LOCUS       sequence-263174-        5094 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Gateway entry clone for AacCas12b sgRNA expression under ZmUbi 
            promoter with ribozyme processing; sgRNA scaffold with MS2 at the 
            third stem loop is used.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 5094)
  AUTHORS   Ming M, Ren Q, Pan C, He Y, Zhang Y, Liu S, Zhong Z, Wang J, Malzahn
            AA, Wu J, Zheng X, Zhang Y, Qi Y
  TITLE     CRISPR-Cas12b enables efficient plant genome engineering.
  JOURNAL   Nat Plants. 2020 Mar;6(3):202-208. doi: 10.1038/s41477-020-0614-6. 
            Epub 2020 Mar 9.
  PUBMED    32170285
REFERENCE   2  (bases 1 to 5094)
  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"; doi: 
            "10.1038/s41477-020-0614-6"; journalName: "Nat Plants"; date: 
            "2020-03"; volume: "6"; issue: "3"; pages: "202-208"
COMMENT     Sequence Label: pYPQ141-ZmUbi-RZ-Aac.3
FEATURES             Location/Qualifiers
     source          1..5094
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     4..26
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     primer_bind     18..35
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     19..35
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     protein_bind    51..146
                     /gene="mutant version of attL"
                     /label=attL5
                     /bound_moiety="LR Clonase(TM)"
                     /note="recombination site for the Gateway(R) LR reaction"
     ncRNA           2142..2178
                     /product="self-cleaving hammerhead ribozyme (Ferre-D'Amare 
                     and Scott, 2010)"
                     /label=hammerhead ribozyme
                     /ncRNA_class="hammerhead_ribozyme"
     misc_RNA        2259..2277
                     /label=MS2 stem loop
                     /note="stem loop that binds the bacteriophage MS2 coat 
                     protein"
     ncRNA           2333..2400
                     /product="hepatitis delta virus self-cleaving ribozyme 
                     (Ferre-D'Amare and Scott, 2010)"
                     /label=HDV ribozyme
                     /ncRNA_class="ribozyme"
     protein_bind    complement(2420..2519)
                     /gene="mutant version of attL"
                     /label=attL2
                     /bound_moiety="LR Clonase(TM)"
                     /note="recombination site for the Gateway(R) LR reaction"
     promoter        complement(2537..2555)
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     promoter        complement(2572..2590)
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     primer_bind     complement(2595..2611)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(2595..2611)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     CDS             3042..3833
                     /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      3926..4514
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     4415..4434
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     4668..4685
                     /label=L4440
                     /note="L4440 vector, forward primer"
     terminator      complement(4844..4871)
                     /label=rrnB T2 terminator
                     /note="transcription terminator T2 from the E. coli rrnB 
                     gene"
     terminator      complement(4963..5049)
                     /gene="Escherichia coli rrnB"
                     /label=rrnB T1 terminator
                     /note="transcription terminator T1 from the E. coli rrnB 
                     gene"
ORIGIN
        1 tttcccagtc acgacgttgt aaaacgacgg ccagtcttaa gctcgggccc aaataatgat
       61 tttattttga ctgatagtga cctgttcgtt gcaacaaatt gatgagcaat gcttttttat
      121 aatgccaact ttgtatacaa aagttgcccc atggcgttcc ctctagagag ataatgagca
      181 ttgcatgtct aagttataaa aaattaccac atattttttt tgtcacactt gtttgaagtg
      241 cagtttatct atctttatac atatatttaa actttactct acgaataata taatctatag
      301 tactacaata atatcagtgt tttagagaat catataaatg aacagttaga catggtctaa
      361 aggacaattg agtattttga caacaggact ctacagtttt atctttttag tgtgcatgtg
      421 ttctcctttt tttttgcaaa tagcttcacc tatataatac ttcatccatt ttattagtac
      481 atccatttag ggtttagggt taatggtttt tatagactaa tttttttagt acatctattt
      541 tattctattt tagcctctaa attaagaaaa ctaaaactct attttagttt ttttatttaa
      601 taatttagat ataaaataga ataaaataaa gtgactaaaa attaaacaaa taccctttaa
      661 gaaattaaaa aaactaagga aacatttttc ttgtttcgag tagataatgc cagcctgtta
      721 aacgccgtcg acgagtctaa cggacaccaa ccagcgaacc agcagcgtcg cgtcgggcca
      781 agcgaagcag acggcacggc atctctgtcg ctgcctctgg acccctctcg agagttccgc
      841 tccaccgttg gacttgctcc gctgtcggca tccagaaatt gcgtggcgga gcggcagacg
      901 tgagccggca cggcaggcgg cctcctcctc ctctcacggc accggcagct acgggggatt
      961 cctttcccac cgctccttcg ctttcccttc ctcgcccgcc gtaataaata gacaccccct
     1021 ccacaccctc tttccccaac ctcgtgttgt tcggagcgca cacacacaca accagatctc
     1081 ccccaaatcc acccgtcggc acctccgctt caaggtacgc cgctcgtcct cccccccccc
     1141 cccctctcta ccttctctag atcggcgttc cggtccatgg ttagggcccg gtagttctac
     1201 ttctgttcat gtttgtgtta gatccgtgtt tgtgttagat ccgtgctgct agcgttcgta
     1261 cacggatgcg acctgtacgt cagacacgtt ctgattgcta acttgccagt gtttctcttt
     1321 ggggaatcct gggatggctc tagccgttcc gcagacggga tcgatttcat gatttttttt
     1381 gtttcgttgc atagggtttg gtttgccctt ttcctttatt tcaatatatg ccgtgcactt
     1441 gtttgtcggg tcatcttttc atgctttttt ttgtcttggt tgtgatgatg tggtctggtt
     1501 gggcggtcgt tctagatcgg agtagaatta attctgtttc aaactacctg gtggatttat
     1561 taattttgga tctgtatgtg tgtgccatac atattcatag ttacgaattg aagatgatgg
     1621 atggaaatat cgatctagga taggtataca tgttgatgcg ggttttactg atgcatatac
     1681 agagatgctt tttgttcgct tggttgtgat gatgtggtgt ggttgggcgg tcgttcattc
     1741 gttctagatc ggagtagaat actgtttcaa actacctggt gtatttatta attttggaac
     1801 tgtatgtgtg tgtcatacat cttcatagtt acgagtttaa gatggatgga aatatcgatc
     1861 taggataggt atacatgttg atgtgggttt tactgatgca tatacatgat ggcatatgca
     1921 gcatctattc atatgctcta accttgagta cctatctatt ataataaaca agtatgtttt
     1981 ataattattt tgatcttgat atacttggat gatggcatat gcagcagcta tatgtggatt
     2041 tttttagccc tgccttcata cgctatttat ttgcttggta ctgtttcttt tgtcgatgct
     2101 caccctgttg tttggtgtta cttctgcagg gatcctagac cctgatgagt ccgtgaggac
     2161 gaaacgagta agctcgtcgg tctagaggac agaatttttc aacgggtgtg ccaatggcca
     2221 ctttccaggt ggcaaagccc gttgagcttc tcaggccaac atgaggatca cccatgtctg
     2281 cagggcctga gaagtggcac gagacggagc tcagtctgac cgcggcgtct ctggccggca
     2341 tggtcccagc ctcctcgctg gcgccggctg ggcaacatgc ttcggcatgg cgaatgggac
     2401 ggtacccggc cggaattcga cccagctttc ttgtacaaag ttggcattat aaaaaataat
     2461 tgctcatcaa tttgttgcaa cgaacaggtc actatcagtc aaaataaaat cattatttgc
     2521 catccagctg atatccccta tagtgagtcg tattacatgg tcatagcatc ccctatagtg
     2581 agtcgtatta catggtcata gctgtttcct ggcagctctg gcccgtgtct caaaatctct
     2641 gatgttacat tgcacaagat aaaaatatat catcatgcct cctctggacc agccaggaca
     2701 gaaatgcctc gacttcgctg ctgcccaagg ttgccgggtg acgcacaccg tggaaacgga
     2761 tgaaggcacg aacccagtgg acataagcct gttcggttcg taagctgtaa tgcaagtagc
     2821 gtatgcgctc acgcaactgg tccagaacct tgaccgaacg cagcggtggt aacggcgcag
     2881 tggcggtttt catggcttgt tatgactgtt tttttggggt acagtctatg cctcgggcat
     2941 ccaagcagca agcgcgttac gccgtgggtc gatgtttgat gttatggagc agcaacgatg
     3001 ttacgcagca gggcagtcgc cctaaaacaa agttaaacat catgagggaa gcggtgatcg
     3061 ccgaagtatc gactcaacta tcagaggtag ttggcgtcat cgagcgccat ctcgaaccga
     3121 cgttgctggc cgtacatttg tacggctccg cagtggatgg cggcctgaag ccacacagtg
     3181 atattgattt gctggttacg gtgaccgtaa ggcttgatga aacaacgcgg cgagctttga
     3241 tcaacgacct tttggaaact tcggcttccc ctggagagag cgagattctc cgcgctgtag
     3301 aagtcaccat tgttgtgcac gacgacatca ttccgtggcg ttatccagct aagcgcgaac
     3361 tgcaatttgg agaatggcag cgcaatgaca ttcttgcagg tatcttcgag ccagccacga
     3421 tcgacattga tctggctatc ttgctgacaa aagcaagaga acatagcgtt gccttggtag
     3481 gtccagcggc ggaggaactc tttgatccgg ttcctgaaca ggatctattt gaggcgctaa
     3541 atgaaacctt aacgctatgg aactcgccgc ccgactgggc tggcgatgag cgaaatgtag
     3601 tgcttacgtt gtcccgcatt tggtacagcg cagtaaccgg caaaatcgcg ccgaaggatg
     3661 tcgctgccga ctgggcaatg gagcgcctgc cggcccagta tcagcccgtc atacttgaag
     3721 ctagacaggc ttatcttgga caagaagaag atcgcttggc ctcgcgcgca gatcagttgg
     3781 aagaatttgt ccactacgtg aaaggcgaga tcaccaaggt agtcggcaaa taaccctcga
     3841 gccacccatg accaaaatcc cttaacgtga gttacgcgtc gttccactga gcgtcagacc
     3901 ccgtagaaaa gatcaaagga tcttcttgag atcctttttt tctgcgcgta atctgctgct
     3961 tgcaaacaaa aaaaccaccg ctaccagcgg tggtttgttt gccggatcaa gagctaccaa
     4021 ctctttttcc gaaggtaact ggcttcagca gagcgcagat accaaatact gtccttctag
     4081 tgtagccgta gttaggccac cacttcaaga actctgtagc accgcctaca tacctcgctc
     4141 tgctaatcct gttaccagtg gctgctgcca gtggcgataa gtcgtgtctt accgggttgg
     4201 actcaagacg atagttaccg gataaggcgc agcggtcggg ctgaacgggg ggttcgtgca
     4261 cacagcccag cttggagcga acgacctaca ccgaactgag atacctacag cgtgagcatt
     4321 gagaaagcgc cacgcttccc gaagggagaa aggcggacag gtatccggta agcggcaggg
     4381 tcggaacagg agagcgcacg agggagcttc cagggggaaa cgcctggtat ctttatagtc
     4441 ctgtcgggtt tcgccacctc tgacttgagc gtcgattttt gtgatgctcg tcaggggggc
     4501 ggagcctatg gaaaaacgcc agcaacgcgg cctttttacg gttcctggcc ttttgctggc
     4561 cttttgctca catgttcttt cctgcgttat cccctgattc tgtggataac cgtattaccg
     4621 cctttgagtg agctgatacc gctcgccgca gccgaacgac cgagcgcagc gagtcagtga
     4681 gcgaggaagc ggaagagcgc ccaatacgca aaccgcctct ccccgcgcgt tggccgattc
     4741 attaatgcag ctggcacgac aggtttcccg actggaaagc gggcagtgag cgcaacgcaa
     4801 ttaatacgcg taccgctcgc caggaagagt ttgtagaaac gcaaaaaggc catccgtcag
     4861 gatggccttc tgcttagttt gatgcctggc agtttatggc gggcgtcctg cccgccaccc
     4921 tccgggccgt tgcttcacaa cgttcaaatc cgctcccggc ggatttgtcc tactcaggag
     4981 agcgttcacc gacaaacaac agataaaacg aaaggcccag tcttccgact gagcctttcg
     5041 ttttatttga tgcctggcag ttccctactc tcgcgttaac gcttgcatgg atgt
//
