LOCUS       sequence-331331-        5387 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Gateway entry clone and Golden Gate recipient for pYPQ131-tRNA2.0 
            and pYPQ132-tRNA2.0; assembly of 2 gRNAs.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 5387)
  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 5387)
  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: pYPQ142-ZmUbi-tRNA
FEATURES             Location/Qualifiers
     source          1..5387
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     promoter        13..2010
                     /label=Ubi promoter
                     /note="maize polyubiquitin gene promoter"
     CDS             complement(1937..2335)
                     /codon_start=1
                     /gene="lacZ fragment"
                     /product="LacZ-alpha fragment of beta-galactosidase"
                     /label=lacZ-alpha
                     /translation="MTMITPSAQLTLTKGNKSWRTSRGGPVPNSPYSESYYARSLAVVL
                     QRRDWENPGVTQLNRLAAHPPFASWRNSEEARTDRPSQQLRSLNGEWDAPCSGAGLDSH
                     HLGIPAEVTPNNRVSIDKRNSTKQINSV"
     primer_bind     2200..2216
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     promoter        2226..2244
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     primer_bind     complement(2292..2312)
                     /label=T3
                     /note="T3 promoter, forward primer"
     promoter        complement(2292..2310)
                     /label=T3 promoter
                     /note="promoter for bacteriophage T3 RNA polymerase"
     primer_bind     complement(2331..2347)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(2344..2366)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    2355..2371
                     /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(2379..2385,2386..2403,2404..2409))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    2424..2445
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     protein_bind    complement(2587..2686)
                     /gene="mutant version of attL"
                     /label=attL2
                     /bound_moiety="LR Clonase(TM)"
                     /note="recombination site for the Gateway(R) LR reaction"
     promoter        complement(2704..2722)
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     primer_bind     complement(2727..2743)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     CDS             3174..3965
                     /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      4058..4646
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     4547..4566
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     4800..4817
                     /label=L4440
                     /note="L4440 vector, forward primer"
     terminator      complement(4976..5003)
                     /label=rrnB T2 terminator
                     /note="transcription terminator T2 from the E. coli rrnB 
                     gene"
     terminator      complement(5095..5181)
                     /gene="Escherichia coli rrnB"
                     /label=rrnB T1 terminator
                     /note="transcription terminator T1 from the E. coli rrnB 
                     gene"
     primer_bind     5245..5261
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     protein_bind    5276..5371
                     /gene="mutant version of attL"
                     /label=attL5
                     /bound_moiety="LR Clonase(TM)"
                     /note="recombination site for the Gateway(R) LR reaction"
ORIGIN
        1 ctagataacg cactgcagtg cagcgtgacc cggtcgtgcc cctctctaga gataatgagc
       61 attgcatgtc taagttataa aaaattacca catatttttt ttgtcacact tgtttgaagt
      121 gcagtttatc tatctttata catatattta aactttactc tacgaataat ataatctata
      181 gtactacaat aatatcagtg ttttagagaa tcatataaat gaacagttag acatggtcta
      241 aaggacaatt gagtattttg acaacaggac tctacagttt tatcttttta gtgtgcatgt
      301 gttctccttt ttttttgcaa atagcttcac ctatataata cttcatccat tttattagta
      361 catccattta gggtttaggg ttaatggttt ttatagacta atttttttag tacatctatt
      421 ttattctatt ttagcctcta aattaagaaa actaaaactc tattttagtt tttttattta
      481 ataatttaga tataaaatag aataaaataa agtgactaaa aattaaacaa atacccttta
      541 agaaattaaa aaaactaagg aaacattttt cttgtttcga gtagataatg ccagcctgtt
      601 aaacgccgtc gacgagtcta acggacacca accagcgaac cagcagcgtc gcgtcgggcc
      661 aagcgaagca gacggcacgg catctctgtc gctgcctctg gacccctctc gagagttccg
      721 ctccaccgtt ggacttgctc cgctgtcggc atccagaaat tgcgtggcgg agcggcagac
      781 gtgagccggc acggcaggcg gcctcctcct cctctcacgg caccggcagc tacgggggat
      841 tcctttccca ccgctccttc gctttccctt cctcgcccgc cgtaataaat agacaccccc
      901 tccacaccct ctttccccaa cctcgtgttg ttcggagcgc acacacacac aaccagatct
      961 cccccaaatc cacccgtcgg cacctccgct tcaaggtacg ccgctcgtcc tccccccccc
     1021 ccccctctct accttctcta gatcggcgtt ccggtccatg gttagggccc ggtagttcta
     1081 cttctgttca tgtttgtgtt agatccgtgt ttgtgttaga tccgtgctgc tagcgttcgt
     1141 acacggatgc gacctgtacg tcagacacgt tctgattgct aacttgccag tgtttctctt
     1201 tggggaatcc tgggatggct ctagccgttc cgcagacggg atcgatttca tgattttttt
     1261 tgtttcgttg catagggttt ggtttgccct tttcctttat ttcaatatat gccgtgcact
     1321 tgtttgtcgg gtcatctttt catgcttttt tttgtcttgg ttgtgatgat gtggtctggt
     1381 tgggcggtcg ttctagatcg gagtagaatt aattctgttt caaactacct ggtggattta
     1441 ttaattttgg atctgtatgt gtgtgccata catattcata gttacgaatt gaagatgatg
     1501 gatggaaata tcgatctagg ataggtatac atgttgatgc gggttttact gatgcatata
     1561 cagagatgct ttttgttcgc ttggttgtga tgatgtggtg tggttgggcg gtcgttcatt
     1621 cgttctagat cggagtagaa tactgtttca aactacctgg tgtatttatt aattttggaa
     1681 ctgtatgtgt gtgtcataca tcttcatagt tacgagttta agatggatgg aaatatcgat
     1741 ctaggatagg tatacatgtt gatgtgggtt ttactgatgc atatacatga tggcatatgc
     1801 agcatctatt catatgctct aaccttgagt acctatctat tataataaac aagtatgttt
     1861 tataattatt ttgatcttga tatacttgga tgatggcata tgcagcagct atatgtggat
     1921 ttttttagcc ctgccttcat acgctattta tttgcttggt actgtttctt ttgtcgatgc
     1981 tcaccctgtt gtttggtgtt acttctgcag ggatccccaa gtggtggcta tcgagaccgg
     2041 cgccgctaca gggcgcgtcc cattcgccat tcaggctgcg caactgttgg gaagggcgat
     2101 cggtgcgggc ctcttcgcta ttacgccagc tggcgaaagg gggatgtgct gcaaggcgat
     2161 taagttgggt aacgccaggg ttttcccagt cacgacgttg taaaacgacg gccagtgagc
     2221 gcgcgtaata cgactcacta tagggcgaat tgggtaccgg gccccccctc gaggtcctcc
     2281 agcttttgtt ccctttagtg agggttaatt gcgcgcttgg cgtaatcatg gtcatagctg
     2341 tttcctgtgt gaaattgtta tccgctcaca attccacaca acatacgagc cggaagcata
     2401 aagtgtaaag cctggggtgc ctaatgagtg agctaactca cattaattgc gttgcgctca
     2461 ctgcccgctt tccaccggtg gtctctggac aacaaagcac cagtggtcta gtggtagaat
     2521 agtaccctgc cacggtacag acccgggttc gattcccggc tggtgcaact agtaagggcg
     2581 aattcgaccc agctttcttg tacaaagttg gcattataaa aaataattgc tcatcaattt
     2641 gttgcaacga acaggtcact atcagtcaaa ataaaatcat tatttgccat ccagctgata
     2701 tcccctatag tgagtcgtat tacatggtca tagctgtttc ctggcagctc tggcccgtgt
     2761 ctcaaaatct ctgatgttac attgcacaag ataaaaatat atcatcatgc ctcctctgga
     2821 ccagccagga cagaaatgcc tcgacttcgc tgctacccaa ggttgccggg tgacgcacac
     2881 cgtggaaacg gatgaaggca cgaacccagt ggacataagc ctgttcggtt cgtaagctgt
     2941 aatgcaagta gcgtatgcgc tcacgcaact ggtccagaac cttgaccgaa cgcagcggtg
     3001 gtaacggcgc agtggcggtt ttcatggctt gttatgactg tttttttggg gtacagtcta
     3061 tgcctcgggc atccaagcag caagcgcgtt acgccgtggg tcgatgtttg atgttatgga
     3121 gcagcaacga tgttacgcag cagggcagtc gccctaaaac aaagttaaac attatgaggg
     3181 aagcggtgat cgccgaagta tcgactcaac tatcagaggt agttggcgtc atcgagcgcc
     3241 atctcgaacc gacgttgctg gccgtacatt tgtacggctc cgcagtggat ggcggcctga
     3301 agccacacag tgatattgat ttgctggtta cggtgaccgt aaggcttgat gaaacaacgc
     3361 ggcgagcttt gatcaacgac cttttggaaa cttcggcttc ccctggagag agcgagattc
     3421 tccgcgctgt agaagtcacc attgttgtgc acgacgacat cattccgtgg cgttatccag
     3481 ctaagcgcga actgcaattt ggagaatggc agcgcaatga cattcttgca ggtatcttcg
     3541 agccagccac gatcgacatt gatctggcta tcttgctgac aaaagcaaga gaacatagcg
     3601 ttgccttggt aggtccagcg gcggaggaac tctttgatcc ggttcctgaa caggatctat
     3661 ttgaggcgct aaatgaaacc ttaacgctat ggaactcgcc gcccgactgg gctggcgatg
     3721 agcgaaatgt agtgcttacg ttgtcccgca tttggtacag cgcagtaacc ggcaaaatcg
     3781 cgccgaagga tgtcgctgcc gactgggcaa tggagcgcct gccggcccag tatcagcccg
     3841 tcatacttga agctagacag gcttatcttg gacaagaaga agatcgcttg gcctcgcgcg
     3901 cagatcagtt ggaagaattt gtccactacg tgaaaggcga gatcaccaag gtagtcggca
     3961 aataaccctc gagccaccca tgaccaaaat cccttaacgt gagttacgcg tcgttccact
     4021 gagcgtcaga ccccgtagaa aagatcaaag gatcttcttg agatcctttt tttctgcgcg
     4081 taatctgctg cttgcaaaca aaaaaaccac cgctaccagc ggtggtttgt ttgccggatc
     4141 aagagctacc aactcttttt ccgaaggtaa ctggcttcag cagagcgcag ataccaaata
     4201 ctgttcttct agtgtagccg tagttaggcc accacttcaa gaactctgta gcaccgccta
     4261 catacctcgc tctgctaatc ctgttaccag tggctgctgc cagtggcgat aagtcgtgtc
     4321 ttaccgggtt ggactcaaga cgatagttac cggataaggc gcagcggtcg ggctgaacgg
     4381 ggggttcgtg cacacagccc agcttggagc gaacgaccta caccgaactg agatacctac
     4441 agcgtgagct atgagaaagc gccacgcttc ccgaagggag aaaggcggac aggtatccgg
     4501 taagcggcag ggtcggaaca ggagagcgca cgagggagct tccaggggga aacgcctggt
     4561 atctttatag tcctgtcggg tttcgccacc tctgacttga gcgtcgattt ttgtgatgct
     4621 cgtcaggggg gcggagccta tggaaaaacg ccagcaacgc ggccttttta cggttcctgg
     4681 ccttttgctg gccttttgct cacatgttct ttcctgcgtt atcccctgat tctgtggata
     4741 accgtattac cgcctttgag tgagctgata ccgctcgccg cagccgaacg accgagcgca
     4801 gcgagtcagt gagcgaggaa gcggaagagc gcccaatacg caaaccgcct ctccccgcgc
     4861 gttggccgat tcattaatgc agctggcacg acaggtttcc cgactggaaa gcgggcagtg
     4921 agcgcaacgc aattaatacg cgtaccgcga gccaggaaga gtttgtagaa acgcaaaaag
     4981 gccatccgtc aggatggcct tctgcttagt ttgatgcctg gcagtttatg gcgggcgtcc
     5041 tgcccgccac cctccgggcc gttgcttcac aacgttcaaa tccgctcccg gcggatttgt
     5101 cctactcagg agagcgttca ccgacaaaca acagataaaa cgaaaggccc agtcttccga
     5161 ctgagccttt cgttttattt gatgcctggc agttccctac tctcgcgtta acgcttgcat
     5221 ggatgttttc ccagtcacga cgttgtaaaa cgacggccag tcttaagctc gggcccaata
     5281 atgattttat tttgactgat agtgacctgt tcgttgcaac aaattgatga gcaatgcttt
     5341 tttataatgc caactttgta tacaaaagtt gccccatggc gttccct
//
