LOCUS       sequence-390460-        3064 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Gateway-compatible gRNA backbone . Template for the addition of 
            target sequences to produce 2x gRNA..
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 3064)
  AUTHORS   Yang L, Machin F, Wang S, Saplaoura E, Kragler F
  TITLE     Heritable transgene-free genome editing in plants by grafting of 
            wild-type shoots to transgenic donor rootstocks.
  JOURNAL   Nat Biotechnol. 2023 Jan 2. doi: 10.1038/s41587-022-01585-8.
  PUBMED    36593415
REFERENCE   2  (bases 1 to 3064)
  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 
            Biotechnol. 2023 Jan 2. doi: 10.1038/s41587-022-01585-8."
COMMENT     Sequence Label: Fragment 1 Null
FEATURES             Location/Qualifiers
     source          1..3064
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     terminator      103..189
                     /gene="Escherichia coli rrnB"
                     /label=rrnB T1 terminator
                     /note="transcription terminator T1 from the E. coli rrnB 
                     gene"
     terminator      281..308
                     /label=rrnB T2 terminator
                     /note="transcription terminator T2 from the E. coli rrnB 
                     gene"
     primer_bind     316..339
                     /label=pENTR-F
                     /note="pENTR vectors, forward primer"
     protein_bind    358..457
                     /gene="mutant version of attL"
                     /label=attL1
                     /bound_moiety="LR Clonase(TM)"
                     /note="recombination site for the Gateway(R) LR reaction"
     misc_RNA        963..1038
                     /label=gRNA scaffold
                     /note="guide RNA scaffold for the Streptococcus pyogenes 
                     CRISPR/Cas9 system"
     protein_bind    complement(1293..1392)
                     /gene="mutant version of attL"
                     /label=attL2
                     /bound_moiety="LR Clonase(TM)"
                     /note="recombination site for the Gateway(R) LR reaction"
     primer_bind     complement(1504..1523)
                     /label=pENTR-R
                     /note="pENTR vectors, reverse primer"
     CDS             1515..2324
                     /codon_start=1
                     /gene="aph(3')-Ia"
                     /product="aminoglycoside phosphotransferase"
                     /label=KanR
                     /note="confers resistance to kanamycin in bacteria or G418 
                     (Geneticin(R)) in eukaryotes"
                     /translation="MSHIQRETSRPRLNSNMDADLYGYKWARDNVGQSGATIYRLYGKP
                     DAPELFLKHGKGSVANDVTDEMVRLNWLTEFMPLPTIKHFIRTPGDAWLLTTAIPGKTA
                     FQVLEEYPDSGENIVDALAVFLRRLHSIPVCNCPFNSDRVFRLAQAQSRMNNGLVDASD
                     FDDERNGWPVEQVWKEMHKLLPFSPDSVVTHGDFSLDNLIFDEGKLIGCIDVGRVGIAD
                     RYQDLAILWNCLGEFSPSLQKRLFQKYGIDNPDMNKLQFHLMLDEFF"
     primer_bind     complement(1582..1601)
                     /label=Kan-R
                     /note="Kanamycin resistance gene, reverse primer"
     rep_origin      2414..3002
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     2903..2922
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
ORIGIN
        1 ctttcctgcg ttatcccctg attctgtgga taaccgtatt accgctagca tggatctcgg
       61 ggacgtctaa ctactaagcg agagtaggga actgccaggc atcaaataaa acgaaaggct
      121 cagtcggaag actgggcctt tcgttttatc tgttgtttgt cggtgaacgc tctcctgagt
      181 aggacaaatc cgccgggagc ggatttgaac gttgtgaagc aacggcccgg agggtggcgg
      241 gcaggacgcc cgccataaac tgccaggcat caaactaagc agaaggccat cctgacggat
      301 ggcctttttg cgtttctaca aactcttcct gttagttagt tacttaagct cgggccccaa
      361 ataatgattt tattttgact gatagtgacc tgttcgttgc aacaaattga taagcaatgc
      421 ttttttataa tgccaacttt gtacaaaaaa gcaggctcca ccatgggaac caattcagtc
      481 gactggatcc ggtaccgagc tcgcggccgc aagcgcggga tcccgacttg ccttccgcac
      541 aatacatcat ttcttcttag ctttttttct tcttcttcgt tcatacagtt tttttttgtt
      601 tatcagctta cattttcttg aaccgtagct ttcgttttct tctttttaac tttccattcg
      661 gagtttttgt atcttgtttc atagtttgtc ccaggattag aatgattagg catcgaacct
      721 tcaagaattt gattgaataa aacatcttca ttcttaagat atgaagataa tcttcaaaag
      781 gcccctggga atctgaaaga agagaagcag gcccatttat atgggaaaga acaatagtat
      841 ttcttatata ggcccattta agttgaaaac aatcttcaaa agtcccacat cgcttagata
      901 agaaaacgaa gctgagttta tatacagcta gagtcgaagt agtgattgag agaccggtct
      961 ccgttttaga gctagaaata gcaagttaaa ataaggctag tccgttatca acttgaaaaa
     1021 gtggcaccga gtcggtgctt ttttttgcaa aattttccag atcgatttct tcttcctctg
     1081 ttcttcggcg ttcaatttct ggggttttct cttcgttttc tgtaactgaa acctaaaatt
     1141 tgacctaaaa aaaatctcaa ataatatgat tcagtggttt tgtacttttc agttagttga
     1201 gttttgcagt tccgatgaga taaaccaata ctgcagcgca cctgcttttg ctcgcttgga
     1261 tccgaattcg cggccgcact cgagatatct agacccagct ttcttgtaca aagttggcat
     1321 tataagaaag cattgcttat caatttgttg caacgaacag gtcactatca gtcaaaataa
     1381 aatcattatt tgccatccag ctgcagctct ggcccgtgtc tcaaaatctc tgatgttaca
     1441 ttgcacaaga taaaaatata tcatcatgaa caataaaact gtctgcttac ataaacagta
     1501 atacaagggg tgttatgagc catattcaac gggaaacgtc gaggccgcga ttaaattcca
     1561 acatggatgc tgatttatat gggtataaat gggctcgcga taatgtcggg caatcaggtg
     1621 cgacaatcta tcgcttgtat gggaagcccg atgcgccaga gttgtttctg aaacatggca
     1681 aaggtagcgt tgccaatgat gttacagatg agatggtcag actaaactgg ctgacggaat
     1741 ttatgcctct tccgaccatc aagcatttta tccgtactcc tggtgatgca tggttactca
     1801 ccactgcgat ccccggaaaa acagcattcc aggtattaga agaatatcct gattcaggtg
     1861 aaaatattgt tgatgcgctg gcagtgttcc tgcgccggtt gcattcgatt cctgtttgta
     1921 attgtccttt taacagcgat cgcgtatttc gtctcgctca ggcgcaatca cgaatgaata
     1981 acggtttggt tgatgcgagt gattttgatg acgagcgtaa tggctggcct gttgaacaag
     2041 tctggaaaga aatgcataaa cttttgccat tctcaccgga ttcagtcgtc actcatggtg
     2101 atttctcact tgataacctt atttttgacg aggggaaatt aataggttgt attgatgttg
     2161 gacgagtcgg aatcgcagac cgataccagg atcttgccat cctatggaac tgcctcggtg
     2221 agttttctcc ttcattacag aaacggcttt ttcaaaaata tggtattgat aatcctgata
     2281 tgaataaatt gcagtttcat ttgatgctcg atgagttttt ctaatcagaa ttggttaatt
     2341 ggttgtaaca ttattcagat tgggccccgt tccactgagc gtcagacccc gtagaaaaga
     2401 tcaaaggatc ttcttgagat cctttttttc tgcgcgtaat ctgctgcttg caaacaaaaa
     2461 aaccaccgct accagcggtg gtttgtttgc cggatcaaga gctaccaact ctttttccga
     2521 aggtaactgg cttcagcaga gcgcagatac caaatactgt tcttctagtg tagccgtagt
     2581 taggccacca cttcaagaac tctgtagcac cgcctacata cctcgctctg ctaatcctgt
     2641 taccagtggc tgctgccagt ggcgataagt cgtgtcttac cgggttggac tcaagacgat
     2701 agttaccgga taaggcgcag cggtcgggct gaacgggggg ttcgtgcaca cagcccagct
     2761 tggagcgaac gacctacacc gaactgagat acctacagcg tgagctatga gaaagcgcca
     2821 cgcttcccga agggagaaag gcggacaggt atccggtaag cggcagggtc ggaacaggag
     2881 agcgcacgag ggagcttcca gggggaaacg cctggtatct ttatagtcct gtcgggtttc
     2941 gccacctctg acttgagcgt cgatttttgt gatgctcgtc aggggggcgg agcctatgga
     3001 aaaacgccag caacgcggcc tttttacggt tcctggcctt ttgctggcct tttgctcaca
     3061 tgtt
//
