LOCUS       sequence-310925-        3228 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 3228)
  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 3228)
  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..3228
                     /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        1966..2041
                     /label=gRNA scaffold
                     /note="guide RNA scaffold for the Streptococcus pyogenes 
                     CRISPR/Cas9 system"
     promoter        complement(2136..2154)
                     /label=SP6 promoter
                     /note="promoter for bacteriophage SP6 RNA polymerase"
     primer_bind     complement(2137..2154)
                     /label=SP6
                     /note="SP6 promoter, forward primer"
     primer_bind     complement(2172..2188)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(2172..2188)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(2185..2207)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    2196..2212
                     /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(2220..2226,2227..2244,2245..2250))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    2265..2286
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(2403..2420)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(2574..3162)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(2654..2673)
                     /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 gatcccgtct cgctcgcgag
     1861 tcggtgcaac aaagcaccag tggtctagtg gtagaatagt accctgccac ggtacagacc
     1921 cgggttcgat tcccggctgg tgcagagacg gcgcgcgccg tctcagtttt agagctagaa
     1981 atagcaagtt aaaataaggc tagtccgtta tcaacttgaa aaagtggcac cgagtcggtg
     2041 caatgagcga gacgggatca ctagtgaatt cgcggccgcc tgcaggtcga ccatatggga
     2101 gagctcccaa cgcgttggat gcatagcttg agtattctat agtgtcacct aaatagcttg
     2161 gcgtaatcat ggtcatagct gtttcctgtg tgaaattgtt atccgctcac aattccacac
     2221 aacatacgag ccggaagcat aaagtgtaaa gcctggggtg cctaatgagt gagctaactc
     2281 acattaattg cgttgcgctc actgcccgct ttccagtcgg gaaacctgtc gtgccagctg
     2341 cattaatgaa tcggccaacg cgcggggaga ggcggtttgc gtattgggcg ctcttccgct
     2401 tcctcgctca ctgactcgct gcgctcggtc gttcggctgc ggcgagcggt atcagctcac
     2461 tcaaaggcgg taatacggtt atccacagaa tcaggggata acgcaggaaa gaacatgtga
     2521 gcaaaaggcc agcaaaaggc caggaaccgt aaaaaggccg cgttgctggc gtttttccat
     2581 aggctccgcc cccctgacga gcatcacaaa aatcgacgct caagtcagag gtggcgaaac
     2641 ccgacaggac tataaagata ccaggcgttt ccccctggaa gctccctcgt gcgctctcct
     2701 gttccgaccc tgccgcttac cggatacctg tccgcctttc tcccttcggg aagcgtggcg
     2761 ctttctcata gctcacgctg taggtatctc agttcggtgt aggtcgttcg ctccaagctg
     2821 ggctgtgtgc acgaaccccc cgttcagccc gaccgctgcg ccttatccgg taactatcgt
     2881 cttgagtcca acccggtaag acacgactta tcgccactgg cagcagccac tggtaacagg
     2941 attagcagag cgaggtatgt aggcggtgct acagagttct tgaagtggtg gcctaactac
     3001 ggctacacta gaagaacagt atttggtatc tgcgctctgc tgaagccagt taccttcgga
     3061 aaaagagttg gtagctcttg atccggcaaa caaaccaccg ctggtagcgg tggttttttt
     3121 gtttgcaagc agcagattac gcgcagaaaa aaaggatctc aagaagatcc tttgatcttt
     3181 tctacggggt ctgacgctca gtggaacgaa aactcacgtt aagggatt
//
