LOCUS       sequence-330909-        5419 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 5419)
  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 5419)
  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..5419
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     CDS             complement(917..1315)
                     /codon_start=1
                     /gene="lacZ fragment"
                     /product="LacZ-alpha fragment of beta-galactosidase"
                     /label=lacZ-alpha
                     /translation="MTMITPSAQLTLTKGNKSWRTSRGGPVPNSPYSESYYARSLAVVL
                     QRRDWENPGVTQLNRLAAHPPFASWRNSEEARTDRPSQQLRSLNGEWDAPCSGAGLDSH
                     HLGIPAEVTPNNRVSIDKRNSTKQINSV"
     primer_bind     1180..1196
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     promoter        1206..1224
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     primer_bind     complement(1272..1292)
                     /label=T3
                     /note="T3 promoter, forward primer"
     promoter        complement(1272..1290)
                     /label=T3 promoter
                     /note="promoter for bacteriophage T3 RNA polymerase"
     primer_bind     complement(1311..1327)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(1324..1346)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    1335..1351
                     /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(1359..1365,1366..1383,1384..1389))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    1404..1425
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     protein_bind    complement(1567..1666)
                     /gene="mutant version of attL"
                     /label=attL2
                     /bound_moiety="LR Clonase(TM)"
                     /note="recombination site for the Gateway(R) LR reaction"
     promoter        complement(1684..1702)
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     promoter        complement(1719..1737)
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     primer_bind     complement(1742..1758)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     CDS             2189..2980
                     /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      3073..3661
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     3562..3581
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     3815..3832
                     /label=L4440
                     /note="L4440 vector, forward primer"
     terminator      complement(3991..4018)
                     /label=rrnB T2 terminator
                     /note="transcription terminator T2 from the E. coli rrnB 
                     gene"
     terminator      complement(4110..4196)
                     /gene="Escherichia coli rrnB"
                     /label=rrnB T1 terminator
                     /note="transcription terminator T1 from the E. coli rrnB 
                     gene"
     primer_bind     4260..4276
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     protein_bind    4291..4386
                     /gene="mutant version of attL"
                     /label=attL5
                     /bound_moiety="LR Clonase(TM)"
                     /note="recombination site for the Gateway(R) LR reaction"
     promoter        join(4415..5419,1..990)
                     /label=Ubi promoter
                     /note="maize polyubiquitin gene promoter"
ORIGIN
        1 ccccctctct accttctcta gatcggcgtt ccggtccatg gttagggccc ggtagttcta
       61 cttctgttca tgtttgtgtt agatccgtgt ttgtgttaga tccgtgctgc tagcgttcgt
      121 acacggatgc gacctgtacg tcagacacgt tctgattgct aacttgccag tgtttctctt
      181 tggggaatcc tgggatggct ctagccgttc cgcagacggg atcgatttca tgattttttt
      241 tgtttcgttg catagggttt ggtttgccct tttcctttat ttcaatatat gccgtgcact
      301 tgtttgtcgg gtcatctttt catgcttttt tttgtcttgg ttgtgatgat gtggtctggt
      361 tgggcggtcg ttctagatcg gagtagaatt aattctgttt caaactacct ggtggattta
      421 ttaattttgg atctgtatgt gtgtgccata catattcata gttacgaatt gaagatgatg
      481 gatggaaata tcgatctagg ataggtatac atgttgatgc gggttttact gatgcatata
      541 cagagatgct ttttgttcgc ttggttgtga tgatgtggtg tggttgggcg gtcgttcatt
      601 cgttctagat cggagtagaa tactgtttca aactacctgg tgtatttatt aattttggaa
      661 ctgtatgtgt gtgtcataca tcttcatagt tacgagttta agatggatgg aaatatcgat
      721 ctaggatagg tatacatgtt gatgtgggtt ttactgatgc atatacatga tggcatatgc
      781 agcatctatt catatgctct aaccttgagt acctatctat tataataaac aagtatgttt
      841 tataattatt ttgatcttga tatacttgga tgatggcata tgcagcagct atatgtggat
      901 ttttttagcc ctgccttcat acgctattta tttgcttggt actgtttctt ttgtcgatgc
      961 tcaccctgtt gtttggtgtt acttctgcag ggatccccaa gtggtggcta tcgagaccgg
     1021 cgccgctaca gggcgcgtcc cattcgccat tcaggctgcg caactgttgg gaagggcgat
     1081 cggtgcgggc ctcttcgcta ttacgccagc tggcgaaagg gggatgtgct gcaaggcgat
     1141 taagttgggt aacgccaggg ttttcccagt cacgacgttg taaaacgacg gccagtgagc
     1201 gcgcgtaata cgactcacta tagggcgaat tgggtaccgg gccccccctc gaggtcctcc
     1261 agcttttgtt ccctttagtg agggttaatt gcgcgcttgg cgtaatcatg gtcatagctg
     1321 tttcctgtgt gaaattgtta tccgctcaca attccacaca acatacgagc cggaagcata
     1381 aagtgtaaag cctggggtgc ctaatgagtg agctaactca cattaattgc gttgcgctca
     1441 ctgcccgctt tccaccggtg gtctctccag aacaaagcac cagtggtcta gtggtagaat
     1501 agtaccctgc cacggtacag acccgggttc gattcccggc tggtgcaact agtaagggca
     1561 aattcgaccc agctttcttg tacaaagttg gcattataaa aaataattgc tcatcaattt
     1621 gttgcaacga acaggtcact atcagtcaaa ataaaatcat tatttgccat ccagctgata
     1681 tcccctatag tgagtcgtat tacatggtca tagcatcccc tatagtgagt cgtattacat
     1741 ggtcatagct gtttcctggc agctctggcc cgtgtctcaa aatctctgat gttacattgc
     1801 acaagataaa aatatatcat catgcctcct ctggaccagc caggacagaa atgcctcgac
     1861 ttcgctgcta cccaaggttg ccgggtgacg cacaccgtgg aaacggatga aggcacgaac
     1921 ccagtggaca taagcctgtt cggttcgtaa gctgtaatgc aagtagcgta tgcgctcacg
     1981 caactggtcc agaaccttga ccgaacgcag cggtggtaac ggcgcagtgg cggttttcat
     2041 ggcttgttat gactgttttt ttggggtaca gtctatgcct cgggcatcca agcagcaagc
     2101 gcgttacgcc gtgggtcgat gtttgatgtt atggagcagc aacgatgtta cgcagcaggg
     2161 cagtcgccct aaaacaaagt taaacattat gagggaagcg gtgatcgccg aagtatcgac
     2221 tcaactatca gaggtagttg gcgtcatcga gcgccatctc gaaccgacgt tgctggccgt
     2281 acatttgtac ggctccgcag tggatggcgg cctgaagcca cacagtgata ttgatttgct
     2341 ggttacggtg accgtaaggc ttgatgaaac aacgcggcga gctttgatca acgacctttt
     2401 ggaaacttcg gcttcccctg gagagagcga gattctccgc gctgtagaag tcaccattgt
     2461 tgtgcacgac gacatcattc cgtggcgtta tccagctaag cgcgaactgc aatttggaga
     2521 atggcagcgc aatgacattc ttgcaggtat cttcgagcca gccacgatcg acattgatct
     2581 ggctatcttg ctgacaaaag caagagaaca tagcgttgcc ttggtaggtc cagcggcgga
     2641 ggaactcttt gatccggttc ctgaacagga tctatttgag gcgctaaatg aaaccttaac
     2701 gctatggaac tcgccgcccg actgggctgg cgatgagcga aatgtagtgc ttacgttgtc
     2761 ccgcatttgg tacagcgcag taaccggcaa aatcgcgccg aaggatgtcg ctgccgactg
     2821 ggcaatggag cgcctgccgg cccagtatca gcccgtcata cttgaagcta gacaggctta
     2881 tcttggacaa gaagaagatc gcttggcctc gcgcgcagat cagttggaag aatttgtcca
     2941 ctacgtgaaa ggcgagatca ccaaggtagt cggcaaataa ccctcgagcc acccatgacc
     3001 aaaatccctt aacgtgagtt acgcgtcgtt ccactgagcg tcagaccccg tagaaaagat
     3061 caaaggatct tcttgagatc ctttttttct gcgcgtaatc tgctgcttgc aaacaaaaaa
     3121 accaccgcta ccagcggtgg tttgtttgcc ggatcaagag ctaccaactc tttttccgaa
     3181 ggtaactggc ttcagcagag cgcagatacc aaatactgtt cttctagtgt agccgtagtt
     3241 aggccaccac ttcaagaact ctgtagcacc gcctacatac ctcgctctgc taatcctgtt
     3301 accagtggct gctgccagtg gcgataagtc gtgtcttacc gggttggact caagacgata
     3361 gttaccggat aaggcgcagc ggtcgggctg aacggggggt tcgtgcacac agcccagctt
     3421 ggagcgaacg acctacaccg aactgagata cctacagcgt gagctatgag aaagcgccac
     3481 gcttcccgaa gggagaaagg cggacaggta tccggtaagc ggcagggtcg gaacaggaga
     3541 gcgcacgagg gagcttccag ggggaaacgc ctggtatctt tatagtcctg tcgggtttcg
     3601 ccacctctga cttgagcgtc gatttttgtg atgctcgtca ggggggcgga gcctatggaa
     3661 aaacgccagc aacgcggcct ttttacggtt cctggccttt tgctggcctt ttgctcacat
     3721 gttctttcct gcgttatccc ctgattctgt ggataaccgt attaccgcct ttgagtgagc
     3781 tgataccgct cgccgcagcc gaacgaccga gcgcagcgag tcagtgagcg aggaagcgga
     3841 agagcgccca atacgcaaac cgcctctccc cgcgcgttgg ccgattcatt aatgcagctg
     3901 gcacgacagg tttcccgact ggaaagcggg cagtgagcgc aacgcaatta atacgcgtac
     3961 cgcgagccag gaagagtttg tagaaacgca aaaaggccat ccgtcaggat ggccttctgc
     4021 ttagtttgat gcctggcagt ttatggcggg cgtcctgccc gccaccctcc gggccgttgc
     4081 ttcacaacgt tcaaatccgc tcccggcgga tttgtcctac tcaggagagc gttcaccgac
     4141 aaacaacaga taaaacgaaa ggcccagtct tccgactgag cctttcgttt tatttgatgc
     4201 ctggcagttc cctactctcg cgttaacgct tgcatggatg ttttcccagt cacgacgttg
     4261 taaaacgacg gccagtctta agctcgggcc caataatgat tttattttga ctgatagtga
     4321 cctgttcgtt gcaacaaatt gatgagcaat gcttttttat aatgccaact ttgtatacaa
     4381 aagttgcccc atggcgttcc ctctagataa cgcactgcag tgcagcgtga cccggtcgtg
     4441 cccctctcta gagataatga gcattgcatg tctaagttat aaaaaattac cacatatttt
     4501 ttttgtcaca cttgtttgaa gtgcagttta tctatcttta tacatatatt taaactttac
     4561 tctacgaata atataatcta tagtactaca ataatatcag tgttttagag aatcatataa
     4621 atgaacagtt agacatggtc taaaggacaa ttgagtattt tgacaacagg actctacagt
     4681 tttatctttt tagtgtgcat gtgttctcct ttttttttgc aaatagcttc acctatataa
     4741 tacttcatcc attttattag tacatccatt tagggtttag ggttaatggt ttttatagac
     4801 taattttttt agtacatcta ttttattcta ttttagcctc taaattaaga aaactaaaac
     4861 tctattttag tttttttatt taataattta gatataaaat agaataaaat aaagtgacta
     4921 aaaattaaac aaataccctt taagaaatta aaaaaactaa ggaaacattt ttcttgtttc
     4981 gagtagataa tgccagcctg ttaaacgccg tcgacgagtc taacggacac caaccagcga
     5041 accagcagcg tcgcgtcggg ccaagcgaag cagacggcac ggcatctctg tcgctgcctc
     5101 tggacccctc tcgagagttc cgctccaccg ttggacttgc tccgctgtcg gcatccagaa
     5161 attgcgtggc ggagcggcag acgtgagccg gcacggcagg cggcctcctc ctcctctcac
     5221 ggcaccggca gctacggggg attcctttcc caccgctcct tcgctttccc ttcctcgccc
     5281 gccgtaataa atagacaccc cctccacacc ctctttcccc aacctcgtgt tgttcggagc
     5341 gcacacacac acaaccagat ctcccccaaa tccacccgtc ggcacctccg cttcaaggta
     5401 cgccgctcgt cctcccccc
//
