LOCUS       sequence-263169-        5147 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 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 5147)
  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 5147)
  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-Aa1.2.3
FEATURES             Location/Qualifiers
     source          1..5147
                     /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        2291..2309
                     /label=MS2 stem loop
                     /note="stem loop that binds the bacteriophage MS2 coat 
                     protein"
     ncRNA           2386..2453
                     /product="hepatitis delta virus self-cleaving ribozyme 
                     (Ferre-D'Amare and Scott, 2010)"
                     /label=HDV ribozyme
                     /ncRNA_class="ribozyme"
     protein_bind    complement(2473..2572)
                     /gene="mutant version of attL"
                     /label=attL2
                     /bound_moiety="LR Clonase(TM)"
                     /note="recombination site for the Gateway(R) LR reaction"
     promoter        complement(2590..2608)
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     promoter        complement(2625..2643)
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     primer_bind     complement(2648..2664)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(2648..2664)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     CDS             3095..3886
                     /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      3979..4567
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     4468..4487
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     4721..4738
                     /label=L4440
                     /note="L4440 vector, forward primer"
     terminator      complement(4897..4924)
                     /label=rrnB T2 terminator
                     /note="transcription terminator T2 from the E. coli rrnB 
                     gene"
     terminator      complement(5016..5102)
                     /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 ttctgggcca acatgaggat cacccatgtc tgcagggccc ggatcactga gcgagcgatc
     2341 tgagaagtgg cacgagacgg agctcagtct gaccgcggcg tctctggccg gcatggtccc
     2401 agcctcctcg ctggcgccgg ctgggcaaca tgcttcggca tggcgaatgg gacggtaccc
     2461 ggccggaatt cgacccagct ttcttgtaca aagttggcat tataaaaaat aattgctcat
     2521 caatttgttg caacgaacag gtcactatca gtcaaaataa aatcattatt tgccatccag
     2581 ctgatatccc ctatagtgag tcgtattaca tggtcatagc atcccctata gtgagtcgta
     2641 ttacatggtc atagctgttt cctggcagct ctggcccgtg tctcaaaatc tctgatgtta
     2701 cattgcacaa gataaaaata tatcatcatg cctcctctgg accagccagg acagaaatgc
     2761 ctcgacttcg ctgctgccca aggttgccgg gtgacgcaca ccgtggaaac ggatgaaggc
     2821 acgaacccag tggacataag cctgttcggt tcgtaagctg taatgcaagt agcgtatgcg
     2881 ctcacgcaac tggtccagaa ccttgaccga acgcagcggt ggtaacggcg cagtggcggt
     2941 tttcatggct tgttatgact gtttttttgg ggtacagtct atgcctcggg catccaagca
     3001 gcaagcgcgt tacgccgtgg gtcgatgttt gatgttatgg agcagcaacg atgttacgca
     3061 gcagggcagt cgccctaaaa caaagttaaa catcatgagg gaagcggtga tcgccgaagt
     3121 atcgactcaa ctatcagagg tagttggcgt catcgagcgc catctcgaac cgacgttgct
     3181 ggccgtacat ttgtacggct ccgcagtgga tggcggcctg aagccacaca gtgatattga
     3241 tttgctggtt acggtgaccg taaggcttga tgaaacaacg cggcgagctt tgatcaacga
     3301 ccttttggaa acttcggctt cccctggaga gagcgagatt ctccgcgctg tagaagtcac
     3361 cattgttgtg cacgacgaca tcattccgtg gcgttatcca gctaagcgcg aactgcaatt
     3421 tggagaatgg cagcgcaatg acattcttgc aggtatcttc gagccagcca cgatcgacat
     3481 tgatctggct atcttgctga caaaagcaag agaacatagc gttgccttgg taggtccagc
     3541 ggcggaggaa ctctttgatc cggttcctga acaggatcta tttgaggcgc taaatgaaac
     3601 cttaacgcta tggaactcgc cgcccgactg ggctggcgat gagcgaaatg tagtgcttac
     3661 gttgtcccgc atttggtaca gcgcagtaac cggcaaaatc gcgccgaagg atgtcgctgc
     3721 cgactgggca atggagcgcc tgccggccca gtatcagccc gtcatacttg aagctagaca
     3781 ggcttatctt ggacaagaag aagatcgctt ggcctcgcgc gcagatcagt tggaagaatt
     3841 tgtccactac gtgaaaggcg agatcaccaa ggtagtcggc aaataaccct cgagccaccc
     3901 atgaccaaaa tcccttaacg tgagttacgc gtcgttccac tgagcgtcag accccgtaga
     3961 aaagatcaaa ggatcttctt gagatccttt ttttctgcgc gtaatctgct gcttgcaaac
     4021 aaaaaaacca ccgctaccag cggtggtttg tttgccggat caagagctac caactctttt
     4081 tccgaaggta actggcttca gcagagcgca gataccaaat actgtccttc tagtgtagcc
     4141 gtagttaggc caccacttca agaactctgt agcaccgcct acatacctcg ctctgctaat
     4201 cctgttacca gtggctgctg ccagtggcga taagtcgtgt cttaccgggt tggactcaag
     4261 acgatagtta ccggataagg cgcagcggtc gggctgaacg gggggttcgt gcacacagcc
     4321 cagcttggag cgaacgacct acaccgaact gagataccta cagcgtgagc attgagaaag
     4381 cgccacgctt cccgaaggga gaaaggcgga caggtatccg gtaagcggca gggtcggaac
     4441 aggagagcgc acgagggagc ttccaggggg aaacgcctgg tatctttata gtcctgtcgg
     4501 gtttcgccac ctctgacttg agcgtcgatt tttgtgatgc tcgtcagggg ggcggagcct
     4561 atggaaaaac gccagcaacg cggccttttt acggttcctg gccttttgct ggccttttgc
     4621 tcacatgttc tttcctgcgt tatcccctga ttctgtggat aaccgtatta ccgcctttga
     4681 gtgagctgat accgctcgcc gcagccgaac gaccgagcgc agcgagtcag tgagcgagga
     4741 agcggaagag cgcccaatac gcaaaccgcc tctccccgcg cgttggccga ttcattaatg
     4801 cagctggcac gacaggtttc ccgactggaa agcgggcagt gagcgcaacg caattaatac
     4861 gcgtaccgct cgccaggaag agtttgtaga aacgcaaaaa ggccatccgt caggatggcc
     4921 ttctgcttag tttgatgcct ggcagtttat ggcgggcgtc ctgcccgcca ccctccgggc
     4981 cgttgcttca caacgttcaa atccgctccc ggcggatttg tcctactcag gagagcgttc
     5041 accgacaaac aacagataaa acgaaaggcc cagtcttccg actgagcctt tcgttttatt
     5101 tgatgcctgg cagttcccta ctctcgcgtt aacgcttgca tggatgt
//
