LOCUS       sequence-319680-        3225 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  GBoligomers for the position [3_(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 3225)
  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 3225)
  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 E3-E4-En-1 (GB2244)
FEATURES             Location/Qualifiers
     source          1..3225
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     394..413
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(632..736)
                     /gene="bla"
                     /label=AmpR promoter
     rep_origin      complement(814..1269)
                     /direction=LEFT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(951..972)
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     1163..1182
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     1395..1417
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     primer_bind     1409..1426
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     1410..1426
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     1433..1452
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        1433..1451
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     regulatory      1482..1491
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     regulatory      1486..1495
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     misc_RNA        1629..1704
                     /label=gRNA scaffold
                     /note="guide RNA scaffold for the Streptococcus pyogenes 
                     CRISPR/Cas9 system"
     promoter        complement(1799..1817)
                     /label=SP6 promoter
                     /note="promoter for bacteriophage SP6 RNA polymerase"
     primer_bind     complement(1800..1817)
                     /label=SP6
                     /note="SP6 promoter, forward primer"
     primer_bind     complement(1835..1851)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(1835..1851)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(1848..1870)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    1859..1875
                     /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(1883..1889,1890..1907,1908..1913))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    1928..1949
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(2066..2083)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(2237..2825)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(2317..2336)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(2996..3225,1..562,563..631))
                     /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"
ORIGIN
        1 cctccatcca gtctattaat tgttgccggg aagctagagt aagtagttcg ccagttaata
       61 gtttgcgcaa cgttgttgcc attgctacag gcatcgtggt gtcacgctcg tcgtttggta
      121 tggcttcatt cagctccggt tcccaacgat caaggcgagt tacatgatcc cccatgttgt
      181 gcaaaaaagc ggttagctcc ttcggtcctc cgatcgttgt cagaagtaag ttggccgcag
      241 tgttatcact catggttatg gcagcactgc ataattctct tactgtcatg ccatccgtaa
      301 gatgcttttc tgtgactggt gagtactcaa ccaagtcatt ctgagaatag tgtatgcggc
      361 gaccgagttg ctcttgcccg gcgtcaatac gggataatac cgcgccacat agcagaactt
      421 taaaagtgct catcattgga aaacgttctt cggggcgaaa actctcaagg atcttaccgc
      481 tgttgagatc cagttcgatg taacccactc gtgcacccaa ctgatcttca gcatctttta
      541 ctttcaccag cgtttctggg tgagcaaaaa caggaaggca aaatgccgca aaaaagggaa
      601 taagggcgac acggaaatgt tgaatactca tactcttcct ttttcaatat tattgaagca
      661 tttatcaggg ttattgtctc atgagcggat acatatttga atgtatttag aaaaataaac
      721 aaataggggt tccgcgcaca tttccccgaa aagtgccacc tgatgcggtg tgaaataccg
      781 cacagatgcg taaggagaaa ataccgcatc aggaaattgt aagcgttaat attttgttaa
      841 aattcgcgtt aaatttttgt taaatcagct cattttttaa ccaataggcc gaaatcggca
      901 aaatccctta taaatcaaaa gaatagaccg agatagggtt gagtgttgtt ccagtttgga
      961 acaagagtcc actattaaag aacgtggact ccaacgtcaa agggcgaaaa accgtctatc
     1021 agggcgatgg cccactacgt gaaccatcac cctaatcaag ttttttgggg tcgaggtgcc
     1081 gtaaagcact aaatcggaac cctaaaggga gcccccgatt tagagcttga cggggaaagc
     1141 cggcgaacgt ggcgagaaag gaagggaaga aagcgaaagg agcgggcgct agggcgctgg
     1201 caagtgtagc ggtcacgctg cgcgtaacca ccacacccgc cgcgcttaat gcgccgctac
     1261 agggcgcgtc cattcgccat tcaggctgcg caactgttgg gaagggcgat cggtgcgggc
     1321 ctcttcgcta ttacgccagc tggcgaaagg gggatgtgct gcaaggcgat taagttgggt
     1381 aacgccaggg ttttcccagt cacgacgttg taaaacgacg gccagtgaat tgtaatacga
     1441 ctcactatag ggcgaattgg gcccgacgtc gcatgctccc ggccgccatg gcggccgcgg
     1501 gaattcgatt cccgtctcgc tcgtcggtgc aacaaagcac cagtggtcta gtggtagaat
     1561 agtaccctgc cacggtacag acccgggttc gattcccggc tggtgcagag acggcgcgcg
     1621 ccgtctcagt tttagagcta gaaatagcaa gttaaaataa ggctagtccg ttatcaactt
     1681 gaaaaagtgg caccgagtcg gtgcaatgag cgagacggga tcactagtga attcgcggcc
     1741 gcctgcaggt cgaccatatg ggagagctcc caacgcgttg gatgcatagc ttgagtattc
     1801 tatagtgtca cctaaatagc ttggcgtaat catggtcata gctgtttcct gtgtgaaatt
     1861 gttatccgct cacaattcca cacaacatac gagccggaag cataaagtgt aaagcctggg
     1921 gtgcctaatg agtgagctaa ctcacattaa ttgcgttgcg ctcactgccc gctttccagt
     1981 cgggaaacct gtcgtgccag ctgcattaat gaatcggcca acgcgcgggg agaggcggtt
     2041 tgcgtattgg gcgctcttcc gcttcctcgc tcactgactc gctgcgctcg gtcgttcggc
     2101 tgcggcgagc ggtatcagct cactcaaagg cggtaatacg gttatccaca gaatcagggg
     2161 ataacgcagg aaagaacatg tgagcaaaag gccagcaaaa ggccaggaac cgtaaaaagg
     2221 ccgcgttgct ggcgtttttc cataggctcc gcccccctga cgagcatcac aaaaatcgac
     2281 gctcaagtca gaggtggcga aacccgacag gactataaag ataccaggcg tttccccctg
     2341 gaagctccct cgtgcgctct cctgttccga ccctgccgct taccggatac ctgtccgcct
     2401 ttctcccttc gggaagcgtg gcgctttctc atagctcacg ctgtaggtat ctcagttcgg
     2461 tgtaggtcgt tcgctccaag ctgggctgtg tgcacgaacc ccccgttcag cccgaccgct
     2521 gcgccttatc cggtaactat cgtcttgagt ccaacccggt aagacacgac ttatcgccac
     2581 tggcagcagc cactggtaac aggattagca gagcgaggta tgtaggcggt gctacagagt
     2641 tcttgaagtg gtggcctaac tacggctaca ctagaagaac agtatttggt atctgcgctc
     2701 tgctgaagcc agttaccttc ggaaaaagag ttggtagctc ttgatccggc aaacaaacca
     2761 ccgctggtag cggtggtttt tttgtttgca agcagcagat tacgcgcaga aaaaaaggat
     2821 ctcaagaaga tcctttgatc ttttctacgg ggtctgacgc tcagtggaac gaaaactcac
     2881 gttaagggat tttggtcatg agattatcaa aaaggatctt cacctagatc cttttaaatt
     2941 aaaaatgaag ttttaaatca atctaaagta tatatgagta aacttggtct gacagttacc
     3001 aatgcttaat cagtgaggca cctatctcag cgatctgtct atttcgttca tccatagttg
     3061 cctgactccc cgtcgtgtag ataactacga tacgggaggg cttaccatct ggccccagtg
     3121 ctgcaatgat accgcgagac ccacgctcac cggctccaga tttatcagca ataaaccagc
     3181 cagccggaag ggccgagcgc agaagtggtc ctgcaacttt atccg
//
