LOCUS       sequence-271512-        5116 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 5116)
  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 5116)
  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..5116
                     /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           2355..2422
                     /product="hepatitis delta virus self-cleaving ribozyme 
                     (Ferre-D'Amare and Scott, 2010)"
                     /label=HDV ribozyme
                     /ncRNA_class="ribozyme"
     protein_bind    complement(2442..2541)
                     /gene="mutant version of attL"
                     /label=attL2
                     /bound_moiety="LR Clonase(TM)"
                     /note="recombination site for the Gateway(R) LR reaction"
     promoter        complement(2559..2577)
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     promoter        complement(2594..2612)
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     primer_bind     complement(2617..2633)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(2617..2633)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     CDS             3064..3855
                     /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      3948..4536
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     4437..4456
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     4690..4707
                     /label=L4440
                     /note="L4440 vector, forward primer"
     terminator      complement(4866..4893)
                     /label=rrnB T2 terminator
                     /note="transcription terminator T2 from the E. coli rrnB 
                     gene"
     terminator      complement(4985..5071)
                     /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 atccaaatct ctgatgagtc cgtgaggacg
     2161 aaacgagtaa gctcgtcaga tttctgtcta aaggacagaa tttttcaacg ggtgtgccaa
     2221 tggccacttt ccaggtggca aagcccgttg aacttctcaa aaagaacgct cgctcagtgt
     2281 tctgacgtcg gatcactgag cgagcgatct gagaagtggc acgagacgga gctcagtctg
     2341 accgcggcgt ctctggccgg catggtccca gcctcctcgc tggcgccggc tgggcaacat
     2401 gcttcggcat ggcgaatggg acggtacccg gccggaattc gacccagctt tcttgtacaa
     2461 agttggcatt ataaaaaata attgctcatc aatttgttgc aacgaacagg tcactatcag
     2521 tcaaaataaa atcattattt gccatccagc tgatatcccc tatagtgagt cgtattacat
     2581 ggtcatagca tcccctatag tgagtcgtat tacatggtca tagctgtttc ctggcagctc
     2641 tggcccgtgt ctcaaaatct ctgatgttac attgcacaag ataaaaatat atcatcatgc
     2701 ctcctctgga ccagccagga cagaaatgcc tcgacttcgc tgctacccaa ggttgccggg
     2761 tgacgcacac cgtggaaacg gatgaaggca cgaacccagt ggacataagc ctgttcggtt
     2821 cgtaagctgt aatgcaagta gcgtatgcgc tcacgcaact ggtccagaac cttgaccgaa
     2881 cgcagcggtg gtaacggcgc agtggcggtt ttcatggctt gttatgactg tttttttggg
     2941 gtacagtcta tgcctcgggc atccaagcag caagcgcgtt acgccgtggg tcgatgtttg
     3001 atgttatgga gcagcaacga tgttacgcag cagggcagtc gccctaaaac aaagttaaac
     3061 attatgaggg aagcggtgat cgccgaagta tcgactcaac tatcagaggt agttggcgtc
     3121 atcgagcgcc atctcgaacc gacgttgctg gccgtacatt tgtacggctc cgcagtggat
     3181 ggcggcctga agccacacag tgatattgat ttgctggtta cggtgaccgt aaggcttgat
     3241 gaaacaacgc ggcgagcttt gatcaacgac cttttggaaa cttcggcttc ccctggagag
     3301 agcgagattc tccgcgctgt agaagtcacc attgttgtgc acgacgacat cattccgtgg
     3361 cgttatccag ctaagcgcga actgcaattt ggagaatggc agcgcaatga cattcttgca
     3421 ggtatcttcg agccagccac gatcgacatt gatctggcta tcttgctgac aaaagcaaga
     3481 gaacatagcg ttgccttggt aggtccagcg gcggaggaac tctttgatcc ggttcctgaa
     3541 caggatctat ttgaggcgct aaatgaaacc ttaacgctat ggaactcgcc gcccgactgg
     3601 gctggcgatg agcgaaatgt agtgcttacg ttgtcccgca tttggtacag cgcagtaacc
     3661 ggcaaaatcg cgccgaagga tgtcgctgcc gactgggcaa tggagcgcct gccggcccag
     3721 tatcagcccg tcatacttga agctagacag gcttatcttg gacaagaaga agatcgcttg
     3781 gcctcgcgcg cagatcagtt ggaagaattt gtccactacg tgaaaggcga gatcaccaag
     3841 gtagtcggca aataaccctc gagccaccca tgaccaaaat cccttaacgt gagttacgcg
     3901 tcgttccact gagcgtcaga ccccgtagaa aagatcaaag gatcttcttg agatcctttt
     3961 tttctgcgcg taatctgctg cttgcaaaca aaaaaaccac cgctaccagc ggtggtttgt
     4021 ttgccggatc aagagctacc aactcttttt ccgaaggtaa ctggcttcag cagagcgcag
     4081 ataccaaata ctgttcttct agtgtagccg tagttaggcc accacttcaa gaactctgta
     4141 gcaccgccta catacctcgc tctgctaatc ctgttaccag tggctgctgc cagtggcgat
     4201 aagtcgtgtc ttaccgggtt ggactcaaga cgatagttac cggataaggc gcagcggtcg
     4261 ggctgaacgg ggggttcgtg cacacagccc agcttggagc gaacgaccta caccgaactg
     4321 agatacctac agcgtgagct atgagaaagc gccacgcttc ccgaagggag aaaggcggac
     4381 aggtatccgg taagcggcag ggtcggaaca ggagagcgca cgagggagct tccaggggga
     4441 aacgcctggt atctttatag tcctgtcggg tttcgccacc tctgacttga gcgtcgattt
     4501 ttgtgatgct cgtcaggggg gcggagccta tggaaaaacg ccagcaacgc ggccttttta
     4561 cggttcctgg ccttttgctg gccttttgct cacatgttct ttcctgcgtt atcccctgat
     4621 tctgtggata accgtattac cgcctttgag tgagctgata ccgctcgccg cagccgaacg
     4681 accgagcgca gcgagtcagt gagcgaggaa gcggaagagc gcccaatacg caaaccgcct
     4741 ctccccgcgc gttggccgat tcattaatgc agctggcacg acaggtttcc cgactggaaa
     4801 gcgggcagtg agcgcaacgc aattaatacg cgtaccgcga gccaggaaga gtttgtagaa
     4861 acgcaaaaag gccatccgtc aggatggcct tctgcttagt ttgatgcctg gcagtttatg
     4921 gcgggcgtcc tgcccgccac cctccgggcc gttgcttcac aacgttcaaa tccgctcccg
     4981 gcggatttgt cctactcagg agagcgttca ccgacaaaca acagataaaa cgaaaggccc
     5041 agtcttccga ctgagccttt cgttttattt gatgcctggc agttccctac tctcgcgtta
     5101 acgcttgcat ggatgt
//
