LOCUS       sequence-271509-        5086 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 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 5086)
  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 5086)
  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-Aa3.8.4
FEATURES             Location/Qualifiers
     source          1..5086
                     /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"
     misc_RNA        2254..2272
                     /label=MS2 stem loop
                     /note="stem loop that binds the bacteriophage MS2 coat 
                     protein"
     ncRNA           2325..2392
                     /product="hepatitis delta virus self-cleaving ribozyme 
                     (Ferre-D'Amare and Scott, 2010)"
                     /label=HDV ribozyme
                     /ncRNA_class="ribozyme"
     protein_bind    complement(2412..2511)
                     /gene="mutant version of attL"
                     /label=attL2
                     /bound_moiety="LR Clonase(TM)"
                     /note="recombination site for the Gateway(R) LR reaction"
     promoter        complement(2529..2547)
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     promoter        complement(2564..2582)
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     primer_bind     complement(2587..2603)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(2587..2603)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     CDS             3034..3825
                     /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      3918..4506
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     4407..4426
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     4660..4677
                     /label=L4440
                     /note="L4440 vector, forward primer"
     terminator      complement(4836..4863)
                     /label=rrnB T2 terminator
                     /note="transcription terminator T2 from the E. coli rrnB 
                     gene"
     terminator      complement(4955..5041)
                     /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 tgaacttcgg ccaacatgag gatcacccat gtctgcaggg
     2281 ccgaagtggc acgagacgga gctcagtctg accgcggcgt ctctggccgg catggtccca
     2341 gcctcctcgc tggcgccggc tgggcaacat gcttcggcat ggcgaatggg acggtacccg
     2401 gccggaattc gacccagctt tcttgtacaa agttggcatt ataaaaaata attgctcatc
     2461 aatttgttgc aacgaacagg tcactatcag tcaaaataaa atcattattt gccatccagc
     2521 tgatatcccc tatagtgagt cgtattacat ggtcatagca tcccctatag tgagtcgtat
     2581 tacatggtca tagctgtttc ctggcagctc tggcccgtgt ctcaaaatct ctgatgttac
     2641 attgcacaag ataaaaatat atcatcatgc ctcctctgga ccagccagga cagaaatgcc
     2701 tcgacttcgc tgctacccaa ggttgccggg tgacgcacac cgtggaaacg gatgaaggca
     2761 cgaacccagt ggacataagc ctgttcggtt cgtaagctgt aatgcaagta gcgtatgcgc
     2821 tcacgcaact ggtccagaac cttgaccgaa cgcagcggtg gtaacggcgc agtggcggtt
     2881 ttcatggctt gttatgactg tttttttggg gtacagtcta tgcctcgggc atccaagcag
     2941 caagcgcgtt acgccgtggg tcgatgtttg atgttatgga gcagcaacga tgttacgcag
     3001 cagggcagtc gccctaaaac aaagttaaac attatgaggg aagcggtgat cgccgaagta
     3061 tcgactcaac tatcagaggt agttggcgtc atcgagcgcc atctcgaacc gacgttgctg
     3121 gccgtacatt tgtacggctc cgcagtggat ggcggcctga agccacacag tgatattgat
     3181 ttgctggtta cggtgaccgt aaggcttgat gaaacaacgc ggcgagcttt gatcaacgac
     3241 cttttggaaa cttcggcttc ccctggagag agcgagattc tccgcgctgt agaagtcacc
     3301 attgttgtgc acgacgacat cattccgtgg cgttatccag ctaagcgcga actgcaattt
     3361 ggagaatggc agcgcaatga cattcttgca ggtatcttcg agccagccac gatcgacatt
     3421 gatctggcta tcttgctgac aaaagcaaga gaacatagcg ttgccttggt aggtccagcg
     3481 gcggaggaac tctttgatcc ggttcctgaa caggatctat ttgaggcgct aaatgaaacc
     3541 ttaacgctat ggaactcgcc gcccgactgg gctggcgatg agcgaaatgt agtgcttacg
     3601 ttgtcccgca tttggtacag cgcagtaacc ggcaaaatcg cgccgaagga tgtcgctgcc
     3661 gactgggcaa tggagcgcct gccggcccag tatcagcccg tcatacttga agctagacag
     3721 gcttatcttg gacaagaaga agatcgcttg gcctcgcgcg cagatcagtt ggaagaattt
     3781 gtccactacg tgaaaggcga gatcaccaag gtagtcggca aataaccctc gagccaccca
     3841 tgaccaaaat cccttaacgt gagttacgcg tcgttccact gagcgtcaga ccccgtagaa
     3901 aagatcaaag gatcttcttg agatcctttt tttctgcgcg taatctgctg cttgcaaaca
     3961 aaaaaaccac cgctaccagc ggtggtttgt ttgccggatc aagagctacc aactcttttt
     4021 ccgaaggtaa ctggcttcag cagagcgcag ataccaaata ctgttcttct agtgtagccg
     4081 tagttaggcc accacttcaa gaactctgta gcaccgccta catacctcgc tctgctaatc
     4141 ctgttaccag tggctgctgc cagtggcgat aagtcgtgtc ttaccgggtt ggactcaaga
     4201 cgatagttac cggataaggc gcagcggtcg ggctgaacgg ggggttcgtg cacacagccc
     4261 agcttggagc gaacgaccta caccgaactg agatacctac agcgtgagct atgagaaagc
     4321 gccacgcttc ccgaagggag aaaggcggac aggtatccgg taagcggcag ggtcggaaca
     4381 ggagagcgca cgagggagct tccaggggga aacgcctggt atctttatag tcctgtcggg
     4441 tttcgccacc tctgacttga gcgtcgattt ttgtgatgct cgtcaggggg gcggagccta
     4501 tggaaaaacg ccagcaacgc ggccttttta cggttcctgg ccttttgctg gccttttgct
     4561 cacatgttct ttcctgcgtt atcccctgat tctgtggata accgtattac cgcctttgag
     4621 tgagctgata ccgctcgccg cagccgaacg accgagcgca gcgagtcagt gagcgaggaa
     4681 gcggaagagc gcccaatacg caaaccgcct ctccccgcgc gttggccgat tcattaatgc
     4741 agctggcacg acaggtttcc cgactggaaa gcgggcagtg agcgcaacgc aattaatacg
     4801 cgtaccgcga gccaggaaga gtttgtagaa acgcaaaaag gccatccgtc aggatggcct
     4861 tctgcttagt ttgatgcctg gcagtttatg gcgggcgtcc tgcccgccac cctccgggcc
     4921 gttgcttcac aacgttcaaa tccgctcccg gcggatttgt cctactcagg agagcgttca
     4981 ccgacaaaca acagataaaa cgaaaggccc agtcttccga ctgagccttt cgttttattt
     5041 gatgcctggc agttccctac tctcgcgtta acgcttgcat ggatgt
//
