LOCUS       sequence-263170-        5117 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Gateway entry clone for AaCas12b sgRNA expression under ZmUbi 
            promoter with ribozyme processing; sgRNA scaffold 1.2 is used.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 5117)
  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 5117)
  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-Aa1.2
FEATURES             Location/Qualifiers
     source          1..5117
                     /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"
     ncRNA           2356..2423
                     /product="hepatitis delta virus self-cleaving ribozyme 
                     (Ferre-D'Amare and Scott, 2010)"
                     /label=HDV ribozyme
                     /ncRNA_class="ribozyme"
     protein_bind    complement(2443..2542)
                     /gene="mutant version of attL"
                     /label=attL2
                     /bound_moiety="LR Clonase(TM)"
                     /note="recombination site for the Gateway(R) LR reaction"
     promoter        complement(2560..2578)
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     promoter        complement(2595..2613)
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     primer_bind     complement(2618..2634)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(2618..2634)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     CDS             3065..3856
                     /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      3949..4537
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     4438..4457
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     4691..4708
                     /label=L4440
                     /note="L4440 vector, forward primer"
     terminator      complement(4867..4894)
                     /label=rrnB T2 terminator
                     /note="transcription terminator T2 from the E. coli rrnB 
                     gene"
     terminator      complement(4986..5072)
                     /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 gatccaaatc tctgatgagt ccgtgaggac
     2161 gaaacgagta agctcgtcag atttctgtct aaaggacaga atttttcaac gggtgtgcca
     2221 atggccactt tccaggtggc aaagcccgtt gaacttctca aaaagaacgc tcgctcagtg
     2281 ttctgacgtc ggatcactga gcgagcgatc tgagaagtgg cacgagacgg agctcagtct
     2341 gaccgcggcg tctctggccg gcatggtccc agcctcctcg ctggcgccgg ctgggcaaca
     2401 tgcttcggca tggcgaatgg gacggtaccc ggccggaatt cgacccagct ttcttgtaca
     2461 aagttggcat tataaaaaat aattgctcat caatttgttg caacgaacag gtcactatca
     2521 gtcaaaataa aatcattatt tgccatccag ctgatatccc ctatagtgag tcgtattaca
     2581 tggtcatagc atcccctata gtgagtcgta ttacatggtc atagctgttt cctggcagct
     2641 ctggcccgtg tctcaaaatc tctgatgtta cattgcacaa gataaaaata tatcatcatg
     2701 cctcctctgg accagccagg acagaaatgc ctcgacttcg ctgctgccca aggttgccgg
     2761 gtgacgcaca ccgtggaaac ggatgaaggc acgaacccag tggacataag cctgttcggt
     2821 tcgtaagctg taatgcaagt agcgtatgcg ctcacgcaac tggtccagaa ccttgaccga
     2881 acgcagcggt ggtaacggcg cagtggcggt tttcatggct tgttatgact gtttttttgg
     2941 ggtacagtct atgcctcggg catccaagca gcaagcgcgt tacgccgtgg gtcgatgttt
     3001 gatgttatgg agcagcaacg atgttacgca gcagggcagt cgccctaaaa caaagttaaa
     3061 catcatgagg gaagcggtga tcgccgaagt atcgactcaa ctatcagagg tagttggcgt
     3121 catcgagcgc catctcgaac cgacgttgct ggccgtacat ttgtacggct ccgcagtgga
     3181 tggcggcctg aagccacaca gtgatattga tttgctggtt acggtgaccg taaggcttga
     3241 tgaaacaacg cggcgagctt tgatcaacga ccttttggaa acttcggctt cccctggaga
     3301 gagcgagatt ctccgcgctg tagaagtcac cattgttgtg cacgacgaca tcattccgtg
     3361 gcgttatcca gctaagcgcg aactgcaatt tggagaatgg cagcgcaatg acattcttgc
     3421 aggtatcttc gagccagcca cgatcgacat tgatctggct atcttgctga caaaagcaag
     3481 agaacatagc gttgccttgg taggtccagc ggcggaggaa ctctttgatc cggttcctga
     3541 acaggatcta tttgaggcgc taaatgaaac cttaacgcta tggaactcgc cgcccgactg
     3601 ggctggcgat gagcgaaatg tagtgcttac gttgtcccgc atttggtaca gcgcagtaac
     3661 cggcaaaatc gcgccgaagg atgtcgctgc cgactgggca atggagcgcc tgccggccca
     3721 gtatcagccc gtcatacttg aagctagaca ggcttatctt ggacaagaag aagatcgctt
     3781 ggcctcgcgc gcagatcagt tggaagaatt tgtccactac gtgaaaggcg agatcaccaa
     3841 ggtagtcggc aaataaccct cgagccaccc atgaccaaaa tcccttaacg tgagttacgc
     3901 gtcgttccac tgagcgtcag accccgtaga aaagatcaaa ggatcttctt gagatccttt
     3961 ttttctgcgc gtaatctgct gcttgcaaac aaaaaaacca ccgctaccag cggtggtttg
     4021 tttgccggat caagagctac caactctttt tccgaaggta actggcttca gcagagcgca
     4081 gataccaaat actgtccttc tagtgtagcc gtagttaggc caccacttca agaactctgt
     4141 agcaccgcct acatacctcg ctctgctaat cctgttacca gtggctgctg ccagtggcga
     4201 taagtcgtgt cttaccgggt tggactcaag acgatagtta ccggataagg cgcagcggtc
     4261 gggctgaacg gggggttcgt gcacacagcc cagcttggag cgaacgacct acaccgaact
     4321 gagataccta cagcgtgagc attgagaaag cgccacgctt cccgaaggga gaaaggcgga
     4381 caggtatccg gtaagcggca gggtcggaac aggagagcgc acgagggagc ttccaggggg
     4441 aaacgcctgg tatctttata gtcctgtcgg gtttcgccac ctctgacttg agcgtcgatt
     4501 tttgtgatgc tcgtcagggg ggcggagcct atggaaaaac gccagcaacg cggccttttt
     4561 acggttcctg gccttttgct ggccttttgc tcacatgttc tttcctgcgt tatcccctga
     4621 ttctgtggat aaccgtatta ccgcctttga gtgagctgat accgctcgcc gcagccgaac
     4681 gaccgagcgc agcgagtcag tgagcgagga agcggaagag cgcccaatac gcaaaccgcc
     4741 tctccccgcg cgttggccga ttcattaatg cagctggcac gacaggtttc ccgactggaa
     4801 agcgggcagt gagcgcaacg caattaatac gcgtaccgct cgccaggaag agtttgtaga
     4861 aacgcaaaaa ggccatccgt caggatggcc ttctgcttag tttgatgcct ggcagtttat
     4921 ggcgggcgtc ctgcccgcca ccctccgggc cgttgcttca caacgttcaa atccgctccc
     4981 ggcggatttg tcctactcag gagagcgttc accgacaaac aacagataaa acgaaaggcc
     5041 cagtcttccg actgagcctt tcgttttatt tgatgcctgg cagttcccta ctctcgcgtt
     5101 aacgcttgca tggatgt
//
