LOCUS       sequence-390457-        3113 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Gateway-compatible gRNA backbone with TLS2 mobility signal. Template
            for the addition of target sequences to produce 2x graft-mobile 
            gRNA-TLS2..
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 3113)
  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 3113)
  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: Fragment1 with TLS2
FEATURES             Location/Qualifiers
     source          1..3113
                     /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(1342..1441)
                     /gene="mutant version of attL"
                     /label=attL2
                     /bound_moiety="LR Clonase(TM)"
                     /note="recombination site for the Gateway(R) LR reaction"
     primer_bind     complement(1553..1572)
                     /label=pENTR-R
                     /note="pENTR vectors, reverse primer"
     CDS             1564..2373
                     /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(1631..1650)
                     /label=Kan-R
                     /note="Kanamycin resistance gene, reverse primer"
     rep_origin      2463..3051
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     2952..2971
                     /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 gtcggtgcat tatcagagtg gtggtgggcc cataacccac aggtccgctc
     1081 tgataaattt tttttgcaaa attttccaga tcgatttctt cttcctctgt tcttcggcgt
     1141 tcaatttctg gggttttctc ttcgttttct gtaactgaaa cctaaaattt gacctaaaaa
     1201 aaatctcaaa taatatgatt cagtggtttt gtacttttca gttagttgag ttttgcagtt
     1261 ccgatgagat aaaccaatac tgcagcgcac ctgcttttgc tcgcttggat ccgaattcgc
     1321 ggccgcactc gagatatcta gacccagctt tcttgtacaa agttggcatt ataagaaagc
     1381 attgcttatc aatttgttgc aacgaacagg tcactatcag tcaaaataaa atcattattt
     1441 gccatccagc tgcagctctg gcccgtgtct caaaatctct gatgttacat tgcacaagat
     1501 aaaaatatat catcatgaac aataaaactg tctgcttaca taaacagtaa tacaaggggt
     1561 gttatgagcc atattcaacg ggaaacgtcg aggccgcgat taaattccaa catggatgct
     1621 gatttatatg ggtataaatg ggctcgcgat aatgtcgggc aatcaggtgc gacaatctat
     1681 cgcttgtatg ggaagcccga tgcgccagag ttgtttctga aacatggcaa aggtagcgtt
     1741 gccaatgatg ttacagatga gatggtcaga ctaaactggc tgacggaatt tatgcctctt
     1801 ccgaccatca agcattttat ccgtactcct ggtgatgcat ggttactcac cactgcgatc
     1861 cccggaaaaa cagcattcca ggtattagaa gaatatcctg attcaggtga aaatattgtt
     1921 gatgcgctgg cagtgttcct gcgccggttg cattcgattc ctgtttgtaa ttgtcctttt
     1981 aacagcgatc gcgtatttcg tctcgctcag gcgcaatcac gaatgaataa cggtttggtt
     2041 gatgcgagtg attttgatga cgagcgtaat ggctggcctg ttgaacaagt ctggaaagaa
     2101 atgcataaac ttttgccatt ctcaccggat tcagtcgtca ctcatggtga tttctcactt
     2161 gataacctta tttttgacga ggggaaatta ataggttgta ttgatgttgg acgagtcgga
     2221 atcgcagacc gataccagga tcttgccatc ctatggaact gcctcggtga gttttctcct
     2281 tcattacaga aacggctttt tcaaaaatat ggtattgata atcctgatat gaataaattg
     2341 cagtttcatt tgatgctcga tgagtttttc taatcagaat tggttaattg gttgtaacat
     2401 tattcagatt gggccccgtt ccactgagcg tcagaccccg tagaaaagat caaaggatct
     2461 tcttgagatc ctttttttct gcgcgtaatc tgctgcttgc aaacaaaaaa accaccgcta
     2521 ccagcggtgg tttgtttgcc ggatcaagag ctaccaactc tttttccgaa ggtaactggc
     2581 ttcagcagag cgcagatacc aaatactgtt cttctagtgt agccgtagtt aggccaccac
     2641 ttcaagaact ctgtagcacc gcctacatac ctcgctctgc taatcctgtt accagtggct
     2701 gctgccagtg gcgataagtc gtgtcttacc gggttggact caagacgata gttaccggat
     2761 aaggcgcagc ggtcgggctg aacggggggt tcgtgcacac agcccagctt ggagcgaacg
     2821 acctacaccg aactgagata cctacagcgt gagctatgag aaagcgccac gcttcccgaa
     2881 gggagaaagg cggacaggta tccggtaagc ggcagggtcg gaacaggaga gcgcacgagg
     2941 gagcttccag ggggaaacgc ctggtatctt tatagtcctg tcgggtttcg ccacctctga
     3001 cttgagcgtc gatttttgtg atgctcgtca ggggggcgga gcctatggaa aaacgccagc
     3061 aacgcggcct ttttacggtt cctggccttt tgctggcctt ttgctcacat gtt
//
