LOCUS       sequence-266595-        5311 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Golden Gate recipient and Gateway entry vector; assembly of 3 gRNAs 
            driven by Zea mays Ubi promoter.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 5311)
  AUTHORS   Zhang Y, Ren Q, Tang X, Liu S, Malzahn AA, Zhou J, Wang J, Yin D, 
            Pan C, Yuan M, Huang L, Yang H, Zhao Y, Fang Q, Zheng X, Tian L, 
            Cheng Y, Le Y, McCoy B, Franklin L, Selengut JD, Mount SM, Que Q, 
            Zhang Y, Qi Y
  TITLE     Expanding the scope of plant genome engineering with Cas12a 
            orthologs and highly multiplexable editing systems.
  JOURNAL   Nat Commun. 2021 Mar 29;12(1):1944. doi: 10.1038/s41467-021-22330-w.
  PUBMED    33782402
REFERENCE   2  (bases 1 to 5311)
  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"; journalName: "Nat 
            Commun."; date: "2021-03-29"; volume: "12(1):1944. doi"; pages: " 
            10.1038/s41467-021-22330-w"
COMMENT     Sequence Label: pYPQ143-ZmUbi
FEATURES             Location/Qualifiers
     source          1..5311
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     185..201
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     promoter        211..229
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     primer_bind     complement(316..332)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(329..351)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    340..356
                     /label=lac operator
                     /bound_moiety="lac repressor encoded by lacI"
                     /note="The lac repressor binds to the lac operator to 
                     inhibit transcription in E. coli. This inhibition can be 
                     relieved by adding lactose or 
                     isopropyl-beta-D-thiogalactopyranoside (IPTG)."
     promoter        complement(join(364..370,371..388,389..394))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    409..430
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     protein_bind    complement(495..594)
                     /gene="mutant version of attL"
                     /label=attL2
                     /bound_moiety="LR Clonase(TM)"
                     /note="recombination site for the Gateway(R) LR reaction"
     promoter        complement(612..630)
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     primer_bind     complement(635..651)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     CDS             1082..1873
                     /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      1966..2554
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     2455..2474
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     2708..2725
                     /label=L4440
                     /note="L4440 vector, forward primer"
     terminator      complement(2884..2911)
                     /label=rrnB T2 terminator
                     /note="transcription terminator T2 from the E. coli rrnB 
                     gene"
     terminator      complement(3003..3089)
                     /gene="Escherichia coli rrnB"
                     /label=rrnB T1 terminator
                     /note="transcription terminator T1 from the E. coli rrnB 
                     gene"
     primer_bind     3153..3169
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     protein_bind    3185..3280
                     /gene="mutant version of attL"
                     /label=attL5
                     /bound_moiety="LR Clonase(TM)"
                     /note="recombination site for the Gateway(R) LR reaction"
     promoter        3309..5306
                     /label=Ubi promoter
                     /note="maize polyubiquitin gene promoter"
     CDS             complement(join(5233..5311,1..320))
                     /codon_start=1
                     /gene="lacZ fragment"
                     /product="LacZ-alpha fragment of beta-galactosidase"
                     /label=lacZ-alpha
                     /translation="MTMITPSAQLTLNKGNKSWRTSRGGPVPNSPYSESYYARSLAVVL
                     QRRDWENPGVTQLNRLAAHPPFASWRNSEEARTDRPSQQLRSLNGEWDAPCSGAGLDSH
                     HLGIPAEVTPNNRVSIDKRNSTKQINSV"
ORIGIN
        1 cccaagtggt ggctatcgag accggcgccg ctacagggcg cgtcccattc gccattcagg
       61 ctgcgcaact gttgggaagg gcgatcggtg cgggcctctt cgctattacg ccagctggcg
      121 aaagggggat gtgctgcaag gcgattaagt tgggtaacgc cagggttttc ccagtcacga
      181 cgttgtaaaa cgacggccag tgagcgcgcg taatacgact cactataggg cgaattgggt
      241 accgggcccc ccctcgaggt cctccagctt ttgttccctt tattgagggt taattgcgcg
      301 cttggcgtaa tcatggtcat agctgtttcc tgtgtgaaat tgttatccgc tcacaattcc
      361 acacaacata ccagccggaa gcataaagtg taaagcctgg ggtgcctaat gagtgagcta
      421 actcacatta attgcgttgc gctcactgcc cgctttccac cggtggtctc tccagactag
      481 taagggcaaa ttcgacccag ctttcttgta caaagttggc attataaaaa ataattgctc
      541 atcaatttgt tgcaacgaac aggtcactat cagtcaaaat aaaatcatta tttgccatcc
      601 agctgatatc ccctatagtg agtcgtatta catggtcata gctgtttcct ggcagctctg
      661 gcccgtgtct caaaatctct gatgttacat tgcacaagat aaaaatatat catcatgcct
      721 cctctggacc agccaggaca gaaatgcctc gacttcgctg ctgcccaagg ttgccgggtg
      781 acgcacaccg tggaaacgga tgaaggcacg aacccagtgg acataagcct gttcggttcg
      841 taagctgtaa tgcaagtagc gtatgcgctc acgcaactgg tccagaacct tgaccgaacg
      901 cagcggtggt aacggcgcag tggcggtttt catggcttgt tatgactgtt tttttggggt
      961 acagtctatg cctcgggcat ccaagcagca agcgcgttac gccgtgggtc gatgtttgat
     1021 gttatggagc agcaacgatg ttacgcagca gggcagtcgc cctaaaacaa agttaaacat
     1081 catgagggaa gcggtgatcg ccgaagtatc gactcaacta tcagaggtag ttggcgtcat
     1141 cgagcgccat ctcgaaccga cgttgctggc cgtacatttg tacggctccg cagtggatgg
     1201 cggcctgaag ccacacagtg atattgattt gctggttacg gtgaccgtaa ggcttgatga
     1261 aacaacgcgg cgagctttga tcaacgacct tttggaaact tcggcttccc ctggagagag
     1321 cgagattctc cgcgctgtag aagtcaccat tgttgtgcac gacgacatca ttccgtggcg
     1381 ttatccagct aagcgcgaac tgcaatttgg agaatggcag cgcaatgaca ttcttgcagg
     1441 tatcttcgag ccagccacga tcgacattga tctggctatc ttgctgacaa aagcaagaga
     1501 acatagcgtt gccttggtag gtccagcggc ggaggaactc tttgatccgg ttcctgaaca
     1561 ggatctattt gaggcgctaa atgaaacctt aacgctatgg aactcgccgc ccgactgggc
     1621 tggcgatgag cgaaatgtag tgcttacgtt gtcccgcatt tggtacagcg cagtaaccgg
     1681 caaaatcgcg ccgaaggatg tcgctgccga ctgggcaatg gagcgcctgc cggcccagta
     1741 tcagcccgtc atacttgaag ctagacaggc ttatcttgga caagaagaag atcgcttggc
     1801 ctcgcgcgca gatcagttgg aagaatttgt ccactacgtg aaaggcgaga tcaccaaggt
     1861 agtcggcaaa taaccctcga gccacccatg accaaaatcc cttaacgtga gttacgcgtc
     1921 gttccactga gcgtcagacc ccgtagaaaa gatcaaagga tcttcttgag atcctttttt
     1981 tctgcgcgta atctgctgct tgcaaacaaa aaaaccaccg ctaccagcgg tggtttgttt
     2041 gccggatcaa gagctaccaa ctctttttcc gaaggtaact ggcttcagca gagcgcagat
     2101 accaaatact gtccttctag tgtagccgta gttaggccac cacttcaaga actctgtagc
     2161 accgcctaca tacctcgctc tgctaatcct gttaccagtg gctgctgcca gtggcgataa
     2221 gtcgtgtctt accgggttgg actcaagacg atagttaccg gataaggcgc agcggtcggg
     2281 ctgaacgggg ggttcgtgca cacagcccag cttggagcga acgacctaca ccgaactgag
     2341 atacctacag cgtgagcatt gagaaagcgc cacgcttccc gaagggagaa aggcggacag
     2401 gtatccggta agcggcaggg tcggaacagg agagcgcacg agggagcttc cagggggaaa
     2461 cgcctggtat ctttatagtc ctgtcgggtt tcgccacctc tgacttgagc gtcgattttt
     2521 gtgatgctcg tcaggggggc ggagcctatg gaaaaacgcc agcaacgcgg cctttttacg
     2581 gttcctggcc ttttgctggc cttttgctca catgttcttt cctgcgttat cccctgattc
     2641 tgtggataac cgtattaccg cctttgagtg agctgatacc gctcgccgca gccgaacgac
     2701 cgagcgcagc gagtcagtga gcgaggaagc ggaagagcgc ccaatacgca aaccgcctct
     2761 ccccgcgcgt tggccgattc attaatgcag ctggcacgac aggtttcccg actggaaagc
     2821 gggcagtgag cgcaacgcaa ttaatacgcg taccgctcgc caggaagagt ttgtagaaac
     2881 gcaaaaaggc catccgtcag gatggccttc tgcttagttt gatgcctggc agtttatggc
     2941 gggcgtcctg cccgccaccc tccgggccgt tgcttcacaa cgttcaaatc cgctcccggc
     3001 ggatttgtcc tactcaggag agcgttcacc gacaaacaac agataaaacg aaaggcccag
     3061 tcttccgact gagcctttcg ttttatttga tgcctggcag ttccctactc tcgcgttaac
     3121 gcttgcatgg atgttttccc agtcacgacg ttgtaaaacg acggccagtc ttaagctcgg
     3181 gcccaaataa tgattttatt ttgactgata gtgacctgtt cgttgcaaca aattgatgag
     3241 caatgctttt ttataatgcc aactttgtat acaaaagttg ccccatggcg ttccctctag
     3301 ataacgcact gcagtgcagc gtgacccggt cgtgcccctc tctagagata atgagcattg
     3361 catgtctaag ttataaaaaa ttaccacata ttttttttgt cacacttgtt tgaagtgcag
     3421 tttatctatc tttatacata tatttaaact ttactctacg aataatataa tctatagtac
     3481 tacaataata tcagtgtttt agagaatcat ataaatgaac agttagacat ggtctaaagg
     3541 acaattgagt attttgacaa caggactcta cagttttatc tttttagtgt gcatgtgttc
     3601 tccttttttt ttgcaaatag cttcacctat ataatacttc atccatttta ttagtacatc
     3661 catttagggt ttagggttaa tggtttttat agactaattt ttttagtaca tctattttat
     3721 tctattttag cctctaaatt aagaaaacta aaactctatt ttagtttttt tatttaataa
     3781 tttagatata aaatagaata aaataaagtg actaaaaatt aaacaaatac cctttaagaa
     3841 attaaaaaaa ctaaggaaac atttttcttg tttcgagtag ataatgccag cctgttaaac
     3901 gccgtcgacg agtctaacgg acaccaacca gcgaaccagc agcgtcgcgt cgggccaagc
     3961 gaagcagacg gcacggcatc tctgtcgctg cctctggacc cctctcgaga gttccgctcc
     4021 accgttggac ttgctccgct gtcggcatcc agaaattgcg tggcggagcg gcagacgtga
     4081 gccggcacgg caggcggcct cctcctcctc tcacggcacc ggcagctacg ggggattcct
     4141 ttcccaccgc tccttcgctt tcccttcctc gcccgccgta ataaatagac accccctcca
     4201 caccctcttt ccccaacctc gtgttgttcg gagcgcacac acacacaacc agatctcccc
     4261 caaatccacc cgtcggcacc tccgcttcaa ggtacgccgc tcgtcctccc cccccccccc
     4321 ctctctacct tctctagatc ggcgttccgg tccatggtta gggcccggta gttctacttc
     4381 tgttcatgtt tgtgttagat ccgtgtttgt gttagatccg tgctgctagc gttcgtacac
     4441 ggatgcgacc tgtacgtcag acacgttctg attgctaact tgccagtgtt tctctttggg
     4501 gaatcctggg atggctctag ccgttccgca gacgggatcg atttcatgat tttttttgtt
     4561 tcgttgcata gggtttggtt tgcccttttc ctttatttca atatatgccg tgcacttgtt
     4621 tgtcgggtca tcttttcatg cttttttttg tcttggttgt gatgatgtgg tctggttggg
     4681 cggtcgttct agatcggagt agaattaatt ctgtttcaaa ctacctggtg gatttattaa
     4741 ttttggatct gtatgtgtgt gccatacata ttcatagtta cgaattgaag atgatggatg
     4801 gaaatatcga tctaggatag gtatacatgt tgatgcgggt tttactgatg catatacaga
     4861 gatgcttttt gttcgcttgg ttgtgatgat gtggtgtggt tgggcggtcg ttcattcgtt
     4921 ctagatcgga gtagaatact gtttcaaact acctggtgta tttattaatt ttggaactgt
     4981 atgtgtgtgt catacatctt catagttacg agtttaagat ggatggaaat atcgatctag
     5041 gataggtata catgttgatg tgggttttac tgatgcatat acatgatggc atatgcagca
     5101 tctattcata tgctctaacc ttgagtacct atctattata ataaacaagt atgttttata
     5161 attattttga tcttgatata cttggatgat ggcatatgca gcagctatat gtggattttt
     5221 ttagccctgc cttcatacgc tatttatttg cttggtactg tttcttttgt cgatgctcac
     5281 cctgttgttt ggtgttactt ctgcagggat c
//
