LOCUS       sequence-500621-        4652 bp    DNA     circular SYN 20-FEB-2026
DEFINITION  gRNA (guide RNA) expression cassette.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 4652)
  TITLE     The combination of morphogenic regulators BABY BOOM and GRF-GIF 
            improves maize transformation efficiency
  JOURNAL   Unpublished
REFERENCE   2  (bases 1 to 4652)
  AUTHORS   .
  TITLE     Direct Submission
  JOURNAL   Exported Feb 20, 2026 from SnapGene Server 8.0.1
            https://www.snapgene.com
COMMENT     SGRef: number: 1; type: "Journal Article"; journalName: 
            "Unpublished"
COMMENT     Sequence Label: pENTR-sfi_OsU6P2_gRNA_SpecR_OsU3Ter
FEATURES             Location/Qualifiers
     source          1..4652
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     CDS             535..1326
                     /codon_start=1
                     /gene="aadA"
                     /product="aminoglycoside adenylyltransferase (Murphy, 
                     1985)"
                     /label=SmR
                     /note="confers resistance to spectinomycin and 
                     streptomycin"
                     /translation="MGEAVIAEVSTQLSEVVGVIERHLEPTLLAVHLYGSAVDGGLKPH
                     SDIDLLVTVTVRLDETTRRALINDLLETSASPGESEILRAVEVTIVVHDDIIPWRYPAK
                     RELQFGEWQRNDILAGIFEPATIDIDLAILLTKAREHSVALVGPAAEELFDPVPEQDLF
                     EALNETLTLWNSPPDWAGDERNVVLTLSRIWYSAVTGKIAPKDVAADWAMERLPAQYQP
                     VILEARQAYLGQEEDRLASRADQLEEFVHYVKGEITKVVGK"
     misc_RNA        1478..1553
                     /label=gRNA scaffold
                     /note="guide RNA scaffold for the Streptococcus pyogenes 
                     CRISPR/Cas9 system"
     protein_bind    complement(1855..1954)
                     /gene="mutant version of attL"
                     /label=attL2
                     /bound_moiety="LR Clonase(TM)"
                     /note="recombination site for the Gateway(R) LR reaction"
     primer_bind     complement(1971..1990)
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        complement(1972..1990)
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     primer_bind     complement(1995..2011)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(1995..2011)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     CDS             2442..3233
                     /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      3326..3914
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     3815..3834
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     4068..4085
                     /label=L4440
                     /note="L4440 vector, forward primer"
     terminator      complement(4244..4271)
                     /label=rrnB T2 terminator
                     /note="transcription terminator T2 from the E. coli rrnB 
                     gene"
     terminator      complement(4363..4449)
                     /gene="Escherichia coli rrnB"
                     /label=rrnB T1 terminator
                     /note="transcription terminator T1 from the E. coli rrnB 
                     gene"
     primer_bind     4498..4520
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     primer_bind     4512..4529
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     4513..4529
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     protein_bind    4545..4644
                     /gene="mutant version of attL"
                     /label=attL1
                     /bound_moiety="LR Clonase(TM)"
                     /note="recombination site for the Gateway(R) LR reaction"
ORIGIN
        1 aggccaggat catgaaccaa cggcctggct gtatttggtg gttgtgtagg gagatgggga
       61 gaagaaaagc ccgattctct tcgctgtgat gggctggatg catgcggggg agcgggaggc
      121 ccaagtacgt gcacggtgag cggcccacag ggcgagtgtg agcgcgagag gcgggaggaa
      181 cagtttagta ccacattgcc cagctaactc gaacgcgacc aacttataaa cccgcgcgct
      241 gtcgcttgtg ttggcgtgag accaacccag tggacataag cctgttcggt tcgtaagctg
      301 taatgcaagt agcgtatgcg ctcacgcaac tggtccagaa ccttgaccga acgcagcggt
      361 ggtaacggcg cagtggcggt tttcatggct tgttatgact gtttttttgg ggtacagtct
      421 atgcctcggg catccaagca gcaagcgcgt tacgccgtgg gtcgatgttt gatgttatgg
      481 agcagcaacg atgttacgca gcagggcagt cgccctaaaa caaagttaaa catcatgggg
      541 gaagcggtga tcgccgaagt atcgactcaa ctatcagagg tagttggcgt catcgagcgc
      601 catctcgaac cgacgttgct ggccgtacat ttgtacggct ccgcagtgga tggcggcctg
      661 aagccacaca gtgatattga tttgctggtt acggtgaccg taaggcttga tgaaacaacg
      721 cggcgagctt tgatcaacga ccttttggaa acttcggctt cccctggaga gagcgagatt
      781 ctccgcgctg tagaagtcac cattgttgtg cacgacgaca tcattccgtg gcgttatcca
      841 gctaagcgcg aactgcaatt tggagaatgg cagcgcaatg acattcttgc aggtatcttc
      901 gagccagcca cgatcgacat tgatctggct atcttgctga caaaagcaag agaacatagc
      961 gttgccttgg taggtccagc ggcggaggaa ctctttgatc cggttcctga acaggatcta
     1021 tttgaggcgc taaatgaaac cttaacgcta tggaactcgc cgcccgactg ggctggcgat
     1081 gagcgaaatg tagtgcttac gttgtcccgc atttggtaca gcgcagtaac cggcaaaatc
     1141 gcgccgaagg atgtcgctgc cgactgggca atggagcgcc tgccggccca gtatcagccc
     1201 gtcatacttg aagctagaca ggcttatctt ggacaagaag aagatcgctt ggcctcgcgc
     1261 gcagatcagt tggaagaatt tgtccactac gtgaaaggcg agatcaccaa ggtagtcggc
     1321 aaataatgtc tagctagaaa ttcgttcaag ccgacgccgc ttcgcggcgc ggcttaactc
     1381 aagcgttaga tgcactaagc acataattgc tcacagccaa actatcaggt caagtctgct
     1441 tttattattt ttaagcgtgc ataataagcc ggtctcggtt ttagagctag aaatagcaag
     1501 ttaaaataag gctagtccgt tatcaacttg aaaaagtggc accgagtcgg tgcttttttt
     1561 tttcgttttg cattgagttt tctccgtcgc atgtttgcag ttttattttc cgttttgcat
     1621 tgaaatttct ccgtctcatg tttgcagcgt gttcaaaaag tacgcagctg tatttcactt
     1681 atttacggcg ccacattttc atgccgtttg tgccaactat cccgagctag tgaatacagc
     1741 ttggcttcac acaacactgg tgacccgctg acctgctcgt acctcgtacc gtcgtacggc
     1801 acagcatttg gaattaaagg gtgtgatcga tactgcttgc tgctggcctc atgggcccag
     1861 ctttcttgta caaagttggc attataaaaa ataattgctc atcaatttgt tgcaacgaac
     1921 aggtcactat cagtcaaaat aaaatcatta tttgccatcc agctgatatc ccctatagtg
     1981 agtcgtatta catggtcata gctgtttcct ggcagctctg gcccgtgtct caaaatctct
     2041 gatgttacat tgcacaagat aaaaatatat catcatgcct cctctagacc agccaggaca
     2101 gaaatgcctc gacttcgctg ctacccaagg ttgccgggtg acgcacaccg tggaaacgga
     2161 tgaaggcacg aacccagtgg acataagcct gttcggttcg taagctgtaa tgcaagtagc
     2221 gtatgcgctc acgcaactgg tccagaacct tgaccgaacg cagcggtggt aacggcgcag
     2281 tggcggtttt catggcttgt tatgactgtt tttttggggt acagtctatg cctcgggcat
     2341 ccaagcagca agcgcgttac gccgtgggtc gatgtttgat gttatggagc agcaacgatg
     2401 ttacgcagca gggcagtcgc cctaaaacaa agttaaacat tatgagggaa gcggtgatcg
     2461 ccgaagtatc gactcaacta tcagaggtag ttggcgtcat cgagcgccat ctcgaaccga
     2521 cgttgctggc cgtacatttg tacggctccg cagtggatgg cggcctgaag ccacacagtg
     2581 atattgattt gctggttacg gtgaccgtaa ggcttgatga aacaacgcgg cgagctttga
     2641 tcaacgacct tttggaaact tcggcttccc ctggagagag cgagattctc cgcgctgtag
     2701 aagtcaccat tgttgtgcac gacgacatca ttccgtggcg ttatccagct aagcgcgaac
     2761 tgcaatttgg agaatggcag cgcaatgaca ttcttgcagg tatcttcgag ccagccacga
     2821 tcgacattga tctggctatc ttgctgacaa aagcaagaga acatagcgtt gccttggtag
     2881 gtccagcggc ggaggaactc tttgatccgg ttcctgaaca ggatctattt gaggcgctaa
     2941 atgaaacctt aacgctatgg aactcgccgc ccgactgggc tggcgatgag cgaaatgtag
     3001 tgcttacgtt gtcccgcatt tggtacagcg cagtaaccgg caaaatcgcg ccgaaggatg
     3061 tcgctgccga ctgggcaatg gagcgcctgc cggcccagta tcagcccgtc atacttgaag
     3121 ctagacaggc ttatcttgga caagaagaag atcgcttggc ctcgcgcgca gatcagttgg
     3181 aagaatttgt ccactacgtg aaaggcgaga tcaccaaggt agtcggcaaa taaccctcga
     3241 gccacccatg accaaaatcc cttaacgtga gttacgcgtc gttccactga gcgtcagacc
     3301 ccgtagaaaa gatcaaagga tcttcttgag atcctttttt tctgcgcgta atctgctgct
     3361 tgcaaacaaa aaaaccaccg ctaccagcgg tggtttgttt gccggatcaa gagctaccaa
     3421 ctctttttcc gaaggtaact ggcttcagca gagcgcagat accaaatact gttcttctag
     3481 tgtagccgta gttaggccac cacttcaaga actctgtagc accgcctaca tacctcgctc
     3541 tgctaatcct gttaccagtg gctgctgcca gtggcgataa gtcgtgtctt accgggttgg
     3601 actcaagacg atagttaccg gataaggcgc agcggtcggg ctgaacgggg ggttcgtgca
     3661 cacagcccag cttggagcga acgacctaca ccgaactgag atacctacag cgtgagctat
     3721 gagaaagcgc cacgcttccc gaagggagaa aggcggacag gtatccggta agcggcaggg
     3781 tcggaacagg agagcgcacg agggagcttc cagggggaaa cgcctggtat ctttatagtc
     3841 ctgtcgggtt tcgccacctc tgacttgagc gtcgattttt gtgatgctcg tcaggggggc
     3901 ggagcctatg gaaaaacgcc agcaacgcgg cctttttacg gttcctggcc ttttgctggc
     3961 cttttgctca catgttcttt cctgcgttat cccctgattc tgtggataac cgtattaccg
     4021 cctttgagtg agctgatacc gctcgccgca gccgaacgac cgagcgcagc gagtcagtga
     4081 gcgaggaagc ggaagagcgc ccaatacgca aaccgcctct ccccgcgcgt tggccgattc
     4141 attaatgcag ctggcacgac aggtttcccg actggaaagc gggcagtgag cgcaacgcaa
     4201 ttaatacgcg taccgctagc caggaagagt ttgtagaaac gcaaaaaggc catccgtcag
     4261 gatggccttc tgcttagttt gatgcctggc agtttatggc gggcgtcctg cccgccaccc
     4321 tccgggccgt tgcttcacaa cgttcaaatc cgctcccggc ggatttgtcc tactcaggag
     4381 agcgttcacc gacaaacaac agataaaacg aaaggcccag tcttccgact gagcctttcg
     4441 ttttatttga tgcctggcag ttccctactc tcgcgttaac gctagcatgg atgttttccc
     4501 agtcacgacg ttgtaaaacg acggccagtc ttaagctcgg gccccaaata atgattttat
     4561 tttgactgat agtgacctgt tcgttgcaac aaattgatga gcaatgcttt tttataatgc
     4621 caactttgta caaaaaagca gaagggccgt ca
//
