtagcg gtcacgctgc gcgtaaccac
      781 cacacccgcc gcgcttaatg cgccgctaca gggcgcgtcc attcgccatt caggctgcgc
      841 aactgttggg aagggcgatc ggtgcgggcc tcttcgctat tacgccagct ggcgaaaggg
      901 ggatgtgctg caaggcgatt aagttgggta acgccagggt tttcccagtc acgacgttgt
      961 aaaacgacgg ccagtgaatt gtaatacgac tcactatagg gcgaattggg cccgacgtcg
     1021 catgctcccg gccgccatgg cggccgcggg aattcgattc ccgtctcgct cgtcggtgca
     1081 acaaagcacc agtggtctag tggtagaata gtaccctgcc acggtacaga cccgggttcg
     1141 attcccggct ggtgcagaga cggcgcgcgc cgtctcagtt ttagagctag aaatagcaag
     1201 ttaaaataag gctagtccgt tatcaacttg aaaaagtggc accgagtctg agcgagacgg
     1261 gatcactagt gaattcgcgg ccgcctgcag gtcgaccata tgggagagct cccaacgcgt
     1321 tggatgcata gcttgagtat tctatagtgt cacctaaata gcttggcgta atcatggtca
     1381 tagctgtttc ctgtgtgaaa ttgttatccg ctcacaattc cacacaacat acgagccgga
     1441 agcataaagt gtaaagcctg gggtgcctaa tgagtgagct aactcacatt aattgcgttg
     1501 cgctcactgc ccgctttcca gtcgggaaac ctgtcgtgcc agctgcatta atgaatcggc
     1561 caacgcgcgg ggagaggcgg tttgcgtatt gggcgctctt ccgcttcctc gctcactgac
     1621 tcgctgcgct cggtcgttcg gctgcggcga gcggtatcag ctcactcaaa ggcggtaata
     1681 cggttatcca cagaatcagg ggataacgca ggaaagaaca tgtgagcaaa aggccagcaa
     1741 aaggccagga accgtaaaaa ggccgcgttg ctggcgtttt tccataggct ccgcccccct
     1801 gacgagcatc acaaaaatcg acgctcaagt cagaggtggc gaaacccgac aggactataa
     1861 agataccagg cgtttccccc tggaagctcc ctcgtgcgct ctcctgttcc gaccctgccg
     1921 cttaccggat acctgtccgc ctttctccct tcgggaagcg tggcgctttc tcatagctca
     1981 cgctgtaggt atctcagttc ggtgtaggtc gttcgctcca agctgggctg tgtgcacgaa
     2041 ccccccgttc agcccgaccg ctgcgcctta tccggtaact atcgtcttga gtccaacccg
     2101 gtaagacacg acttatcgcc actggcagca gccactggta acaggattag cagagcgagg
     2161 tatgtaggcg gtgctacaga gttcttgaag tggtggccta actacggcta cactagaaga
     2221 acagtatttg gtatctgcgc tctgctgaag ccagttacct tcggaaaaag agttggtagc
     2281 tcttgatccg gcaaacaaac caccgctggt agcggtggtt tttttgtttg caagcagcag
     2341 attacgcgca gaaaaaaagg atctcaagaa gatcctttga tcttttctac ggggtctgac
     2401 gctcagtgga acgaaaactc acgttaaggg attttggtca tgagattatc aaaaaggatc
     2461 ttcacctaga tccttttaaa ttaaaaatga agttttaaat caatctaaag tatatatgag
     2521 taaacttggt ctgacagtta ccaatgctta atcagtgagg cacctatctc agcgatctgt
     2581 ctatttcgtt catccatagt tgcctgactc cccgtcgtgt agataactac gatacgggag
     2641 ggcttaccat ctggccccag tgctgcaatg ataccgcgag acccacgctc accggctcca
     2701 gatttatcag caataaacca gccagccgga agggccgagc gcagaagtgg tcctgcaact
     2761 ttatccgcct ccatccagtc tattaattgt tgccgggaag ctagagtaag tagttcgcca
     2821 gttaatagtt tgcgcaacgt tgttgccatt gctacaggca tcgtggtgtc acgctcgtcg
     2881 tttggtatgg cttcattcag ctccggttcc caacgatcaa ggcgagttac atgatccccc
     2941 atgttgtgca aaaaagcggt tagctccttc ggtcctccga tcgttgtcag aagtaagttg
     3001 gccgcagtgt tatcactcat ggttatggca gcactgcata attctcttac tgtcatgcca
     3061 tccgtaagat gcttttctgt gactggtgag tactcaacca agtcattctg agaatagtgt
     3121 atgcggcgac cgagttgctc ttgcccggcg tcaatacggg ataataccgc gccacatagc
     3181 agaactttaa aagtgctcat cattggaaaa cgttcttc
//
