LOCUS       sequence-337076-        5420 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Gateway entry clone and Golden Gate recipient for pYPQ131-tRNA2.0 to
            pYPQ136-tRNA2.0; assembly of 6 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: pYPQ146-ZmUbi-tRNA
FEATURES             Location/Qualifiers
     source          1..5420
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     CDS             complement(921..1319)
                     /codon_start=1
                     /gene="lacZ fragment"
                     /product="LacZ-alpha fragment of beta-galactosidase"
                     /label=lacZ-alpha
                     /translation="MTMITPSAQLTLTKGNKSWRTSRGGPVPNSPYSESYYARSLAVVL
                     QRRDWENPGVTQLNRLAAHPPFASWRNSEEARTDRPSQQLRSLNGEWDAPCSGAGLDSH
                     HLGIPAEVTPNNRVSIDKRNSTKQINSV"
     primer_bind     1184..1200
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     promoter        1210..1228
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     primer_bind     complement(1276..1296)
                     /label=T3
                     /note="T3 promoter, forward primer"
     promoter        complement(1276..1294)
                     /label=T3 promoter
                     /note="promoter for bacteriophage T3 RNA polymerase"
     primer_bind     complement(1315..1331)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(1328..1350)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    1339..1355
                     /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(1363..1369,1370..1387,1388..1393))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    1408..1429
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     protein_bind    complement(1571..1670)
                     /gene="mutant version of attL"
                     /label=attL2
                     /bound_moiety="LR Clonase(TM)"
                     /note="recombination site for the Gateway(R) LR reaction"
     promoter        complement(1688..1706)
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     promoter        complement(1723..1741)
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     primer_bind     complement(1746..1762)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     CDS             2193..2984
                     /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      3077..3665
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     3566..3585
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     3819..3836
                     /label=L4440
                     /note="L4440 vector, forward primer"
     terminator      complement(3995..4022)
                     /label=rrnB T2 terminator
                     /note="transcription terminator T2 from the E. coli rrnB 
                     gene"
     terminator      complement(4114..4200)
                     /gene="Escherichia coli rrnB"
                     /label=rrnB T1 terminator
                     /note="transcription terminator T1 from the E. coli rrnB 
                     gene"
     primer_bind     4264..4280
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     protein_bind    4295..4390
                     /gene="mutant version of attL"
                     /label=attL5
                     /bound_moiety="LR Clonase(TM)"
                     /note="recombination site for the Gateway(R) LR reaction"
     promoter        join(4419..5420,1..994)
                     /label=Ubi promoter
                     /note="maize polyubiquitin gene promoter"
ORIGIN
        1 ccccccccct ctctaccttc tctagatcgg cgttccggtc catggttagg gcccggtagt
       61 tctacttctg ttcatgtttg tgttagatcc gtgtttgtgt tagatccgtg ctgctagcgt
      121 tcgtacacgg atgcgacctg tacgtcagac acgttctgat tgctaacttg ccagtgtttc
      181 tctttgggga atcctgggat ggctctagcc gttccgcaga cgggatcgat ttcatgattt
      241 tttttgtttc gttgcatagg gtttggtttg cccttttcct ttatttcaat atatgccgtg
      301 cacttgtttg tcgggtcatc ttttcatgct tttttttgtc ttggttgtga tgatgtggtc
      361 tggttgggcg gtcgttctag atcggagtag aattaattct gtttcaaact acctggtgga
      421 tttattaatt ttggatctgt atgtgtgtgc catacatatt catagttacg aattgaagat
      481 gatggatgga aatatcgatc taggataggt atacatgttg atgcgggttt tactgatgca
      541 tatacagaga tgctttttgt tcgcttggtt gtgatgatgt ggtgtggttg ggcggtcgtt
      601 cattcgttct agatcggagt agaatactgt ttcaaactac ctggtgtatt tattaatttt
      661 ggaactgtat gtgtgtgtca tacatcttca tagttacgag tttaagatgg atggaaatat
      721 cgatctagga taggtataca tgttgatgtg ggttttactg atgcatatac atgatggcat
      781 atgcagcatc tattcatatg ctctaacctt gagtacctat ctattataat aaacaagtat
      841 gttttataat tattttgatc ttgatatact tggatgatgg catatgcagc agctatatgt
      901 ggattttttt agccctgcct tcatacgcta tttatttgct tggtactgtt tcttttgtcg
      961 atgctcaccc tgttgtttgg tgttacttct gcagggatcc ccaagtggtg gctatcgaga
     1021 ccggcgccgc tacagggcgc gtcccattcg ccattcaggc tgcgcaactg ttgggaaggg
     1081 cgatcggtgc gggcctcttc gctattacgc cagctggcga aagggggatg tgctgcaagg
     1141 cgattaagtt gggtaacgcc agggttttcc cagtcacgac gttgtaaaac gacggccagt
     1201 gagcgcgcgt aatacgactc actatagggc gaattgggta ccgggccccc cctcgaggtc
     1261 ctccagcttt tgttcccttt agtgagggtt aattgcgcgc ttggcgtaat catggtcata
     1321 gctgtttcct gtgtgaaatt gttatccgct cacaattcca cacaacatac gagccggaag
     1381 cataaagtgt aaagcctggg gtgcctaatg agtgagctaa ctcacattaa ttgcgttgcg
     1441 ctcactgccc gctttccacc ggtggtctct cggtaacaaa gcaccagtgg tctagtggta
     1501 gaatagtacc ctgccacggt acagacccgg gttcgattcc cggctggtgc aactagtaag
     1561 ggcgaattcg acccagcttt cttgtacaaa gttggcatta taaaaaataa ttgctcatca
     1621 atttgttgca acgaacaggt cactatcagt caaaataaaa tcattatttg ccatccagct
     1681 gatatcccct atagtgagtc gtattacatg gtcatagcat cccctatagt gagtcgtatt
     1741 acatggtcat agctgtttcc tggcagctct ggcccgtgtc tcaaaatctc tgatgttaca
     1801 ttgcacaaga taaaaatata tcatcatgcc tcctctggac cagccaggac agaaatgcct
     1861 cgacttcgct gctacccaag gttgccgggt gacgcacacc gtggaaacgg atgaaggcac
     1921 gaacccagtg gacataagcc tgttcggttc gtaagctgta atgcaagtag cgtatgcgct
     1981 cacgcaactg gtccagaacc ttgaccgaac gcagcggtgg taacggcgca gtggcggttt
     2041 tcatggcttg ttatgactgt ttttttgggg tacagtctat gcctcgggca tccaagcagc
     2101 aagcgcgtta cgccgtgggt cgatgtttga tgttatggag cagcaacgat gttacgcagc
     2161 agggcagtcg ccctaaaaca aagttaaaca ttatgaggga agcggtgatc gccgaagtat
     2221 cgactcaact atcagaggta gttggcgtca tcgagcgcca tctcgaaccg acgttgctgg
     2281 ccgtacattt gtacggctcc gcagtggatg gcggcctgaa gccacacagt gatattgatt
     2341 tgctggttac ggtgaccgta aggcttgatg aaacaacgcg gcgagctttg atcaacgacc
     2401 ttttggaaac ttcggcttcc cctggagaga gcgagattct ccgcgctgta gaagtcacca
     2461 ttgttgtgca cgacgacatc attccgtggc gttatccagc taagcgcgaa ctgcaatttg
     2521 gagaatggca gcgcaatgac attcttgcag gtatcttcga gccagccacg atcgacattg
     2581 atctggctat cttgctgaca aaagcaagag aacatagcgt tgccttggta ggtccagcgg
     2641 cggaggaact ctttgatccg gttcctgaac aggatctatt tgaggcgcta aatgaaacct
     2701 taacgctatg gaactcgccg cccgactggg ctggcgatga gcgaaatgta gtgcttacgt
     2761 tgtcccgcat ttggtacagc gcagtaaccg gcaaaatcgc gccgaaggat gtcgctgccg
     2821 actgggcaat ggagcgcctg ccggcccagt atcagcccgt catacttgaa gctagacagg
     2881 cttatcttgg acaagaagaa gatcgcttgg cctcgcgcgc agatcagttg gaagaatttg
     2941 tccactacgt gaaaggcgag atcaccaagg tagtcggcaa ataaccctcg agccacccat
     3001 gaccaaaatc ccttaacgtg agttacgcgt cgttccactg agcgtcagac cccgtagaaa
     3061 agatcaaagg atcttcttga gatccttttt ttctgcgcgt aatctgctgc ttgcaaacaa
     3121 aaaaaccacc gctaccagcg gtggtttgtt tgccggatca agagctacca actctttttc
     3181 cgaaggtaac tggcttcagc agagcgcaga taccaaatac tgttcttcta gtgtagccgt
     3241 agttaggcca ccacttcaag aactctgtag caccgcctac atacctcgct ctgctaatcc
     3301 tgttaccagt ggctgctgcc agtggcgata agtcgtgtct taccgggttg gactcaagac
     3361 gatagttacc ggataaggcg cagcggtcgg gctgaacggg gggttcgtgc acacagccca
     3421 gcttggagcg aacgacctac accgaactga gatacctaca gcgtgagcta tgagaaagcg
     3481 ccacgcttcc cgaagggaga aaggcggaca ggtatccggt aagcggcagg gtcggaacag
     3541 gagagcgcac gagggagctt ccagggggaa acgcctggta tctttatagt cctgtcgggt
     3601 ttcgccacct ctgacttgag cgtcgatttt tgtgatgctc gtcagggggg cggagcctat
     3661 ggaaaaacgc cagcaacgcg gcctttttac ggttcctggc cttttgctgg ccttttgctc
     3721 acatgttctt tcctgcgtta tcccctgatt ctgtggataa ccgtattacc gcctttgagt
     3781 gagctgatac cgctcgccgc agccgaacga ccgagcgcag cgagtcagtg agcgaggaag
     3841 cggaagagcg cccaatacgc aaaccgcctc tccccgcgcg ttggccgatt cattaatgca
     3901 gctggcacga caggtttccc gactggaaag cgggcagtga gcgcaacgca attaatacgc
     3961 gtaccgcgag ccaggaagag tttgtagaaa cgcaaaaagg ccatccgtca ggatggcctt
     4021 ctgcttagtt tgatgcctgg cagtttatgg cgggcgtcct gcccgccacc ctccgggccg
     4081 ttgcttcaca acgttcaaat ccgctcccgg cggatttgtc ctactcagga gagcgttcac
     4141 cgacaaacaa cagataaaac gaaaggccca gtcttccgac tgagcctttc gttttatttg
     4201 atgcctggca gttccctact ctcgcgttaa cgcttgcatg gatgttttcc cagtcacgac
     4261 gttgtaaaac gacggccagt cttaagctcg ggcccaataa tgattttatt ttgactgata
     4321 gtgacctgtt cgttgcaaca aattgatgag caatgctttt ttataatgcc aactttgtat
     4381 acaaaagttg ccccatggcg ttccctctag ataacgcact gcagtgcagc gtgacccggt
     4441 cgtgcccctc tctagagata atgagcattg catgtctaag ttataaaaaa ttaccacata
     4501 ttttttttgt cacacttgtt tgaagtgcag tttatctatc tttatacata tatttaaact
     4561 ttactctacg aataatataa tctatagtac tacaataata tcagtgtttt agagaatcat
     4621 ataaatgaac agttagacat ggtctaaagg acaattgagt attttgacaa caggactcta
     4681 cagttttatc tttttagtgt gcatgtgttc tccttttttt ttgcaaatag cttcacctat
     4741 ataatacttc atccatttta ttagtacatc catttagggt ttagggttaa tggtttttat
     4801 agactaattt ttttagtaca tctattttat tctattttag cctctaaatt aagaaaacta
     4861 aaactctatt ttagtttttt tatttaataa tttagatata aaatagaata aaataaagtg
     4921 actaaaaatt aaacaaatac cctttaagaa attaaaaaaa ctaaggaaac atttttcttg
     4981 tttcgagtag ataatgccag cctgttaaac gccgtcgacg agtctaacgg acaccaacca
     5041 gcgaaccagc agcgtcgcgt cgggccaagc gaagcagacg gcacggcatc tctgtcgctg
     5101 cctctggacc cctctcgaga gttccgctcc accgttggac ttgctccgct gtcggcatcc
     5161 agaaattgcg tggcggagcg gcagacgtga gccggcacgg caggcggcct cctcctcctc
     5221 tcacggcacc ggcagctacg ggggattcct ttcccaccgc tccttcgctt tcccttcctc
     5281 gcccgccgta ataaatagac accccctcca caccctcttt ccccaacctc gtgttgttcg
     5341 gagcgcacac acacacaacc agatctcccc caaatccacc cgtcggcacc tccgcttcaa
     5401 ggtacgccgc tcgtcctccc
//
