LOCUS       sequence-336013-        5420 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Gateway entry clone and Golden Gate recipient for pYPQ131-tRNA2.0 to
            pYPQ135-tRNA2.0; assembly of 5 gRNAs.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 5420)
  AUTHORS   Pan C, Wu X, Markel K, Malzahn AA, Kundagrami N, Sretenovic S, Zhang
            Y, Cheng Y, Shih PM, Qi Y
  TITLE     CRISPR-Act3.0 for highly efficient multiplexed gene activation in 
            plants.
  JOURNAL   Nat Plants. 2021 Jul;7(7):942-953. doi: 10.1038/s41477-021-00953-7. 
            Epub 2021 Jun 24.
  PUBMED    34168320
REFERENCE   2  (bases 1 to 5420)
  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/s41477-021-00953-7"; journalName: "Nat Plants"; date: 
            "2021-07"; volume: "7"; issue: "7"; pages: "942-953"
COMMENT     Sequence Label: pYPQ145-ZmUbi-tRNA
FEATURES             Location/Qualifiers
     source          1..5420
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     promoter        23..2018
                     /label=Ubi promoter
                     /note="maize polyubiquitin gene promoter"
     CDS             complement(1945..2343)
                     /codon_start=1
                     /gene="lacZ fragment"
                     /product="LacZ-alpha fragment of beta-galactosidase"
                     /label=lacZ-alpha
                     /translation="MTMITPSAQLTLTKGNKSWRTSRGGPVPNSPYSESYYARSLAVVL
                     QRRDWENPGVTQLNRLAAHPPFASWRNSEEARTDRPSQQLRSLNGEWDAPCSGAGLDSH
                     HLGIPAEVTPNNRVSIDKRNSTKQINSV"
     primer_bind     2208..2224
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     promoter        2234..2252
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     primer_bind     complement(2300..2320)
                     /label=T3
                     /note="T3 promoter, forward primer"
     promoter        complement(2300..2318)
                     /label=T3 promoter
                     /note="promoter for bacteriophage T3 RNA polymerase"
     primer_bind     complement(2339..2355)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(2352..2374)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    2363..2379
                     /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(2387..2393,2394..2411,2412..2417))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    2432..2453
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     protein_bind    complement(2595..2694)
                     /gene="mutant version of attL"
                     /label=attL2
                     /bound_moiety="LR Clonase(TM)"
                     /note="recombination site for the Gateway(R) LR reaction"
     promoter        complement(2712..2730)
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     promoter        complement(2747..2765)
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     primer_bind     complement(2770..2786)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     CDS             3217..4008
                     /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      4101..4689
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     4590..4609
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     4843..4860
                     /label=L4440
                     /note="L4440 vector, forward primer"
     terminator      complement(5019..5046)
                     /label=rrnB T2 terminator
                     /note="transcription terminator T2 from the E. coli rrnB 
                     gene"
     terminator      complement(5138..5224)
                     /gene="Escherichia coli rrnB"
                     /label=rrnB T1 terminator
                     /note="transcription terminator T1 from the E. coli rrnB 
                     gene"
     primer_bind     5288..5304
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     protein_bind    5319..5414
                     /gene="mutant version of attL"
                     /label=attL5
                     /bound_moiety="LR Clonase(TM)"
                     /note="recombination site for the Gateway(R) LR reaction"
ORIGIN
        1 ggcgttccct ctagataacg cactgcagtg cagcgtgacc cggtcgtgcc cctctctaga
       61 gataatgagc attgcatgtc taagttataa aaaattacca catatttttt ttgtcacact
      121 tgtttgaagt gcagtttatc tatctttata catatattta aactttactc tacgaataat
      181 ataatctata gtactacaat aatatcagtg ttttagagaa tcatataaat gaacagttag
      241 acatggtcta aaggacaatt gagtattttg acaacaggac tctacagttt tatcttttta
      301 gtgtgcatgt gttctccttt ttttttgcaa atagcttcac ctatataata cttcatccat
      361 tttattagta catccattta gggtttaggg ttaatggttt ttatagacta atttttttag
      421 tacatctatt ttattctatt ttagcctcta aattaagaaa actaaaactc tattttagtt
      481 tttttattta ataatttaga tataaaatag aataaaataa agtgactaaa aattaaacaa
      541 atacccttta agaaattaaa aaaactaagg aaacattttt cttgtttcga gtagataatg
      601 ccagcctgtt aaacgccgtc gacgagtcta acggacacca accagcgaac cagcagcgtc
      661 gcgtcgggcc aagcgaagca gacggcacgg catctctgtc gctgcctctg gacccctctc
      721 gagagttccg ctccaccgtt ggacttgctc cgctgtcggc atccagaaat tgcgtggcgg
      781 agcggcagac gtgagccggc acggcaggcg gcctcctcct cctctcacgg caccggcagc
      841 tacgggggat tcctttccca ccgctccttc gctttccctt cctcgcccgc cgtaataaat
      901 agacaccccc tccacaccct ctttccccaa cctcgtgttg ttcggagcgc acacacacac
      961 aaccagatct cccccaaatc cacccgtcgg cacctccgct tcaaggtacg ccgctcgtcc
     1021 tccccccccc ccctctctac cttctctaga tcggcgttcc ggtccatggt tagggcccgg
     1081 tagttctact tctgttcatg tttgtgttag atccgtgttt gtgttagatc cgtgctgcta
     1141 gcgttcgtac acggatgcga cctgtacgtc agacacgttc tgattgctaa cttgccagtg
     1201 tttctctttg gggaatcctg ggatggctct agccgttccg cagacgggat cgatttcatg
     1261 attttttttg tttcgttgca tagggtttgg tttgcccttt tcctttattt caatatatgc
     1321 cgtgcacttg tttgtcgggt catcttttca tgcttttttt tgtcttggtt gtgatgatgt
     1381 ggtctggttg ggcggtcgtt ctagatcgga gtagaattaa ttctgtttca aactacctgg
     1441 tggatttatt aattttggat ctgtatgtgt gtgccataca tattcatagt tacgaattga
     1501 agatgatgga tggaaatatc gatctaggat aggtatacat gttgatgcgg gttttactga
     1561 tgcatataca gagatgcttt ttgttcgctt ggttgtgatg atgtggtgtg gttgggcggt
     1621 cgttcattcg ttctagatcg gagtagaata ctgtttcaaa ctacctggtg tatttattaa
     1681 ttttggaact gtatgtgtgt gtcatacatc ttcatagtta cgagtttaag atggatggaa
     1741 atatcgatct aggataggta tacatgttga tgtgggtttt actgatgcat atacatgatg
     1801 gcatatgcag catctattca tatgctctaa ccttgagtac ctatctatta taataaacaa
     1861 gtatgtttta taattatttt gatcttgata tacttggatg atggcatatg cagcagctat
     1921 atgtggattt ttttagccct gccttcatac gctatttatt tgcttggtac tgtttctttt
     1981 gtcgatgctc accctgttgt ttggtgttac ttctgcaggg atccccaagt ggtggctatc
     2041 gagaccggcg ccgctacagg gcgcgtccca ttcgccattc aggctgcgca actgttggga
     2101 agggcgatcg gtgcgggcct cttcgctatt acgccagctg gcgaaagggg gatgtgctgc
     2161 aaggcgatta agttgggtaa cgccagggtt ttcccagtca cgacgttgta aaacgacggc
     2221 cagtgagcgc gcgtaatacg actcactata gggcgaattg ggtaccgggc cccccctcga
     2281 ggtcctccag cttttgttcc ctttagtgag ggttaattgc gcgcttggcg taatcatggt
     2341 catagctgtt tcctgtgtga aattgttatc cgctcacaat tccacacaac atacgagccg
     2401 gaagcataaa gtgtaaagcc tggggtgcct aatgagtgag ctaactcaca ttaattgcgt
     2461 tgcgctcact gcccgctttc caccggtggt ctcttgcaaa caaagcacca gtggtctagt
     2521 ggtagaatag taccctgcca cggtacagac ccgggttcga ttcccggctg gtgcaactag
     2581 taagggcgaa ttcgacccag ctttcttgta caaagttggc attataaaaa ataattgctc
     2641 atcaatttgt tgcaacgaac aggtcactat cagtcaaaat aaaatcatta tttgccatcc
     2701 agctgatatc ccctatagtg agtcgtatta catggtcata gcatccccta tagtgagtcg
     2761 tattacatgg tcatagctgt ttcctggcag ctctggcccg tgtctcaaaa tctctgatgt
     2821 tacattgcac aagataaaaa tatatcatca tgcctcctct ggaccagcca ggacagaaat
     2881 gcctcgactt cgctgctacc caaggttgcc gggtgacgca caccgtggaa acggatgaag
     2941 gcacgaaccc agtggacata agcctgttcg gttcgtaagc tgtaatgcaa gtagcgtatg
     3001 cgctcacgca actggtccag aaccttgacc gaacgcagcg gtggtaacgg cgcagtggcg
     3061 gttttcatgg cttgttatga ctgttttttt ggggtacagt ctatgcctcg ggcatccaag
     3121 cagcaagcgc gttacgccgt gggtcgatgt ttgatgttat ggagcagcaa cgatgttacg
     3181 cagcagggca gtcgccctaa aacaaagtta aacattatga gggaagcggt gatcgccgaa
     3241 gtatcgactc aactatcaga ggtagttggc gtcatcgagc gccatctcga accgacgttg
     3301 ctggccgtac atttgtacgg ctccgcagtg gatggcggcc tgaagccaca cagtgatatt
     3361 gatttgctgg ttacggtgac cgtaaggctt gatgaaacaa cgcggcgagc tttgatcaac
     3421 gaccttttgg aaacttcggc ttcccctgga gagagcgaga ttctccgcgc tgtagaagtc
     3481 accattgttg tgcacgacga catcattccg tggcgttatc cagctaagcg cgaactgcaa
     3541 tttggagaat ggcagcgcaa tgacattctt gcaggtatct tcgagccagc cacgatcgac
     3601 attgatctgg ctatcttgct gacaaaagca agagaacata gcgttgcctt ggtaggtcca
     3661 gcggcggagg aactctttga tccggttcct gaacaggatc tatttgaggc gctaaatgaa
     3721 accttaacgc tatggaactc gccgcccgac tgggctggcg atgagcgaaa tgtagtgctt
     3781 acgttgtccc gcatttggta cagcgcagta accggcaaaa tcgcgccgaa ggatgtcgct
     3841 gccgactggg caatggagcg cctgccggcc cagtatcagc ccgtcatact tgaagctaga
     3901 caggcttatc ttggacaaga agaagatcgc ttggcctcgc gcgcagatca gttggaagaa
     3961 tttgtccact acgtgaaagg cgagatcacc aaggtagtcg gcaaataacc ctcgagccac
     4021 ccatgaccaa aatcccttaa cgtgagttac gcgtcgttcc actgagcgtc agaccccgta
     4081 gaaaagatca aaggatcttc ttgagatcct ttttttctgc gcgtaatctg ctgcttgcaa
     4141 acaaaaaaac caccgctacc agcggtggtt tgtttgccgg atcaagagct accaactctt
     4201 tttccgaagg taactggctt cagcagagcg cagataccaa atactgttct tctagtgtag
     4261 ccgtagttag gccaccactt caagaactct gtagcaccgc ctacatacct cgctctgcta
     4321 atcctgttac cagtggctgc tgccagtggc gataagtcgt gtcttaccgg gttggactca
     4381 agacgatagt taccggataa ggcgcagcgg tcgggctgaa cggggggttc gtgcacacag
     4441 cccagcttgg agcgaacgac ctacaccgaa ctgagatacc tacagcgtga gctatgagaa
     4501 agcgccacgc ttcccgaagg gagaaaggcg gacaggtatc cggtaagcgg cagggtcgga
     4561 acaggagagc gcacgaggga gcttccaggg ggaaacgcct ggtatcttta tagtcctgtc
     4621 gggtttcgcc acctctgact tgagcgtcga tttttgtgat gctcgtcagg ggggcggagc
     4681 ctatggaaaa acgccagcaa cgcggccttt ttacggttcc tggccttttg ctggcctttt
     4741 gctcacatgt tctttcctgc gttatcccct gattctgtgg ataaccgtat taccgccttt
     4801 gagtgagctg ataccgctcg ccgcagccga acgaccgagc gcagcgagtc agtgagcgag
     4861 gaagcggaag agcgcccaat acgcaaaccg cctctccccg cgcgttggcc gattcattaa
     4921 tgcagctggc acgacaggtt tcccgactgg aaagcgggca gtgagcgcaa cgcaattaat
     4981 acgcgtaccg cgagccagga agagtttgta gaaacgcaaa aaggccatcc gtcaggatgg
     5041 ccttctgctt agtttgatgc ctggcagttt atggcgggcg tcctgcccgc caccctccgg
     5101 gccgttgctt cacaacgttc aaatccgctc ccggcggatt tgtcctactc aggagagcgt
     5161 tcaccgacaa acaacagata aaacgaaagg cccagtcttc cgactgagcc tttcgtttta
     5221 tttgatgcct ggcagttccc tactctcgcg ttaacgcttg catggatgtt ttcccagtca
     5281 cgacgttgta aaacgacggc cagtcttaag ctcgggccca ataatgattt tattttgact
     5341 gatagtgacc tgttcgttgc aacaaattga tgagcaatgc ttttttataa tgccaacttt
     5401 gtatacaaaa gttgccccat
//
