LOCUS       sequence-271507-        5056 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Gateway entry clone for AaCas12b sgRNA expression under ZmUbi 
            promoter with ribozyme processing; sgRNA scaffold 3.8 is used.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 5056)
  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 5056)
  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-Aa3.8
FEATURES             Location/Qualifiers
     source          1..5056
                     /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           2295..2362
                     /product="hepatitis delta virus self-cleaving ribozyme 
                     (Ferre-D'Amare and Scott, 2010)"
                     /label=HDV ribozyme
                     /ncRNA_class="ribozyme"
     protein_bind    complement(2382..2481)
                     /gene="mutant version of attL"
                     /label=attL2
                     /bound_moiety="LR Clonase(TM)"
                     /note="recombination site for the Gateway(R) LR reaction"
     promoter        complement(2499..2517)
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     promoter        complement(2534..2552)
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     primer_bind     complement(2557..2573)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(2557..2573)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     CDS             3004..3795
                     /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      3888..4476
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     4377..4396
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     4630..4647
                     /label=L4440
                     /note="L4440 vector, forward primer"
     terminator      complement(4806..4833)
                     /label=rrnB T2 terminator
                     /note="transcription terminator T2 from the E. coli rrnB 
                     gene"
     terminator      complement(4925..5011)
                     /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 atccttagac ctgatgagtc cgtgaggacg
     2161 aaacgagtaa gctcgtcgtc taaaggacag aatttttcaa cgggtgtgcc aatggccact
     2221 ttccaggtgg caaagcccgt tgaacttcaa gcgaagtggc acgagacgga gctcagtctg
     2281 accgcggcgt ctctggccgg catggtccca gcctcctcgc tggcgccggc tgggcaacat
     2341 gcttcggcat ggcgaatggg acggtacccg gccggaattc gacccagctt tcttgtacaa
     2401 agttggcatt ataaaaaata attgctcatc aatttgttgc aacgaacagg tcactatcag
     2461 tcaaaataaa atcattattt gccatccagc tgatatcccc tatagtgagt cgtattacat
     2521 ggtcatagca tcccctatag tgagtcgtat tacatggtca tagctgtttc ctggcagctc
     2581 tggcccgtgt ctcaaaatct ctgatgttac attgcacaag ataaaaatat atcatcatgc
     2641 ctcctctgga ccagccagga cagaaatgcc tcgacttcgc tgctacccaa ggttgccggg
     2701 tgacgcacac cgtggaaacg gatgaaggca cgaacccagt ggacataagc ctgttcggtt
     2761 cgtaagctgt aatgcaagta gcgtatgcgc tcacgcaact ggtccagaac cttgaccgaa
     2821 cgcagcggtg gtaacggcgc agtggcggtt ttcatggctt gttatgactg tttttttggg
     2881 gtacagtcta tgcctcgggc atccaagcag caagcgcgtt acgccgtggg tcgatgtttg
     2941 atgttatgga gcagcaacga tgttacgcag cagggcagtc gccctaaaac aaagttaaac
     3001 attatgaggg aagcggtgat cgccgaagta tcgactcaac tatcagaggt agttggcgtc
     3061 atcgagcgcc atctcgaacc gacgttgctg gccgtacatt tgtacggctc cgcagtggat
     3121 ggcggcctga agccacacag tgatattgat ttgctggtta cggtgaccgt aaggcttgat
     3181 gaaacaacgc ggcgagcttt gatcaacgac cttttggaaa cttcggcttc ccctggagag
     3241 agcgagattc tccgcgctgt agaagtcacc attgttgtgc acgacgacat cattccgtgg
     3301 cgttatccag ctaagcgcga actgcaattt ggagaatggc agcgcaatga cattcttgca
     3361 ggtatcttcg agccagccac gatcgacatt gatctggcta tcttgctgac aaaagcaaga
     3421 gaacatagcg ttgccttggt aggtccagcg gcggaggaac tctttgatcc ggttcctgaa
     3481 caggatctat ttgaggcgct aaatgaaacc ttaacgctat ggaactcgcc gcccgactgg
     3541 gctggcgatg agcgaaatgt agtgcttacg ttgtcccgca tttggtacag cgcagtaacc
     3601 ggcaaaatcg cgccgaagga tgtcgctgcc gactgggcaa tggagcgcct gccggcccag
     3661 tatcagcccg tcatacttga agctagacag gcttatcttg gacaagaaga agatcgcttg
     3721 gcctcgcgcg cagatcagtt ggaagaattt gtccactacg tgaaaggcga gatcaccaag
     3781 gtagtcggca aataaccctc gagccaccca tgaccaaaat cccttaacgt gagttacgcg
     3841 tcgttccact gagcgtcaga ccccgtagaa aagatcaaag gatcttcttg agatcctttt
     3901 tttctgcgcg taatctgctg cttgcaaaca aaaaaaccac cgctaccagc ggtggtttgt
     3961 ttgccggatc aagagctacc aactcttttt ccgaaggtaa ctggcttcag cagagcgcag
     4021 ataccaaata ctgttcttct agtgtagccg tagttaggcc accacttcaa gaactctgta
     4081 gcaccgccta catacctcgc tctgctaatc ctgttaccag tggctgctgc cagtggcgat
     4141 aagtcgtgtc ttaccgggtt ggactcaaga cgatagttac cggataaggc gcagcggtcg
     4201 ggctgaacgg ggggttcgtg cacacagccc agcttggagc gaacgaccta caccgaactg
     4261 agatacctac agcgtgagct atgagaaagc gccacgcttc ccgaagggag aaaggcggac
     4321 aggtatccgg taagcggcag ggtcggaaca ggagagcgca cgagggagct tccaggggga
     4381 aacgcctggt atctttatag tcctgtcggg tttcgccacc tctgacttga gcgtcgattt
     4441 ttgtgatgct cgtcaggggg gcggagccta tggaaaaacg ccagcaacgc ggccttttta
     4501 cggttcctgg ccttttgctg gccttttgct cacatgttct ttcctgcgtt atcccctgat
     4561 tctgtggata accgtattac cgcctttgag tgagctgata ccgctcgccg cagccgaacg
     4621 accgagcgca gcgagtcagt gagcgaggaa gcggaagagc gcccaatacg caaaccgcct
     4681 ctccccgcgc gttggccgat tcattaatgc agctggcacg acaggtttcc cgactggaaa
     4741 gcgggcagtg agcgcaacgc aattaatacg cgtaccgcga gccaggaaga gtttgtagaa
     4801 acgcaaaaag gccatccgtc aggatggcct tctgcttagt ttgatgcctg gcagtttatg
     4861 gcgggcgtcc tgcccgccac cctccgggcc gttgcttcac aacgttcaaa tccgctcccg
     4921 gcggatttgt cctactcagg agagcgttca ccgacaaaca acagataaaa cgaaaggccc
     4981 agtcttccga ctgagccttt cgttttattt gatgcctggc agttccctac tctcgcgtta
     5041 acgcttgcat ggatgt
//
