LOCUS       sequence-319675-        3229 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  tRNA and scaffold for the assembly of GBoligomers for the position 
            [5_(n-1)] 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 3229)
  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 3229)
  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 Multiplexing Edit En-1 (GB2242)
FEATURES             Location/Qualifiers
     source          1..3229
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     CDS             complement(join(105..896,897..965))
                     /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     728..747
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(966..1070)
                     /gene="bla"
                     /label=AmpR promoter
     rep_origin      complement(1148..1603)
                     /direction=LEFT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(1285..1306)
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     1497..1516
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     1729..1751
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     primer_bind     1743..1760
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     1744..1760
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     1767..1786
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        1767..1785
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     regulatory      1816..1825
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     regulatory      1820..1829
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     misc_RNA        complement(1860..1935)
                     /label=gRNA scaffold
                     /note="guide RNA scaffold for the Streptococcus pyogenes 
                     CRISPR/Cas9 system"
     promoter        complement(2137..2155)
                     /label=SP6 promoter
                     /note="promoter for bacteriophage SP6 RNA polymerase"
     primer_bind     complement(2138..2155)
                     /label=SP6
                     /note="SP6 promoter, forward primer"
     primer_bind     complement(2173..2189)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(2173..2189)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(2186..2208)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    2197..2213
                     /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(2221..2227,2228..2245,2246..2251))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    2266..2287
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(2404..2421)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(2575..3163)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(2655..2674)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
ORIGIN
        1 ttggtcatga gattatcaaa aaggatcttc acctagatcc ttttaaatta aaaatgaagt
       61 tttaaatcaa tctaaagtat atatgagtaa acttggtctg acagttacca atgcttaatc
      121 agtgaggcac ctatctcagc gatctgtcta tttcgttcat ccatagttgc ctgactcccc
      181 gtcgtgtaga taactacgat acgggagggc ttaccatctg gccccagtgc tgcaatgata
      241 ccgcgagacc cacgctcacc ggctccagat ttatcagcaa taaaccagcc agccggaagg
      301 gccgagcgca gaagtggtcc tgcaacttta tccgcctcca tccagtctat taattgttgc
      361 cgggaagcta gagtaagtag ttcgccagtt aatagtttgc gcaacgttgt tgccattgct
      421 acaggcatcg tggtgtcacg ctcgtcgttt ggtatggctt cattcagctc cggttcccaa
      481 cgatcaaggc gagttacatg atcccccatg ttgtgcaaaa aagcggttag ctccttcggt
      541 cctccgatcg ttgtcagaag taagttggcc gcagtgttat cactcatggt tatggcagca
      601 ctgcataatt ctcttactgt catgccatcc gtaagatgct tttctgtgac tggtgagtac
      661 tcaaccaagt cattctgaga atagtgtatg cggcgaccga gttgctcttg cccggcgtca
      721 atacgggata ataccgcgcc acatagcaga actttaaaag tgctcatcat tggaaaacgt
      781 tcttcggggc gaaaactctc aaggatctta ccgctgttga gatccagttc gatgtaaccc
      841 actcgtgcac ccaactgatc ttcagcatct tttactttca ccagcgtttc tgggtgagca
      901 aaaacaggaa ggcaaaatgc cgcaaaaaag ggaataaggg cgacacggaa atgttgaata
      961 ctcatactct tcctttttca atattattga agcatttatc agggttattg tctcatgagc
     1021 ggatacatat ttgaatgtat ttagaaaaat aaacaaatag gggttccgcg cacatttccc
     1081 cgaaaagtgc cacctgatgc ggtgtgaaat accgcacaga tgcgtaagga gaaaataccg
     1141 catcaggaaa ttgtaagcgt taatattttg ttaaaattcg cgttaaattt ttgttaaatc
     1201 agctcatttt ttaaccaata ggccgaaatc ggcaaaatcc cttataaatc aaaagaatag
     1261 accgagatag ggttgagtgt tgttccagtt tggaacaaga gtccactatt aaagaacgtg
     1321 gactccaacg tcaaagggcg aaaaaccgtc tatcagggcg atggcccact acgtgaacca
     1381 tcaccctaat caagtttttt ggggtcgagg tgccgtaaag cactaaatcg gaaccctaaa
     1441 gggagccccc gatttagagc ttgacgggga aagccggcga acgtggcgag aaaggaaggg
     1501 aagaaagcga aaggagcggg cgctagggcg ctggcaagtg tagcggtcac gctgcgcgta
     1561 accaccacac ccgccgcgct taatgcgccg ctacagggcg cgtccattcg ccattcaggc
     1621 tgcgcaactg ttgggaaggg cgatcggtgc gggcctcttc gctattacgc cagctggcga
     1681 aagggggatg tgctgcaagg cgattaagtt gggtaacgcc agggttttcc cagtcacgac
     1741 gttgtaaaac gacggccagt gaattgtaat acgactcact atagggcgaa ttgggcccga
     1801 cgtcgcatgc tcccggccgc catggcggcc gcgggaattc gattcccgtc tcgctcattg
     1861 caccgactcg gtgccacttt ttcaagttga taacggacta gccttatttt aacttgctat
     1921 ttctagctct aaaactgaga cggcgcgcgc cgtctctgca ccagccggga atcgaacccg
     1981 ggtctgtacc gtggcagggt actattctac cactagacca ctggtgcttt gttgcaccga
     2041 ctcgcgagcg agacgggatc actagtgaat tcgcggccgc ctgcaggtcg accatatggg
     2101 agagctccca acgcgttgga tgcatagctt gagtattcta tagtgtcacc taaatagctt
     2161 ggcgtaatca tggtcatagc tgtttcctgt gtgaaattgt tatccgctca caattccaca
     2221 caacatacga gccggaagca taaagtgtaa agcctggggt gcctaatgag tgagctaact
     2281 cacattaatt gcgttgcgct cactgcccgc tttccagtcg ggaaacctgt cgtgccagct
     2341 gcattaatga atcggccaac gcgcggggag aggcggtttg cgtattgggc gctcttccgc
     2401 ttcctcgctc actgactcgc tgcgctcggt cgttcggctg cggcgagcgg tatcagctca
     2461 ctcaaaggcg gtaatacggt tatccacaga atcaggggat aacgcaggaa agaacatgtg
     2521 agcaaaaggc cagcaaaagg ccaggaaccg taaaaaggcc gcgttgctgg cgtttttcca
     2581 taggctccgc ccccctgacg agcatcacaa aaatcgacgc tcaagtcaga ggtggcgaaa
     2641 cccgacagga ctataaagat accaggcgtt tccccctgga agctccctcg tgcgctctcc
     2701 tgttccgacc ctgccgctta ccggatacct gtccgccttt ctcccttcgg gaagcgtggc
     2761 gctttctcat agctcacgct gtaggtatct cagttcggtg taggtcgttc gctccaagct
     2821 gggctgtgtg cacgaacccc ccgttcagcc cgaccgctgc gccttatccg gtaactatcg
     2881 tcttgagtcc aacccggtaa gacacgactt atcgccactg gcagcagcca ctggtaacag
     2941 gattagcaga gcgaggtatg taggcggtgc tacagagttc ttgaagtggt ggcctaacta
     3001 cggctacact agaagaacag tatttggtat ctgcgctctg ctgaagccag ttaccttcgg
     3061 aaaaagagtt ggtagctctt gatccggcaa acaaaccacc gctggtagcg gtggtttttt
     3121 tgtttgcaag cagcagatta cgcgcagaaa aaaaggatct caagaagatc ctttgatctt
     3181 ttctacgggg tctgacgctc agtggaacga aaactcacgt taagggatt
//
