LOCUS       sequence-324662-        5388 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Gateway entry clone and Golden Gate recipient for pYPQ131-tRNA2.0 to
            pYPQ133-tRNA2.0; assembly of 3 gRNAs.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 5388)
  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 5388)
  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: pYPQ143-ZmUbi-tRNA
FEATURES             Location/Qualifiers
     source          1..5388
                     /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(572..671)
                     /gene="mutant version of attL"
                     /label=attL2
                     /bound_moiety="LR Clonase(TM)"
                     /note="recombination site for the Gateway(R) LR reaction"
     promoter        complement(689..707)
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     primer_bind     complement(712..728)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     CDS             1159..1950
                     /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      2043..2631
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     2532..2551
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     2785..2802
                     /label=L4440
                     /note="L4440 vector, forward primer"
     terminator      complement(2961..2988)
                     /label=rrnB T2 terminator
                     /note="transcription terminator T2 from the E. coli rrnB 
                     gene"
     terminator      complement(3080..3166)
                     /gene="Escherichia coli rrnB"
                     /label=rrnB T1 terminator
                     /note="transcription terminator T1 from the E. coli rrnB 
                     gene"
     primer_bind     3230..3246
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     protein_bind    3262..3357
                     /gene="mutant version of attL"
                     /label=attL5
                     /bound_moiety="LR Clonase(TM)"
                     /note="recombination site for the Gateway(R) LR reaction"
     promoter        3386..5383
                     /label=Ubi promoter
                     /note="maize polyubiquitin gene promoter"
     CDS             complement(join(5310..5388,1..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 tccagaacaa
      481 agcaccagtg gtctagtggt agaatagtac cctgccacgg tacagacccg ggttcgattc
      541 ccggctggtg caactagtaa gggcaaattc gacccagctt tcttgtacaa agttggcatt
      601 ataaaaaata attgctcatc aatttgttgc aacgaacagg tcactatcag tcaaaataaa
      661 atcattattt gccatccagc tgatatcccc tatagtgagt cgtattacat ggtcatagct
      721 gtttcctggc agctctggcc cgtgtctcaa aatctctgat gttacattgc acaagataaa
      781 aatatatcat catgcctcct ctggaccagc caggacagaa atgcctcgac ttcgctgctg
      841 cccaaggttg ccgggtgacg cacaccgtgg aaacggatga aggcacgaac ccagtggaca
      901 taagcctgtt cggttcgtaa gctgtaatgc aagtagcgta tgcgctcacg caactggtcc
      961 agaaccttga ccgaacgcag cggtggtaac ggcgcagtgg cggttttcat ggcttgttat
     1021 gactgttttt ttggggtaca gtctatgcct cgggcatcca agcagcaagc gcgttacgcc
     1081 gtgggtcgat gtttgatgtt atggagcagc aacgatgtta cgcagcaggg cagtcgccct
     1141 aaaacaaagt taaacatcat gagggaagcg gtgatcgccg aagtatcgac tcaactatca
     1201 gaggtagttg gcgtcatcga gcgccatctc gaaccgacgt tgctggccgt acatttgtac
     1261 ggctccgcag tggatggcgg cctgaagcca cacagtgata ttgatttgct ggttacggtg
     1321 accgtaaggc ttgatgaaac aacgcggcga gctttgatca acgacctttt ggaaacttcg
     1381 gcttcccctg gagagagcga gattctccgc gctgtagaag tcaccattgt tgtgcacgac
     1441 gacatcattc cgtggcgtta tccagctaag cgcgaactgc aatttggaga atggcagcgc
     1501 aatgacattc ttgcaggtat cttcgagcca gccacgatcg acattgatct ggctatcttg
     1561 ctgacaaaag caagagaaca tagcgttgcc ttggtaggtc cagcggcgga ggaactcttt
     1621 gatccggttc ctgaacagga tctatttgag gcgctaaatg aaaccttaac gctatggaac
     1681 tcgccgcccg actgggctgg cgatgagcga aatgtagtgc ttacgttgtc ccgcatttgg
     1741 tacagcgcag taaccggcaa aatcgcgccg aaggatgtcg ctgccgactg ggcaatggag
     1801 cgcctgccgg cccagtatca gcccgtcata cttgaagcta gacaggctta tcttggacaa
     1861 gaagaagatc gcttggcctc gcgcgcagat cagttggaag aatttgtcca ctacgtgaaa
     1921 ggcgagatca ccaaggtagt cggcaaataa ccctcgagcc acccatgacc aaaatccctt
     1981 aacgtgagtt acgcgtcgtt ccactgagcg tcagaccccg tagaaaagat caaaggatct
     2041 tcttgagatc ctttttttct gcgcgtaatc tgctgcttgc aaacaaaaaa accaccgcta
     2101 ccagcggtgg tttgtttgcc ggatcaagag ctaccaactc tttttccgaa ggtaactggc
     2161 ttcagcagag cgcagatacc aaatactgtc cttctagtgt agccgtagtt aggccaccac
     2221 ttcaagaact ctgtagcacc gcctacatac ctcgctctgc taatcctgtt accagtggct
     2281 gctgccagtg gcgataagtc gtgtcttacc gggttggact caagacgata gttaccggat
     2341 aaggcgcagc ggtcgggctg aacggggggt tcgtgcacac agcccagctt ggagcgaacg
     2401 acctacaccg aactgagata cctacagcgt gagcattgag aaagcgccac gcttcccgaa
     2461 gggagaaagg cggacaggta tccggtaagc ggcagggtcg gaacaggaga gcgcacgagg
     2521 gagcttccag ggggaaacgc ctggtatctt tatagtcctg tcgggtttcg ccacctctga
     2581 cttgagcgtc gatttttgtg atgctcgtca ggggggcgga gcctatggaa aaacgccagc
     2641 aacgcggcct ttttacggtt cctggccttt tgctggcctt ttgctcacat gttctttcct
     2701 gcgttatccc ctgattctgt ggataaccgt attaccgcct ttgagtgagc tgataccgct
     2761 cgccgcagcc gaacgaccga gcgcagcgag tcagtgagcg aggaagcgga agagcgccca
     2821 atacgcaaac cgcctctccc cgcgcgttgg ccgattcatt aatgcagctg gcacgacagg
     2881 tttcccgact ggaaagcggg cagtgagcgc aacgcaatta atacgcgtac cgctcgccag
     2941 gaagagtttg tagaaacgca aaaaggccat ccgtcaggat ggccttctgc ttagtttgat
     3001 gcctggcagt ttatggcggg cgtcctgccc gccaccctcc gggccgttgc ttcacaacgt
     3061 tcaaatccgc tcccggcgga tttgtcctac tcaggagagc gttcaccgac aaacaacaga
     3121 taaaacgaaa ggcccagtct tccgactgag cctttcgttt tatttgatgc ctggcagttc
     3181 cctactctcg cgttaacgct tgcatggatg ttttcccagt cacgacgttg taaaacgacg
     3241 gccagtctta agctcgggcc caaataatga ttttattttg actgatagtg acctgttcgt
     3301 tgcaacaaat tgatgagcaa tgctttttta taatgccaac tttgtataca aaagttgccc
     3361 catggcgttc cctctagata acgcactgca gtgcagcgtg acccggtcgt gcccctctct
     3421 agagataatg agcattgcat gtctaagtta taaaaaatta ccacatattt tttttgtcac
     3481 acttgtttga agtgcagttt atctatcttt atacatatat ttaaacttta ctctacgaat
     3541 aatataatct atagtactac aataatatca gtgttttaga gaatcatata aatgaacagt
     3601 tagacatggt ctaaaggaca attgagtatt ttgacaacag gactctacag ttttatcttt
     3661 ttagtgtgca tgtgttctcc tttttttttg caaatagctt cacctatata atacttcatc
     3721 cattttatta gtacatccat ttagggttta gggttaatgg tttttataga ctaatttttt
     3781 tagtacatct attttattct attttagcct ctaaattaag aaaactaaaa ctctatttta
     3841 gtttttttat ttaataattt agatataaaa tagaataaaa taaagtgact aaaaattaaa
     3901 caaataccct ttaagaaatt aaaaaaacta aggaaacatt tttcttgttt cgagtagata
     3961 atgccagcct gttaaacgcc gtcgacgagt ctaacggaca ccaaccagcg aaccagcagc
     4021 gtcgcgtcgg gccaagcgaa gcagacggca cggcatctct gtcgctgcct ctggacccct
     4081 ctcgagagtt ccgctccacc gttggacttg ctccgctgtc ggcatccaga aattgcgtgg
     4141 cggagcggca gacgtgagcc ggcacggcag gcggcctcct cctcctctca cggcaccggc
     4201 agctacgggg gattcctttc ccaccgctcc ttcgctttcc cttcctcgcc cgccgtaata
     4261 aatagacacc ccctccacac cctctttccc caacctcgtg ttgttcggag cgcacacaca
     4321 cacaaccaga tctcccccaa atccacccgt cggcacctcc gcttcaaggt acgccgctcg
     4381 tcctcccccc cccccccctc tctaccttct ctagatcggc gttccggtcc atggttaggg
     4441 cccggtagtt ctacttctgt tcatgtttgt gttagatccg tgtttgtgtt agatccgtgc
     4501 tgctagcgtt cgtacacgga tgcgacctgt acgtcagaca cgttctgatt gctaacttgc
     4561 cagtgtttct ctttggggaa tcctgggatg gctctagccg ttccgcagac gggatcgatt
     4621 tcatgatttt ttttgtttcg ttgcataggg tttggtttgc ccttttcctt tatttcaata
     4681 tatgccgtgc acttgtttgt cgggtcatct tttcatgctt ttttttgtct tggttgtgat
     4741 gatgtggtct ggttgggcgg tcgttctaga tcggagtaga attaattctg tttcaaacta
     4801 cctggtggat ttattaattt tggatctgta tgtgtgtgcc atacatattc atagttacga
     4861 attgaagatg atggatggaa atatcgatct aggataggta tacatgttga tgcgggtttt
     4921 actgatgcat atacagagat gctttttgtt cgcttggttg tgatgatgtg gtgtggttgg
     4981 gcggtcgttc attcgttcta gatcggagta gaatactgtt tcaaactacc tggtgtattt
     5041 attaattttg gaactgtatg tgtgtgtcat acatcttcat agttacgagt ttaagatgga
     5101 tggaaatatc gatctaggat aggtatacat gttgatgtgg gttttactga tgcatataca
     5161 tgatggcata tgcagcatct attcatatgc tctaaccttg agtacctatc tattataata
     5221 aacaagtatg ttttataatt attttgatct tgatatactt ggatgatggc atatgcagca
     5281 gctatatgtg gattttttta gccctgcctt catacgctat ttatttgctt ggtactgttt
     5341 cttttgtcga tgctcaccct gttgtttggt gttacttctg cagggatc
//
