LOCUS       sequence-263175-        5069 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 5069)
  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 5069)
  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..5069
                     /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           2308..2375
                     /product="hepatitis delta virus self-cleaving ribozyme 
                     (Ferre-D'Amare and Scott, 2010)"
                     /label=HDV ribozyme
                     /ncRNA_class="ribozyme"
     protein_bind    complement(2395..2494)
                     /gene="mutant version of attL"
                     /label=attL2
                     /bound_moiety="LR Clonase(TM)"
                     /note="recombination site for the Gateway(R) LR reaction"
     promoter        complement(2512..2530)
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     promoter        complement(2547..2565)
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     primer_bind     complement(2570..2586)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(2570..2586)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     CDS             3017..3808
                     /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      3901..4489
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     4390..4409
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     4643..4660
                     /label=L4440
                     /note="L4440 vector, forward primer"
     terminator      complement(4819..4846)
                     /label=rrnB T2 terminator
                     /note="transcription terminator T2 from the E. coli rrnB 
                     gene"
     terminator      complement(4938..5024)
                     /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 gatcccagaa cctgatgagt ccgtgaggac
     2161 gaaacgagta agctcgtcgt tctgtctttt ggtcaggaca accgtctagc tataagtgct
     2221 gcagggtgtg agaaactcct attgctggac gatgtctctt acgaggcatt agcacgagac
     2281 ggagctcagt ctgaccgcgg cgtctctggc cggcatggtc ccagcctcct cgctggcgcc
     2341 ggctgggcaa catgcttcgg catggcgaat gggacggtac ccggccggaa ttcgacccag
     2401 ctttcttgta caaagttggc attataaaaa ataattgctc atcaatttgt tgcaacgaac
     2461 aggtcactat cagtcaaaat aaaatcatta tttgccatcc agctgatatc ccctatagtg
     2521 agtcgtatta catggtcata gcatccccta tagtgagtcg tattacatgg tcatagctgt
     2581 ttcctggcag ctctggcccg tgtctcaaaa tctctgatgt tacattgcac aagataaaaa
     2641 tatatcatca tgcctcctct ggaccagcca ggacagaaat gcctcgactt cgctgctgcc
     2701 caaggttgcc gggtgacgca caccgtggaa acggatgaag gcacgaaccc agtggacata
     2761 agcctgttcg gttcgtaagc tgtaatgcaa gtagcgtatg cgctcacgca actggtccag
     2821 aaccttgacc gaacgcagcg gtggtaacgg cgcagtggcg gttttcatgg cttgttatga
     2881 ctgttttttt ggggtacagt ctatgcctcg ggcatccaag cagcaagcgc gttacgccgt
     2941 gggtcgatgt ttgatgttat ggagcagcaa cgatgttacg cagcagggca gtcgccctaa
     3001 aacaaagtta aacatcatga gggaagcggt gatcgccgaa gtatcgactc aactatcaga
     3061 ggtagttggc gtcatcgagc gccatctcga accgacgttg ctggccgtac atttgtacgg
     3121 ctccgcagtg gatggcggcc tgaagccaca cagtgatatt gatttgctgg ttacggtgac
     3181 cgtaaggctt gatgaaacaa cgcggcgagc tttgatcaac gaccttttgg aaacttcggc
     3241 ttcccctgga gagagcgaga ttctccgcgc tgtagaagtc accattgttg tgcacgacga
     3301 catcattccg tggcgttatc cagctaagcg cgaactgcaa tttggagaat ggcagcgcaa
     3361 tgacattctt gcaggtatct tcgagccagc cacgatcgac attgatctgg ctatcttgct
     3421 gacaaaagca agagaacata gcgttgcctt ggtaggtcca gcggcggagg aactctttga
     3481 tccggttcct gaacaggatc tatttgaggc gctaaatgaa accttaacgc tatggaactc
     3541 gccgcccgac tgggctggcg atgagcgaaa tgtagtgctt acgttgtccc gcatttggta
     3601 cagcgcagta accggcaaaa tcgcgccgaa ggatgtcgct gccgactggg caatggagcg
     3661 cctgccggcc cagtatcagc ccgtcatact tgaagctaga caggcttatc ttggacaaga
     3721 agaagatcgc ttggcctcgc gcgcagatca gttggaagaa tttgtccact acgtgaaagg
     3781 cgagatcacc aaggtagtcg gcaaataacc ctcgagccac ccatgaccaa aatcccttaa
     3841 cgtgagttac gcgtcgttcc actgagcgtc agaccccgta gaaaagatca aaggatcttc
     3901 ttgagatcct ttttttctgc gcgtaatctg ctgcttgcaa acaaaaaaac caccgctacc
     3961 agcggtggtt tgtttgccgg atcaagagct accaactctt tttccgaagg taactggctt
     4021 cagcagagcg cagataccaa atactgtcct tctagtgtag ccgtagttag gccaccactt
     4081 caagaactct gtagcaccgc ctacatacct cgctctgcta atcctgttac cagtggctgc
     4141 tgccagtggc gataagtcgt gtcttaccgg gttggactca agacgatagt taccggataa
     4201 ggcgcagcgg tcgggctgaa cggggggttc gtgcacacag cccagcttgg agcgaacgac
     4261 ctacaccgaa ctgagatacc tacagcgtga gcattgagaa agcgccacgc ttcccgaagg
     4321 gagaaaggcg gacaggtatc cggtaagcgg cagggtcgga acaggagagc gcacgaggga
     4381 gcttccaggg ggaaacgcct ggtatcttta tagtcctgtc gggtttcgcc acctctgact
     4441 tgagcgtcga tttttgtgat gctcgtcagg ggggcggagc ctatggaaaa acgccagcaa
     4501 cgcggccttt ttacggttcc tggccttttg ctggcctttt gctcacatgt tctttcctgc
     4561 gttatcccct gattctgtgg ataaccgtat taccgccttt gagtgagctg ataccgctcg
     4621 ccgcagccga acgaccgagc gcagcgagtc agtgagcgag gaagcggaag agcgcccaat
     4681 acgcaaaccg cctctccccg cgcgttggcc gattcattaa tgcagctggc acgacaggtt
     4741 tcccgactgg aaagcgggca gtgagcgcaa cgcaattaat acgcgtaccg ctcgccagga
     4801 agagtttgta gaaacgcaaa aaggccatcc gtcaggatgg ccttctgctt agtttgatgc
     4861 ctggcagttt atggcgggcg tcctgcccgc caccctccgg gccgttgctt cacaacgttc
     4921 aaatccgctc ccggcggatt tgtcctactc aggagagcgt tcaccgacaa acaacagata
     4981 aaacgaaagg cccagtcttc cgactgagcc tttcgtttta tttgatgcct ggcagttccc
     5041 tactctcgcg ttaacgcttg catggatgt
//
