LOCUS       sequence-271975-        5310 bp    DNA     circular SYN 14-APR-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 5310)
  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 5310)
  AUTHORS   .
  TITLE     Direct Submission
  JOURNAL   Exported Apr 14, 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..5310
                     /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(277..297)
                     /label=T3
                     /note="T3 promoter, forward primer"
     promoter        complement(277..295)
                     /label=T3 promoter
                     /note="promoter for bacteriophage T3 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    3184..3279
                     /gene="mutant version of attL"
                     /label=attL5
                     /bound_moiety="LR Clonase(TM)"
                     /note="recombination site for the Gateway(R) LR reaction"
     promoter        3308..5305
                     /label=Ubi promoter
                     /note="maize polyubiquitin gene promoter"
     CDS             complement(join(5232..5310,1..320))
                     /codon_start=1
                     /gene="lacZ fragment"
                     /product="LacZ-alpha fragment of beta-galactosidase"
                     /label=lacZ-alpha
                     /translation="MTMITPSAQLTLTKGNKSWRTSRGGPVPNSPYSESYYARSLAVVL
                     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 tagtgagggt taattgcgcg
      301 cttggcgtaa tcatggtcat agctgtttcc tgtgtgaaat tgttatccgc tcacaattcc
      361 acacaacata cgagccggaa gcataaagtg taaagcctgg ggtgcctaat gagtgagcta
      421 actcacatta attgcgttgc gctcactgcc cgctttccac cggtggtctc tccagactag
      481 taagggcgaa 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 ctacccaagg 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 tatgagggaa 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 gttcttctag 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 cgtgagctat 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 taccgcgagc 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 gcccaataat gattttattt tgactgatag tgacctgttc gttgcaacaa attgatgagc
     3241 aatgcttttt tataatgcca actttgtata caaaagttgc cccatggcgt tccctctaga
     3301 taacgcactg cagtgcagcg tgacccggtc gtgcccctct ctagagataa tgagcattgc
     3361 atgtctaagt tataaaaaat taccacatat tttttttgtc acacttgttt gaagtgcagt
     3421 ttatctatct ttatacatat atttaaactt tactctacga ataatataat ctatagtact
     3481 acaataatat cagtgtttta gagaatcata taaatgaaca gttagacatg gtctaaagga
     3541 caattgagta ttttgacaac aggactctac agttttatct ttttagtgtg catgtgttct
     3601 cctttttttt tgcaaatagc ttcacctata taatacttca tccattttat tagtacatcc
     3661 atttagggtt tagggttaat ggtttttata gactaatttt tttagtacat ctattttatt
     3721 ctattttagc ctctaaatta agaaaactaa aactctattt tagttttttt atttaataat
     3781 ttagatataa aatagaataa aataaagtga ctaaaaatta aacaaatacc ctttaagaaa
     3841 ttaaaaaaac taaggaaaca tttttcttgt ttcgagtaga taatgccagc ctgttaaacg
     3901 ccgtcgacga gtctaacgga caccaaccag cgaaccagca gcgtcgcgtc gggccaagcg
     3961 aagcagacgg cacggcatct ctgtcgctgc ctctggaccc ctctcgagag ttccgctcca
     4021 ccgttggact tgctccgctg tcggcatcca gaaattgcgt ggcggagcgg cagacgtgag
     4081 ccggcacggc aggcggcctc ctcctcctct cacggcaccg gcagctacgg gggattcctt
     4141 tcccaccgct ccttcgcttt cccttcctcg cccgccgtaa taaatagaca ccccctccac
     4201 accctctttc cccaacctcg tgttgttcgg agcgcacaca cacacaacca gatctccccc
     4261 aaatccaccc gtcggcacct ccgcttcaag gtacgccgct cgtcctcccc cccccccccc
     4321 tctctacctt ctctagatcg gcgttccggt ccatggttag ggcccggtag ttctacttct
     4381 gttcatgttt gtgttagatc cgtgtttgtg ttagatccgt gctgctagcg ttcgtacacg
     4441 gatgcgacct gtacgtcaga cacgttctga ttgctaactt gccagtgttt ctctttgggg
     4501 aatcctggga tggctctagc cgttccgcag acgggatcga tttcatgatt ttttttgttt
     4561 cgttgcatag ggtttggttt gcccttttcc tttatttcaa tatatgccgt gcacttgttt
     4621 gtcgggtcat cttttcatgc ttttttttgt cttggttgtg atgatgtggt ctggttgggc
     4681 ggtcgttcta gatcggagta gaattaattc tgtttcaaac tacctggtgg atttattaat
     4741 tttggatctg tatgtgtgtg ccatacatat tcatagttac gaattgaaga tgatggatgg
     4801 aaatatcgat ctaggatagg tatacatgtt gatgcgggtt ttactgatgc atatacagag
     4861 atgctttttg ttcgcttggt tgtgatgatg tggtgtggtt gggcggtcgt tcattcgttc
     4921 tagatcggag tagaatactg tttcaaacta cctggtgtat ttattaattt tggaactgta
     4981 tgtgtgtgtc atacatcttc atagttacga gtttaagatg gatggaaata tcgatctagg
     5041 ataggtatac atgttgatgt gggttttact gatgcatata catgatggca tatgcagcat
     5101 ctattcatat gctctaacct tgagtaccta tctattataa taaacaagta tgttttataa
     5161 ttattttgat cttgatatac ttggatgatg gcatatgcag cagctatatg tggatttttt
     5221 tagccctgcc ttcatacgct atttatttgc ttggtactgt ttcttttgtc gatgctcacc
     5281 ctgttgtttg gtgttacttc tgcagggatc
//
