LOCUS       sequence-237999-        3343 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Entry clone expressing gRNA. Target site can be introduced between 
            tRNA and gRNA backbone. For use in plants and compatible with the 
            MultiSite Gateway system.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 3343)
  AUTHORS   Wang X, Ye L, Lyu M, Ursache R, Loytynoja A, Mahonen AP
  TITLE     An inducible genome editing system for plants.
  JOURNAL   Nat Plants. 2020 Jun 29. pii: 10.1038/s41477-020-0695-2. doi: 
            10.1038/s41477-020-0695-2.
  PUBMED    32601420
REFERENCE   2  (bases 1 to 3343)
  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: "Nat Plants.
            2020 Jun 29. pii: 10.1038/s41477-020-0695-2. doi: 
            10.1038/s41477-020-0695-2."
COMMENT     Sequence Label: p2R3z-AtU3b-tRNA-ccdB-gRNA
FEATURES             Location/Qualifiers
     source          1..3343
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     terminator      complement(47..133)
                     /gene="Escherichia coli rrnB"
                     /label=rrnB T1 terminator
                     /note="transcription terminator T1 from the E. coli rrnB 
                     gene"
     primer_bind     182..204
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     primer_bind     196..213
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     197..213
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     protein_bind    complement(284..407)
                     /gene="mutant version of attR"
                     /label=attR2
                     /bound_moiety="LR Clonase(TM)"
                     /note="recombination site for the Gateway(R) LR reaction"
     protein_bind    897..918
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     promoter        join(933..938,939..956,957..963)
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    971..987
                     /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        1029..1047
                     /label=SP6 promoter
                     /note="promoter for bacteriophage SP6 RNA polymerase"
     primer_bind     1029..1046
                     /label=SP6
                     /note="SP6 promoter, forward primer"
     CDS             1121..1423
                     /codon_start=1
                     /gene="ccdB"
                     /product="CcdB, a bacterial toxin that poisons DNA gyrase"
                     /label=ccdB
                     /note="Plasmids containing the ccdB gene cannot be 
                     propagated in standard E. coli strains."
                     /translation="QFKVYTYKRESRYRLFVDVQSDIIDTPGRRMVIPLASARLLSDKV
                     SRELYPVVHIGDGSWRMMTTDMASVPVSVIGEEVADLSHRENDIKNAINLMFWGI"
     primer_bind     1302..1321
                     /label=ccdB-fwd
                     /note="ccdB gene, forward primer"
     misc_RNA        1497..1572
                     /label=gRNA scaffold
                     /note="guide RNA scaffold for the Streptococcus pyogenes 
                     CRISPR/Cas9 system"
     protein_bind    complement(1583..1678)
                     /gene="mutant version of attL"
                     /label=attL3
                     /bound_moiety="LR Clonase(TM)"
                     /note="recombination site for the Gateway(R) LR reaction"
     primer_bind     complement(1715..1734)
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        complement(1716..1734)
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     primer_bind     complement(1739..1755)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     CDS             complement(1823..2197)
                     /codon_start=1
                     /gene="Sh ble from Streptoalloteichus hindustanus"
                     /product="antibiotic-binding protein"
                     /label=BleoR
                     /note="confers resistance to bleomycin, phleomycin, and 
                     Zeocin(TM)"
                     /translation="MAKLTSAVPVLTARDVAGAVEFWTDRLGFSRDFVEDDFAGVVRDD
                     VTLFISAVQDQVVPDNTLAWVWVRGLDELYAEWSEVVSTNFRDASGPAMTEIGEQPWGR
                     EFALRDPAGNCVHFVAEEQD"
     promoter        complement(2216..2263)
                     /label=EM7 promoter
                     /note="synthetic bacterial promoter "
     rep_origin      2353..2941
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     2842..2861
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     3095..3112
                     /label=L4440
                     /note="L4440 vector, forward primer"
     terminator      complement(3271..3298)
                     /label=rrnB T2 terminator
                     /note="transcription terminator T2 from the E. coli rrnB 
                     gene"
ORIGIN
        1 accctccggg ccgttgcttc acaacgttca aatccgctcc cggcggattt gtcctactca
       61 ggagagcgtt caccgacaaa caacagataa aacgaaaggc ccagtcttcc gactgagcct
      121 ttcgttttat ttgatgcctg gcagttccct actctcgcgt taacgctagc atggatgttt
      181 tcccagtcac gacgttgtaa aacgacggcc agtcttaagc tcgggccctg cagctctaga
      241 gctcgaattc tacaggtcac taataccatc taagtagttg gttcatagtg actgcatatg
      301 ttgtgtttta cagtattatg tagtctgttt tttatgcaaa atctaattta atatattgat
      361 atttatatca ttttacgttt ctcgttcaac tttcttgtac aaagtggaat ttactttaaa
      421 ttttttctta tgcagcctgt gatggataac tgaatcaaac aaatggcgtc tgggtttaag
      481 aagatctgtt ttggctatgt tggacgaaac aagtgaactt ttaggatcaa cttcagttta
      541 tatatggagc ttatatcgag caataagata agtgggcttt ttatgtaatt taatgggcta
      601 tcgtccatag attcactaat acccatgccc agtacccatg tatgcgtttc atataagctc
      661 ctaatttctc ccacatcgct caaatctaaa caaatcttgt tgtatatata acactgaggg
      721 agcaacattg gtcaaacaaa gcaccagtgg tctagtggta gaatagtacc ctgccacggt
      781 acagacccgg gttcgattcc cggctggtgc aagagacctc tgaagtggcc gattcattaa
      841 tgcagctggc acgacaggtt tcccgactgg aaagcgggca gtgagcgcaa cgcaattaat
      901 gtgagttagc tcactcatta ggcaccccag gctttacact ttatgcttcc ggctcgtatg
      961 ttgtgtggaa ttgtgagcgg ataacaattt cacacaagaa acagctatga ccatgattac
     1021 gccaagctat ttaggtgaca ctatagaata ctcaagctat gcatcaagct caatgggtct
     1081 agtctgtaga tacccatcac actggcgacc gctcgaacat cagtttaagg tttacaccta
     1141 taaaagagag agccgttatc gtctgtttgt ggatgtacag agtgatatta ttgacacgcc
     1201 ggggcgacgg atggtgatcc ccctggccag tgcacgtctg ctgtcagata aagtctcccg
     1261 tgaactttac ccggtggtgc atatcgggga tggaagctgg cgcatgatga ccaccgatat
     1321 ggccagtgtg cctgtctccg ttatcgggga agaagtggct gatctcagcc accgcgaaaa
     1381 tgacatcaaa aacgccatta acctgatgtt ctggggaata taaatgtcag gcctgaatgg
     1441 cgaatggacg cgccctgtag cggcgcatta agcgcggcgg gtgagcgtgg gtctcggttt
     1501 tagagctaga aatagcaagt taaaataagg ctagtccgtt atcaacttga aaaagtggca
     1561 ccgagtcggt gctttttttt ttcaacttta ttatacaaag ttggcattat aaaaaagcat
     1621 tgcttatcaa tttgttgcaa cgaacaggtc actatcagtc aaaataaaat cattatttgg
     1681 agctccatgg tagcgttaac gcggccgcga tatcccctat agtgagtcgt attacatggt
     1741 catagctgtt tcctggcagc tctggcccgt gtctcaaaat ctctgatgtt acattgcaca
     1801 agataaaaat atatcatcat gatcagtcct gctcctcggc cacgaagtgc acgcagttgc
     1861 cggccgggtc gcgcagggcg aactcccgcc cccacggctg ctcgccgatc tcggtcatgg
     1921 ccggcccgga ggcgtcccgg aagttcgtgg acacgacctc cgaccactcg gcgtacagct
     1981 cgtccaggcc gcgcacccac acccaggcca gggtgttgtc cggcaccacc tggtcctgga
     2041 ccgcgctgat gaacagggtc acgtcgtccc ggaccacacc ggcgaagtcg tcctccacga
     2101 agtcccggga gaacccgagc cggtcggtcc agaactcgac cgctccggcg acgtcgcgcg
     2161 cggtgagcac cggaacggca ctggtcaact tggccatggt ttagttcctc accttgtcgt
     2221 attatactat gccgatatac tatgccgatg attaattgtc aacacgtgct gatcatgacc
     2281 aaaatccctt aacgtgagtt acgcgtcgtt ccactgagcg tcagaccccg tagaaaagat
     2341 caaaggatct tcttgagatc ctttttttct gcgcgtaatc tgctgcttgc aaacaaaaaa
     2401 accaccgcta ccagcggtgg tttgtttgcc ggatcaagag ctaccaactc tttttccgaa
     2461 ggtaactggc ttcagcagag cgcagatacc aaatactgtt cttctagtgt agccgtagtt
     2521 aggccaccac ttcaagaact ctgtagcacc gcctacatac ctcgctctgc taatcctgtt
     2581 accagtggct gctgccagtg gcgataagtc gtgtcttacc gggttggact caagacgata
     2641 gttaccggat aaggcgcagc ggtcgggctg aacggggggt tcgtgcacac agcccagctt
     2701 ggagcgaacg acctacaccg aactgagata cctacagcgt gagctatgag aaagcgccac
     2761 gcttcccgaa gggagaaagg cggacaggta tccggtaagc ggcagggtcg gaacaggaga
     2821 gcgcacgagg gagcttccag ggggaaacgc ctggtatctt tatagtcctg tcgggtttcg
     2881 ccacctctga cttgagcgtc gatttttgtg atgctcgtca ggggggcgga gcctatggaa
     2941 aaacgccagc aacgcggcct ttttacggtt cctggccttt tgctggcctt ttgctcacat
     3001 gttctttcct gcgttatccc ctgattctgt ggataaccgt attaccgcct ttgagtgagc
     3061 tgataccgct cgccgcagcc gaacgaccga gcgcagcgag tcagtgagcg aggaagcgga
     3121 agagcgccca atacgcaaac cgcctctccc cgcgcgttgg ccgattcatt aatgcagctg
     3181 gcacgacagg tttcccgact ggaaagcggg cagtgagcgc aacgcaatta atacgcgtac
     3241 cgctagccag gaagagtttg tagaaacgca aaaaggccat ccgtcaggat ggccttctgc
     3301 ttagtttgat gcctggcagt ttatggcggg cgtcctgccc gcc
//
