LOCUS       sequence-330916-        5395 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Gateway entry clone and Golden Gate recipient for pYPQ131-tRNA2.0 to
            pYPQ134-tRNA2.0; assembly of 4 gRNAs.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 5395)
  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 5395)
  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: pYPQ144-ZmUbi-tRNA
FEATURES             Location/Qualifiers
     source          1..5395
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     196..212
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     promoter        222..240
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     primer_bind     complement(288..308)
                     /label=T3
                     /note="T3 promoter, forward primer"
     promoter        complement(288..306)
                     /label=T3 promoter
                     /note="promoter for bacteriophage T3 RNA polymerase"
     primer_bind     complement(327..343)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(340..362)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    351..367
                     /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(375..381,382..399,400..405))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    420..441
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     protein_bind    complement(601..700)
                     /gene="mutant version of attL"
                     /label=attL2
                     /bound_moiety="LR Clonase(TM)"
                     /note="recombination site for the Gateway(R) LR reaction"
     promoter        complement(718..736)
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     promoter        complement(753..771)
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     primer_bind     complement(776..792)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     CDS             1223..2014
                     /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      2107..2695
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     2596..2615
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     2849..2866
                     /label=L4440
                     /note="L4440 vector, forward primer"
     terminator      complement(3025..3052)
                     /label=rrnB T2 terminator
                     /note="transcription terminator T2 from the E. coli rrnB 
                     gene"
     terminator      complement(3144..3230)
                     /gene="Escherichia coli rrnB"
                     /label=rrnB T1 terminator
                     /note="transcription terminator T1 from the E. coli rrnB 
                     gene"
     primer_bind     3294..3310
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     protein_bind    3325..3420
                     /gene="mutant version of attL"
                     /label=attL5
                     /bound_moiety="LR Clonase(TM)"
                     /note="recombination site for the Gateway(R) LR reaction"
     CDS             complement(join(5328..5395,1..331))
                     /codon_start=1
                     /gene="lacZ fragment"
                     /product="LacZ-alpha fragment of beta-galactosidase"
                     /label=lacZ-alpha
                     /translation="MTMITPSAQLTLTKGNKSWRTSRGGPVPNSPYSESYYARSLAVVL
                     QRRDWENPGVTQLNRLAAHPPFASWRNSEEARTDRPSQQLRSLNGEWDAPCSGAGLDSH
                     HLGIPAEVTPNNRVSIDKRNSTKQINSV"
ORIGIN
        1 ctgcagggat ccccaagtgg tggctatcga gaccggcgcc gctacagggc gcgtcccatt
       61 cgccattcag gctgcgcaac tgttgggaag ggcgatcggt gcgggcctct tcgctattac
      121 gccagctggc gaaaggggga tgtgctgcaa ggcgattaag ttgggtaacg ccagggtttt
      181 cccagtcacg acgttgtaaa acgacggcca gtgagcgcgc gtaatacgac tcactatagg
      241 gcgaattggg taccgggccc cccctcgagg tcctccagct tttgttccct ttagtgaggg
      301 ttaattgcgc gcttggcgta atcatggtca tagctgtttc ctgtgtgaaa ttgttatccg
      361 ctcacaattc cacacaacat acgagccgga agcataaagt gtaaagcctg gggtgcctaa
      421 tgagtgagct aactcacatt aattgcgttg cgctcactgc ccgctttcca ccggtggtct
      481 cttgttaaca aagcaccagt ggtctagtgg tagaatagta ccctgccacg gtacagaccc
      541 gggttcgatt cccggctggt gcaactagtt tttttgctag cagatctaag ggcgaattcg
      601 acccagcttt cttgtacaaa gttggcatta taaaaaataa ttgctcatca atttgttgca
      661 acgaacaggt cactatcagt caaaataaaa tcattatttg ccatccagct gatatcccct
      721 atagtgagtc gtattacatg gtcatagcat cccctatagt gagtcgtatt acatggtcat
      781 agctgtttcc tggcagctct ggcccgtgtc tcaaaatctc tgatgttaca ttgcacaaga
      841 taaaaatata tcatcatgcc tcctctggac cagccaggac agaaatgcct cgacttcgct
      901 gctacccaag gttgccgggt gacgcacacc gtggaaacgg atgaaggcac gaacccagtg
      961 gacataagcc tgttcggttc gtaagctgta atgcaagtag cgtatgcgct cacgcaactg
     1021 gtccagaacc ttgaccgaac gcagcggtgg taacggcgca gtggcggttt tcatggcttg
     1081 ttatgactgt ttttttgggg tacagtctat gcctcgggca tccaagcagc aagcgcgtta
     1141 cgccgtgggt cgatgtttga tgttatggag cagcaacgat gttacgcagc agggcagtcg
     1201 ccctaaaaca aagttaaaca ttatgaggga agcggtgatc gccgaagtat cgactcaact
     1261 atcagaggta gttggcgtca tcgagcgcca tctcgaaccg acgttgctgg ccgtacattt
     1321 gtacggctcc gcagtggatg gcggcctgaa gccacacagt gatattgatt tgctggttac
     1381 ggtgaccgta aggcttgatg aaacaacgcg gcgagctttg atcaacgacc ttttggaaac
     1441 ttcggcttcc cctggagaga gcgagattct ccgcgctgta gaagtcacca ttgttgtgca
     1501 cgacgacatc attccgtggc gttatccagc taagcgcgaa ctgcaatttg gagaatggca
     1561 gcgcaatgac attcttgcag gtatcttcga gccagccacg atcgacattg atctggctat
     1621 cttgctgaca aaagcaagag aacatagcgt tgccttggta ggtccagcgg cggaggaact
     1681 ctttgatccg gttcctgaac aggatctatt tgaggcgcta aatgaaacct taacgctatg
     1741 gaactcgccg cccgactggg ctggcgatga gcgaaatgta gtgcttacgt tgtcccgcat
     1801 ttggtacagc gcagtaaccg gcaaaatcgc gccgaaggat gtcgctgccg actgggcaat
     1861 ggagcgcctg ccggcccagt atcagcccgt catacttgaa gctagacagg cttatcttgg
     1921 acaagaagaa gatcgcttgg cctcgcgcgc agatcagttg gaagaatttg tccactacgt
     1981 gaaaggcgag atcaccaagg tagtcggcaa ataaccctcg agccacccat gaccaaaatc
     2041 ccttaacgtg agttacgcgt cgttccactg agcgtcagac cccgtagaaa agatcaaagg
     2101 atcttcttga gatccttttt ttctgcgcgt aatctgctgc ttgcaaacaa aaaaaccacc
     2161 gctaccagcg gtggtttgtt tgccggatca agagctacca actctttttc cgaaggtaac
     2221 tggcttcagc agagcgcaga taccaaatac tgttcttcta gtgtagccgt agttaggcca
     2281 ccacttcaag aactctgtag caccgcctac atacctcgct ctgctaatcc tgttaccagt
     2341 ggctgctgcc agtggcgata agtcgtgtct taccgggttg gactcaagac gatagttacc
     2401 ggataaggcg cagcggtcgg gctgaacggg gggttcgtgc acacagccca gcttggagcg
     2461 aacgacctac accgaactga gatacctaca gcgtgagcta tgagaaagcg ccacgcttcc
     2521 cgaagggaga aaggcggaca ggtatccggt aagcggcagg gtcggaacag gagagcgcac
     2581 gagggagctt ccagggggaa acgcctggta tctttatagt cctgtcgggt ttcgccacct
     2641 ctgacttgag cgtcgatttt tgtgatgctc gtcagggggg cggagcctat ggaaaaacgc
     2701 cagcaacgcg gcctttttac ggttcctggc cttttgctgg ccttttgctc acatgttctt
     2761 tcctgcgtta tcccctgatt ctgtggataa ccgtattacc gcctttgagt gagctgatac
     2821 cgctcgccgc agccgaacga ccgagcgcag cgagtcagtg agcgaggaag cggaagagcg
     2881 cccaatacgc aaaccgcctc tccccgcgcg ttggccgatt cattaatgca gctggcacga
     2941 caggtttccc gactggaaag cgggcagtga gcgcaacgca attaatacgc gtaccgcgag
     3001 ccaggaagag tttgtagaaa cgcaaaaagg ccatccgtca ggatggcctt ctgcttagtt
     3061 tgatgcctgg cagtttatgg cgggcgtcct gcccgccacc ctccgggccg ttgcttcaca
     3121 acgttcaaat ccgctcccgg cggatttgtc ctactcagga gagcgttcac cgacaaacaa
     3181 cagataaaac gaaaggccca gtcttccgac tgagcctttc gttttatttg atgcctggca
     3241 gttccctact ctcgcgttaa cgcttgcatg gatgttttcc cagtcacgac gttgtaaaac
     3301 gacggccagt cttaagctcg ggcccaataa tgattttatt ttgactgata gtgacctgtt
     3361 cgttgcaaca aattgatgag caatgctttt ttataatgcc aactttgtat acaaaagttg
     3421 ccccatggcg ttccctctag agagataatg agcattgcat gtctaagtta taaaaaatta
     3481 ccacatattt tttttgtcac acttgtttga agtgcagttt atctatcttt atacatatat
     3541 ttaaacttta ctctacgaat aatataatct atagtactac aataatatca gtgttttaga
     3601 gaatcatata aatgaacagt tagacatggt ctaaaggaca attgagtatt ttgacaacag
     3661 gactctacag ttttatcttt ttagtgtgca tgtgttctcc tttttttttg caaatagctt
     3721 cacctatata atacttcatc cattttatta gtacatccat ttagggttta gggttaatgg
     3781 tttttataga ctaatttttt tagtacatct attttattct attttagcct ctaaattaag
     3841 aaaactaaaa ctctatttta gtttttttat ttaataattt agatataaaa tagaataaaa
     3901 taaagtgact aaaaattaaa caaataccct ttaagaaatt aaaaaaacta aggaaacatt
     3961 tttcttgttt cgagtagata atgccagcct gttaaacgcc gtcgacgagt ctaacggaca
     4021 ccaaccagcg aaccagcagc gtcgcgtcgg gccaagcgaa gcagacggca cggcatctct
     4081 gtcgctgcct ctggacccct ctcgagagtt ccgctccacc gttggacttg ctccgctgtc
     4141 ggcatccaga aattgcgtgg cggagcggca gacgtgagcc ggcacggcag gcggcctcct
     4201 cctcctctca cggcaccggc agctacgggg gattcctttc ccaccgctcc ttcgctttcc
     4261 cttcctcgcc cgccgtaata aatagacacc ccctccacac cctctttccc caacctcgtg
     4321 ttgttcggag cgcacacaca cacaaccaga tctcccccaa atccacccgt cggcacctcc
     4381 gcttcaaggt acgccgctcg tcctcccccc cccccctctc taccttctct agatcggcgt
     4441 tccggtccat ggttagggcc cggtagttct acttctgttc atgtttgtgt tagatccgtg
     4501 tttgtgttag atccgtgctg ctagcgttcg tacacggatg cgacctgtac gtcagacacg
     4561 ttctgattgc taacttgcca gtgtttctct ttggggaatc ctgggatggc tctagccgtt
     4621 ccgcagacgg gatcgatttc atgatttttt ttgtttcgtt gcatagggtt tggtttgccc
     4681 ttttccttta tttcaatata tgccgtgcac ttgtttgtcg ggtcatcttt tcatgctttt
     4741 ttttgtcttg gttgtgatga tgtggtctgg ttgggcggtc gttctagatc ggagtagaat
     4801 taattctgtt tcaaactacc tggtggattt attaattttg gatctgtatg tgtgtgccat
     4861 acatattcat agttacgaat tgaagatgat ggatggaaat atcgatctag gataggtata
     4921 catgttgatg cgggttttac tgatgcatat acagagatgc tttttgttcg cttggttgtg
     4981 atgatgtggt gtggttgggc ggtcgttcat tcgttctaga tcggagtaga atactgtttc
     5041 aaactacctg gtgtatttat taattttgga actgtatgtg tgtgtcatac atcttcatag
     5101 ttacgagttt aagatggatg gaaatatcga tctaggatag gtatacatgt tgatgtgggt
     5161 tttactgatg catatacatg atggcatatg cagcatctat tcatatgctc taaccttgag
     5221 tacctatcta ttataataaa caagtatgtt ttataattat tttgatcttg atatacttgg
     5281 atgatggcat atgcagcagc tatatgtgga tttttttagc cctgccttca tacgctattt
     5341 atttgcttgg tactgtttct tttgtcgatg ctcaccctgt tgtttggtgt tactt
//
