LOCUS       sequence-319679-        3227 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  tRNA and scaffold for the assembly of GBoligomers for the position 
            [4_(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 3227)
  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 3227)
  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 E4-En-1 (GB2243)
FEATURES             Location/Qualifiers
     source          1..3227
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     promoter        complement(189..293)
                     /gene="bla"
                     /label=AmpR promoter
     rep_origin      complement(371..826)
                     /direction=LEFT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(508..529)
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     720..739
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     952..974
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     primer_bind     966..983
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     967..983
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     990..1009
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        990..1008
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     regulatory      1039..1048
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     regulatory      1043..1052
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     misc_RNA        1188..1263
                     /label=gRNA scaffold
                     /note="guide RNA scaffold for the Streptococcus pyogenes 
                     CRISPR/Cas9 system"
     promoter        complement(1358..1376)
                     /label=SP6 promoter
                     /note="promoter for bacteriophage SP6 RNA polymerase"
     primer_bind     complement(1359..1376)
                     /label=SP6
                     /note="SP6 promoter, forward primer"
     primer_bind     complement(1394..1410)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(1394..1410)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(1407..1429)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    1418..1434
                     /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(1442..1448,1449..1466,1467..1472))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    1487..1508
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(1625..1642)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(1796..2384)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(1876..1895)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(2555..3227,1..119,120..188))
                     /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     3178..3197
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
ORIGIN
        1 cgttcttcgg ggcgaaaact ctcaaggatc ttaccgctgt tgagatccag ttcgatgtaa
       61 cccactcgtg cacccaactg atcttcagca tcttttactt tcaccagcgt ttctgggtga
      121 gcaaaaacag gaaggcaaaa tgccgcaaaa aagggaataa gggcgacacg gaaatgttga
      181 atactcatac tcttcctttt tcaatattat tgaagcattt atcagggtta ttgtctcatg
      241 agcggataca tatttgaatg tatttagaaa aataaacaaa taggggttcc gcgcacattt
      301 ccccgaaaag tgccacctga tgcggtgtga aataccgcac agatgcgtaa ggagaaaata
      361 ccgcatcagg aaattgtaag cgttaatatt ttgttaaaat tcgcgttaaa tttttgttaa
      421 atcagctcat tttttaacca ataggccgaa atcggcaaaa tcccttataa atcaaaagaa
      481 tagaccgaga tagggttgag tgttgttcca gtttggaaca agagtccact attaaagaac
      541 gtggactcca acgtcaaagg gcgaaaaacc gtctatcagg gcgatggccc actacgtgaa
      601 ccatcaccct aatcaagttt tttggggtcg aggtgccgta aagcactaaa tcggaaccct
      661 aaagggagcc cccgatttag agcttgacgg ggaaagccgg cgaacgtggc gagaaaggaa
      721 gggaagaaag cgaaaggagc gggcgctagg gcgctggcaa gtgtagcggt cacgctgcgc
      781 gtaaccacca cacccgccgc gcttaatgcg ccgctacagg gcgcgtccat tcgccattca
      841 ggctgcgcaa ctgttgggaa gggcgatcgg tgcgggcctc ttcgctatta cgccagctgg
      901 cgaaaggggg atgtgctgca aggcgattaa gttgggtaac gccagggttt tcccagtcac
      961 gacgttgtaa aacgacggcc agtgaattgt aatacgactc actatagggc gaattgggcc
     1021 cgacgtcgca tgctcccggc cgccatggcg gccgcgggaa ttcgattccc gtctcgctcg
     1081 agtcggtgca acaaagcacc agtggtctag tggtagaata gtaccctgcc acggtacaga
     1141 cccgggttcg attcccggct ggtgcagaga cggcgcgcgc cgtctcagtt ttagagctag
     1201 aaatagcaag ttaaaataag gctagtccgt tatcaacttg aaaaagtggc accgagtcgg
     1261 tgcaatgagc gagacgggat cactagtgaa ttcgcggccg cctgcaggtc gaccatatgg
     1321 gagagctccc aacgcgttgg atgcatagct tgagtattct atagtgtcac ctaaatagct
     1381 tggcgtaatc atggtcatag ctgtttcctg tgtgaaattg ttatccgctc acaattccac
     1441 acaacatacg agccggaagc ataaagtgta aagcctgggg tgcctaatga gtgagctaac
     1501 tcacattaat tgcgttgcgc tcactgcccg ctttccagtc gggaaacctg tcgtgccagc
     1561 tgcattaatg aatcggccaa cgcgcgggga gaggcggttt gcgtattggg cgctcttccg
     1621 cttcctcgct cactgactcg ctgcgctcgg tcgttcggct gcggcgagcg gtatcagctc
     1681 actcaaaggc ggtaatacgg ttatccacag aatcagggga taacgcagga aagaacatgt
     1741 gagcaaaagg ccagcaaaag gccaggaacc gtaaaaaggc cgcgttgctg gcgtttttcc
     1801 ataggctccg cccccctgac gagcatcaca aaaatcgacg ctcaagtcag aggtggcgaa
     1861 acccgacagg actataaaga taccaggcgt ttccccctgg aagctccctc gtgcgctctc
     1921 ctgttccgac cctgccgctt accggatacc tgtccgcctt tctcccttcg ggaagcgtgg
     1981 cgctttctca tagctcacgc tgtaggtatc tcagttcggt gtaggtcgtt cgctccaagc
     2041 tgggctgtgt gcacgaaccc cccgttcagc ccgaccgctg cgccttatcc ggtaactatc
     2101 gtcttgagtc caacccggta agacacgact tatcgccact ggcagcagcc actggtaaca
     2161 ggattagcag agcgaggtat gtaggcggtg ctacagagtt cttgaagtgg tggcctaact
     2221 acggctacac tagaagaaca gtatttggta tctgcgctct gctgaagcca gttaccttcg
     2281 gaaaaagagt tggtagctct tgatccggca aacaaaccac cgctggtagc ggtggttttt
     2341 ttgtttgcaa gcagcagatt acgcgcagaa aaaaaggatc tcaagaagat cctttgatct
     2401 tttctacggg gtctgacgct cagtggaacg aaaactcacg ttaagggatt ttggtcatga
     2461 gattatcaaa aaggatcttc acctagatcc ttttaaatta aaaatgaagt tttaaatcaa
     2521 tctaaagtat atatgagtaa acttggtctg acagttacca atgcttaatc agtgaggcac
     2581 ctatctcagc gatctgtcta tttcgttcat ccatagttgc ctgactcccc gtcgtgtaga
     2641 taactacgat acgggagggc ttaccatctg gccccagtgc tgcaatgata ccgcgagacc
     2701 cacgctcacc ggctccagat ttatcagcaa taaaccagcc agccggaagg gccgagcgca
     2761 gaagtggtcc tgcaacttta tccgcctcca tccagtctat taattgttgc cgggaagcta
     2821 gagtaagtag ttcgccagtt aatagtttgc gcaacgttgt tgccattgct acaggcatcg
     2881 tggtgtcacg ctcgtcgttt ggtatggctt cattcagctc cggttcccaa cgatcaaggc
     2941 gagttacatg atcccccatg ttgtgcaaaa aagcggttag ctccttcggt cctccgatcg
     3001 ttgtcagaag taagttggcc gcagtgttat cactcatggt tatggcagca ctgcataatt
     3061 ctcttactgt catgccatcc gtaagatgct tttctgtgac tggtgagtac tcaaccaagt
     3121 cattctgaga atagtgtatg cggcgaccga gttgctcttg cccggcgtca atacgggata
     3181 ataccgcgcc acatagcaga actttaaaag tgctcatcat tggaaaa
//
