LOCUS       sequence-271409-        5068 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Gateway entry clone for BhCas12b sgRNA expression under ZmUbi 
            promoter with ribozyme processing.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 5068)
  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 5068)
  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-Bh
FEATURES             Location/Qualifiers
     source          1..5068
                     /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    50..145
                     /gene="mutant version of attL"
                     /label=attL5
                     /bound_moiety="LR Clonase(TM)"
                     /note="recombination site for the Gateway(R) LR reaction"
     ncRNA           2141..2177
                     /product="self-cleaving hammerhead ribozyme (Ferre-D'Amare 
                     and Scott, 2010)"
                     /label=hammerhead ribozyme
                     /ncRNA_class="hammerhead_ribozyme"
     ncRNA           2307..2374
                     /product="hepatitis delta virus self-cleaving ribozyme 
                     (Ferre-D'Amare and Scott, 2010)"
                     /label=HDV ribozyme
                     /ncRNA_class="ribozyme"
     protein_bind    complement(2394..2493)
                     /gene="mutant version of attL"
                     /label=attL2
                     /bound_moiety="LR Clonase(TM)"
                     /note="recombination site for the Gateway(R) LR reaction"
     promoter        complement(2511..2529)
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     promoter        complement(2546..2564)
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     primer_bind     complement(2569..2585)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(2569..2585)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     CDS             3016..3807
                     /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      3900..4488
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     4389..4408
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     4642..4659
                     /label=L4440
                     /note="L4440 vector, forward primer"
     terminator      complement(4818..4845)
                     /label=rrnB T2 terminator
                     /note="transcription terminator T2 from the E. coli rrnB 
                     gene"
     terminator      complement(4937..5023)
                     /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 aataatgatt
       61 ttattttgac tgatagtgac ctgttcgttg caacaaattg atgagcaatg cttttttata
      121 atgccaactt tgtatacaaa agttgcccca tggcgttccc tctagagaga taatgagcat
      181 tgcatgtcta agttataaaa aattaccaca tatttttttt gtcacacttg tttgaagtgc
      241 agtttatcta tctttataca tatatttaaa ctttactcta cgaataatat aatctatagt
      301 actacaataa tatcagtgtt ttagagaatc atataaatga acagttagac atggtctaaa
      361 ggacaattga gtattttgac aacaggactc tacagtttta tctttttagt gtgcatgtgt
      421 tctccttttt ttttgcaaat agcttcacct atataatact tcatccattt tattagtaca
      481 tccatttagg gtttagggtt aatggttttt atagactaat ttttttagta catctatttt
      541 attctatttt agcctctaaa ttaagaaaac taaaactcta ttttagtttt tttatttaat
      601 aatttagata taaaatagaa taaaataaag tgactaaaaa ttaaacaaat accctttaag
      661 aaattaaaaa aactaaggaa acatttttct tgtttcgagt agataatgcc agcctgttaa
      721 acgccgtcga cgagtctaac ggacaccaac cagcgaacca gcagcgtcgc gtcgggccaa
      781 gcgaagcaga cggcacggca tctctgtcgc tgcctctgga cccctctcga gagttccgct
      841 ccaccgttgg acttgctccg ctgtcggcat ccagaaattg cgtggcggag cggcagacgt
      901 gagccggcac ggcaggcggc ctcctcctcc tctcacggca ccggcagcta cgggggattc
      961 ctttcccacc gctccttcgc tttcccttcc tcgcccgccg taataaatag acaccccctc
     1021 cacaccctct ttccccaacc tcgtgttgtt cggagcgcac acacacacaa ccagatctcc
     1081 cccaaatcca cccgtcggca cctccgcttc aaggtacgcc gctcgtcctc cccccccccc
     1141 ccctctctac cttctctaga tcggcgttcc ggtccatggt tagggcccgg tagttctact
     1201 tctgttcatg tttgtgttag atccgtgttt gtgttagatc cgtgctgcta gcgttcgtac
     1261 acggatgcga cctgtacgtc agacacgttc tgattgctaa cttgccagtg tttctctttg
     1321 gggaatcctg ggatggctct agccgttccg cagacgggat cgatttcatg attttttttg
     1381 tttcgttgca tagggtttgg tttgcccttt tcctttattt caatatatgc cgtgcacttg
     1441 tttgtcgggt catcttttca tgcttttttt tgtcttggtt gtgatgatgt ggtctggttg
     1501 ggcggtcgtt ctagatcgga gtagaattaa ttctgtttca aactacctgg tggatttatt
     1561 aattttggat ctgtatgtgt gtgccataca tattcatagt tacgaattga agatgatgga
     1621 tggaaatatc gatctaggat aggtatacat gttgatgcgg gttttactga tgcatataca
     1681 gagatgcttt ttgttcgctt ggttgtgatg atgtggtgtg gttgggcggt cgttcattcg
     1741 ttctagatcg gagtagaata ctgtttcaaa ctacctggtg tatttattaa ttttggaact
     1801 gtatgtgtgt gtcatacatc ttcatagtta cgagtttaag atggatggaa atatcgatct
     1861 aggataggta tacatgttga tgtgggtttt actgatgcat atacatgatg gcatatgcag
     1921 catctattca tatgctctaa ccttgagtac ctatctatta taataaacaa gtatgtttta
     1981 taattatttt gatcttgata tacttggatg atggcatatg cagcagctat atgtggattt
     2041 ttttagccct gccttcatac gctatttatt tgcttggtac tgtttctttt gtcgatgctc
     2101 accctgttgt ttggtgttac ttctgcaggg atcccagaac ctgatgagtc cgtgaggacg
     2161 aaacgagtaa gctcgtcgtt ctgtcttttg gtcaggacaa ccgtctagct ataagtgctg
     2221 cagggtgtga gaaactccta ttgctggacg atgtctctta cgaggcatta gcacgagacg
     2281 gagctcagtc tgaccgcggc gtctctggcc ggcatggtcc cagcctcctc gctggcgccg
     2341 gctgggcaac atgcttcggc atggcgaatg ggacggtacc cggccggaat tcgacccagc
     2401 tttcttgtac aaagttggca ttataaaaaa taattgctca tcaatttgtt gcaacgaaca
     2461 ggtcactatc agtcaaaata aaatcattat ttgccatcca gctgatatcc cctatagtga
     2521 gtcgtattac atggtcatag catcccctat agtgagtcgt attacatggt catagctgtt
     2581 tcctggcagc tctggcccgt gtctcaaaat ctctgatgtt acattgcaca agataaaaat
     2641 atatcatcat gcctcctctg gaccagccag gacagaaatg cctcgacttc gctgctaccc
     2701 aaggttgccg ggtgacgcac accgtggaaa cggatgaagg cacgaaccca gtggacataa
     2761 gcctgttcgg ttcgtaagct gtaatgcaag tagcgtatgc gctcacgcaa ctggtccaga
     2821 accttgaccg aacgcagcgg tggtaacggc gcagtggcgg ttttcatggc ttgttatgac
     2881 tgtttttttg gggtacagtc tatgcctcgg gcatccaagc agcaagcgcg ttacgccgtg
     2941 ggtcgatgtt tgatgttatg gagcagcaac gatgttacgc agcagggcag tcgccctaaa
     3001 acaaagttaa acattatgag ggaagcggtg atcgccgaag tatcgactca actatcagag
     3061 gtagttggcg tcatcgagcg ccatctcgaa ccgacgttgc tggccgtaca tttgtacggc
     3121 tccgcagtgg atggcggcct gaagccacac agtgatattg atttgctggt tacggtgacc
     3181 gtaaggcttg atgaaacaac gcggcgagct ttgatcaacg accttttgga aacttcggct
     3241 tcccctggag agagcgagat tctccgcgct gtagaagtca ccattgttgt gcacgacgac
     3301 atcattccgt ggcgttatcc agctaagcgc gaactgcaat ttggagaatg gcagcgcaat
     3361 gacattcttg caggtatctt cgagccagcc acgatcgaca ttgatctggc tatcttgctg
     3421 acaaaagcaa gagaacatag cgttgccttg gtaggtccag cggcggagga actctttgat
     3481 ccggttcctg aacaggatct atttgaggcg ctaaatgaaa ccttaacgct atggaactcg
     3541 ccgcccgact gggctggcga tgagcgaaat gtagtgctta cgttgtcccg catttggtac
     3601 agcgcagtaa ccggcaaaat cgcgccgaag gatgtcgctg ccgactgggc aatggagcgc
     3661 ctgccggccc agtatcagcc cgtcatactt gaagctagac aggcttatct tggacaagaa
     3721 gaagatcgct tggcctcgcg cgcagatcag ttggaagaat ttgtccacta cgtgaaaggc
     3781 gagatcacca aggtagtcgg caaataaccc tcgagccacc catgaccaaa atcccttaac
     3841 gtgagttacg cgtcgttcca ctgagcgtca gaccccgtag aaaagatcaa aggatcttct
     3901 tgagatcctt tttttctgcg cgtaatctgc tgcttgcaaa caaaaaaacc accgctacca
     3961 gcggtggttt gtttgccgga tcaagagcta ccaactcttt ttccgaaggt aactggcttc
     4021 agcagagcgc agataccaaa tactgttctt ctagtgtagc cgtagttagg ccaccacttc
     4081 aagaactctg tagcaccgcc tacatacctc gctctgctaa tcctgttacc agtggctgct
     4141 gccagtggcg ataagtcgtg tcttaccggg ttggactcaa gacgatagtt accggataag
     4201 gcgcagcggt cgggctgaac ggggggttcg tgcacacagc ccagcttgga gcgaacgacc
     4261 tacaccgaac tgagatacct acagcgtgag ctatgagaaa gcgccacgct tcccgaaggg
     4321 agaaaggcgg acaggtatcc ggtaagcggc agggtcggaa caggagagcg cacgagggag
     4381 cttccagggg gaaacgcctg gtatctttat agtcctgtcg ggtttcgcca cctctgactt
     4441 gagcgtcgat ttttgtgatg ctcgtcaggg gggcggagcc tatggaaaaa cgccagcaac
     4501 gcggcctttt tacggttcct ggccttttgc tggccttttg ctcacatgtt ctttcctgcg
     4561 ttatcccctg attctgtgga taaccgtatt accgcctttg agtgagctga taccgctcgc
     4621 cgcagccgaa cgaccgagcg cagcgagtca gtgagcgagg aagcggaaga gcgcccaata
     4681 cgcaaaccgc ctctccccgc gcgttggccg attcattaat gcagctggca cgacaggttt
     4741 cccgactgga aagcgggcag tgagcgcaac gcaattaata cgcgtaccgc gagccaggaa
     4801 gagtttgtag aaacgcaaaa aggccatccg tcaggatggc cttctgctta gtttgatgcc
     4861 tggcagttta tggcgggcgt cctgcccgcc accctccggg ccgttgcttc acaacgttca
     4921 aatccgctcc cggcggattt gtcctactca ggagagcgtt caccgacaaa caacagataa
     4981 aacgaaaggc ccagtcttcc gactgagcct ttcgttttat ttgatgcctg gcagttccct
     5041 actctcgcgt taacgcttgc atggatgt
//
