LOCUS       sequence-172939-        3281 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Entry vector containing the TaU6 promoter and an Esp3I Golden Gate 
            cloning site for cloning of the sgRNA, all between attL5 and attL2 
            sites.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 3281)
  AUTHORS   Gil-Humanes J, Wang Y, Liang Z, Shan Q, Ozuna CV, Sanchez-Leon S, 
            Baltes NJ, Starker C, Barro F, Gao C, Voytas DF
  TITLE     High-efficiency gene targeting in hexaploid wheat using DNA 
            replicons and CRISPR/Cas9.
  JOURNAL   Plant J. 2016 Dec 10. doi: 10.1111/tpj.13446.
  PUBMED    27943461
REFERENCE   2  (bases 1 to 3281)
  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: "Plant J. 
            2016 Dec 10. doi: 10.1111/tpj.13446."
COMMENT     Sequence Label: pJG85
FEATURES             Location/Qualifiers
     source          1..3281
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     misc_RNA        399..474
                     /label=gRNA scaffold
                     /note="guide RNA scaffold for the Streptococcus pyogenes 
                     CRISPR/Cas9 system"
     protein_bind    complement(495..594)
                     /gene="mutant version of attL"
                     /label=attL2
                     /bound_moiety="LR Clonase(TM)"
                     /note="recombination site for the Gateway(R) LR reaction"
     primer_bind     complement(611..630)
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        complement(612..630)
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     primer_bind     complement(635..651)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(635..651)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     CDS             1082..1873
                     /codon_start=1
                     /gene="aadA"
                     /product="aminoglycoside adenylyltransferase (Murphy, 
                     1985)"
                     /label=SmR
                     /note="confers resistance to spectinomycin and 
                     streptomycin"
                     /translation="MREAVIAEVSTQLSEVVGVIERHLEPTLLAVHLYGSAVDGGLKPH
                     SDIDLLVTVTVRLDETTRRALINDLLETSASPGESEILRAVEVTIVVHDDIIPWRYPAK
                     RELQFGEWQRNDILAGIFEPATIDIDLAILLTKAREHSVALVGPAAEELFDPVPEQDLF
                     EALNETLTLWNSPPDWAGDERNVVLTLSRIWYSAVTGKIAPKDVAADWAMERLPAQYQP
                     VILEARQAYLGQEEDRLASRADQLEEFVHYVKGEITKVVGK"
     rep_origin      1966..2554
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     2455..2474
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     2708..2725
                     /label=L4440
                     /note="L4440 vector, forward primer"
     terminator      complement(2884..2911)
                     /label=rrnB T2 terminator
                     /note="transcription terminator T2 from the E. coli rrnB 
                     gene"
     terminator      complement(3003..3089)
                     /gene="Escherichia coli rrnB"
                     /label=rrnB T1 terminator
                     /note="transcription terminator T1 from the E. coli rrnB 
                     gene"
     primer_bind     3138..3160
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     primer_bind     3152..3169
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     3153..3169
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     protein_bind    3184..3279
                     /gene="mutant version of attL"
                     /label=attL5
                     /bound_moiety="LR Clonase(TM)"
                     /note="recombination site for the Gateway(R) LR reaction"
ORIGIN
        1 gagctcgcgg ccgcgaccaa gcccgttatt ctgacagttc tggtgctcaa cacatttata
       61 tttatcaagg agcacattgt tactcactgc taggagggaa tcgaactagg aatattgatc
      121 agaggaacta cgagagagct gaagataact gccctctagc tctcactgat ctgggtcgca
      181 tagtgagatg cagcccacgt gagttcagca acggtctagc gctgggcttt taggcccgca
      241 tgatcgggct tttgtcgggt ggtcgacgtg ttcacgattg gggagagcaa cgcagcagtt
      301 cctcttagtt tagtcccacc tcgcctgtcc agcagagttc tgaccggttt ataaactcgc
      361 ttgctgcatc agacttggag acggagtcga ttcgtctcgt tttagagcta gaaatagcaa
      421 gttaaaataa ggctagtccg ttatcaactt gaaaaagtgg caccgagtcg gtgctttttt
      481 tctgcaggtc gacgacccag ctttcttgta caaagttggc attataaaaa ataattgctc
      541 atcaatttgt tgcaacgaac aggtcactat cagtcaaaat aaaatcatta tttgccatcc
      601 agctgatatc ccctatagtg agtcgtatta catggtcata gctgtttcct ggcagctctg
      661 gcccgtgtct caaaatctct gatgttacat tgcacaagat aaaaatatat catcatgcct
      721 cctctggacc agccaggaca gaaatgcctc gacttcgctg ctacccaagg ttgccgggtg
      781 acgcacaccg tggaaacgga tgaaggcacg aacccagtgg acataagcct gttcggttcg
      841 taagctgtaa tgcaagtagc gtatgcgctc acgcaactgg tccagaacct tgaccgaacg
      901 cagcggtggt aacggcgcag tggcggtttt catggcttgt tatgactgtt tttttggggt
      961 acagtctatg cctcgggcat ccaagcagca agcgcgttac gccgtgggtc gatgtttgat
     1021 gttatggagc agcaacgatg ttacgcagca gggcagtcgc cctaaaacaa agttaaacat
     1081 catgagggaa gcggtgatcg ccgaagtatc gactcaacta tcagaggtag ttggcgtcat
     1141 cgagcgccat ctcgaaccga cgttgctggc cgtacatttg tacggctccg cagtggatgg
     1201 cggcctgaag ccacacagtg atattgattt gctggttacg gtgaccgtaa ggcttgatga
     1261 aacaacgcgg cgagctttga tcaacgacct tttggaaact tcggcttccc ctggagagag
     1321 cgagattctc cgcgctgtag aagtcaccat tgttgtgcac gacgacatca ttccgtggcg
     1381 ttatccagct aagcgcgaac tgcaatttgg agaatggcag cgcaatgaca ttcttgcagg
     1441 tatcttcgag ccagccacga tcgacattga tctggctatc ttgctgacaa aagcaagaga
     1501 acatagcgtt gccttggtag gtccagcggc ggaggaactc tttgatccgg ttcctgaaca
     1561 ggatctattt gaggcgctaa atgaaacctt aacgctatgg aactcgccgc ccgactgggc
     1621 tggcgatgag cgaaatgtag tgcttacgtt gtcccgcatt tggtacagcg cagtaaccgg
     1681 caaaatcgcg ccgaaggatg tcgctgccga ctgggcaatg gagcgcctgc cggcccagta
     1741 tcagcccgtc atacttgaag ctagacaggc ttatcttgga caagaagaag atcgcttggc
     1801 ctcgcgcgca gatcagttgg aagaatttgt ccactacgtg aaaggcgaga tcaccaaggt
     1861 agtcggcaaa taaccctcga gccacccatg accaaaatcc cttaacgtga gttacgcgtc
     1921 gttccactga gcgtcagacc ccgtagaaaa gatcaaagga tcttcttgag atcctttttt
     1981 tctgcgcgta atctgctgct tgcaaacaaa aaaaccaccg ctaccagcgg tggtttgttt
     2041 gccggatcaa gagctaccaa ctctttttcc gaaggtaact ggcttcagca gagcgcagat
     2101 accaaatact gtccttctag tgtagccgta gttaggccac cacttcaaga actctgtagc
     2161 accgcctaca tacctcgctc tgctaatcct gttaccagtg gctgctgcca gtggcgataa
     2221 gtcgtgtctt accgggttgg actcaagacg atagttaccg gataaggcgc agcggtcggg
     2281 ctgaacgggg ggttcgtgca cacagcccag cttggagcga acgacctaca ccgaactgag
     2341 atacctacag cgtgagctat gagaaagcgc cacgcttccc gaagggagaa aggcggacag
     2401 gtatccggta agcggcaggg tcggaacagg agagcgcacg agggagcttc cagggggaaa
     2461 cgcctggtat ctttatagtc ctgtcgggtt tcgccacctc tgacttgagc gtcgattttt
     2521 gtgatgctcg tcaggggggc ggagcctatg gaaaaacgcc agcaacgcgg cctttttacg
     2581 gttcctggcc ttttgctggc cttttgctca catgttcttt cctgcgttat cccctgattc
     2641 tgtggataac cgtattaccg cctttgagtg agctgatacc gctcgccgca gccgaacgac
     2701 cgagcgcagc gagtcagtga gcgaggaagc ggaagagcgc ccaatacgca aaccgcctct
     2761 ccccgcgcgt tggccgattc attaatgcag ctggcacgac aggtttcccg actggaaagc
     2821 gggcagtgag cgcaacgcaa ttaatacgcg taccgcgagc caggaagagt ttgtagaaac
     2881 gcaaaaaggc catccgtcag gatggccttc tgcttagttt gatgcctggc agtttatggc
     2941 gggcgtcctg cccgccaccc tccgggccgt tgcttcacaa cgttcaaatc cgctcccggc
     3001 ggatttgtcc tactcaggag agcgttcacc gacaaacaac agataaaacg aaaggcccag
     3061 tcttccgact gagcctttcg ttttatttga tgcctggcag ttccctactc tcgcgttaac
     3121 gcttgcatgg atgttttccc agtcacgacg ttgtaaaacg acggccagtc ttaagctcgg
     3181 gcccaataat gattttattt tgactgatag tgacctgttc gttgcaacaa attgatgagc
     3241 aatgcttttt tataatgcca actttgtata caaaagttgc c
//
