LOCUS       sequence-330367-        3263 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Golden Gate entry vector to express the 3rd gRNA with tRNA2.0 
            scaffold (with four MS2 binding sites) without promoter.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 3263)
  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 3263)
  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: pYPQ133-tRNA2.0
FEATURES             Location/Qualifiers
     source          1..3263
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     promoter        join(196..201,202..218,219..224)
                     /label=tet promoter
                     /note="E. coli promoter for tetracycline efflux protein 
                     gene"
     CDS             272..1462
                     /codon_start=1
                     /gene="tet"
                     /product="tetracycline efflux protein"
                     /label=TcR
                     /note="confers resistance to tetracycline"
                     /translation="MKSNNALIVILGTVTLDAVGIGLVMPVLPGLLRDIVHSDSIASHY
                     GVLLALYALMQFLCAPVLGALSDRFGRRPVLLASLLGATIDYAIMATTPVLWILYAGRI
                     VAGITGATGAVAGAYIADITDGEDRARHFGLMSACFGVGMVAGPVAGGLLGAISLHAPF
                     LAAAVLNGLNLLLGCFLMQESHKGERRPMPLRAFNPVSSFRWARGMTIVAALMTVFFIM
                     QLVGQVPAALWVIFGEDRFRWSATMIGLSLAVFGILHALAQAFVTGPATKRFGEKQAII
                     AGMAADALGYVLLAFATRGWMAFPIMILLASGGIGMPALQAMLSRQVDDDHQGQLQGSL
                     AALTSLTSIIGPLIVTAIYAASASTWNGLAWIVGAALYLVCLPALRRGAWSRATST"
     primer_bind     complement(624..643)
                     /label=pBRrevBam
                     /note="pBR322 vectors, tet region, downstream of BamHI, 
                     reverse primer"
     primer_bind     complement(1636..1655)
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        complement(1637..1655)
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     primer_bind     complement(1660..1676)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(1660..1676)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     misc_RNA        1892..1910
                     /label=MS2 stem loop
                     /note="stem loop that binds the bacteriophage MS2 coat 
                     protein"
     misc_RNA        1962..1980
                     /label=MS2 stem loop
                     /note="stem loop that binds the bacteriophage MS2 coat 
                     protein"
     rep_origin      2112..2700
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     2601..2620
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     terminator      complement(3030..3057)
                     /label=rrnB T2 terminator
                     /note="transcription terminator T2 from the E. coli rrnB 
                     gene"
     terminator      complement(3149..3235)
                     /gene="Escherichia coli rrnB"
                     /label=rrnB T1 terminator
                     /note="transcription terminator T1 from the E. coli rrnB 
                     gene"
ORIGIN
        1 ctgcattaat gaatcggcca acgcgcgggg agaggcggtt tgcgtattgg gcgctcttcc
       61 gcttcctcgc tcactgactc gctgcgctcg gtcgttcggc tgcggcgagc ggtatcagct
      121 cactcaaagg cggtaatacg gttatccaca gaatcagggg ataacgcagg aaagaacatg
      181 aattaattct catgtttgac agcttatcat cgattagctt taatgcggta gtttatcaca
      241 gttaaattgc taacgcagtc aggcaccgtg tatgaaatct aacaatgcgc tcatcgtcat
      301 cctcggcacc gtcaccctgg atgctgtagg cataggcttg gttatgccgg tactgccggg
      361 cctcttgcgg gatatcgtcc attccgacag catcgccagt cactatggcg tgctgctagc
      421 gctatatgcg ttgatgcaat ttctatgcgc acccgttctc ggagcactgt ccgaccgctt
      481 tggccgccgc ccagtcctgc tcgcttcgct acttggagcc actatcgact acgcgatcat
      541 ggcgaccaca cccgtcctgt ggattctcta cgccggacgc atcgtggccg gcatcaccgg
      601 cgccacaggt gcggttgctg gcgcctatat cgccgacatc accgatgggg aagatcgggc
      661 tcgccacttc gggctcatga gcgcttgttt cggcgtgggt atggtggcag gccccgtggc
      721 cgggggactg ttgggcgcca tctccttaca tgcaccattc cttgcggcgg cggtgctcaa
      781 cggcctcaac ctactactgg gctgcttcct aatgcaggag tcgcataagg gagagcgccg
      841 acccatgccc ttgagagcct tcaacccagt cagctccttc cggtgggcgc ggggcatgac
      901 tatcgtcgcc gcacttatga ctgtcttctt tatcatgcaa ctcgtaggac aggtgccggc
      961 agcgctctgg gtcattttcg gcgaggaccg ctttcgctgg agcgcgacga tgatcggcct
     1021 gtcgcttgcg gtattcggaa tcttgcacgc cctcgctcaa gccttcgtca ctggtcccgc
     1081 caccaaacgt ttcggcgaga agcaggccat tatcgccggc atggcggccg acgcgctggg
     1141 ctacgtcttg ctggcgttcg cgacgcgagg ctggatggcc ttccccatta tgattcttct
     1201 cgcttccggc ggcatcggga tgcccgcgtt gcaggccatg ctgtccaggc aggtagatga
     1261 cgaccatcag ggacagcttc aaggatcgct cgcggctctt accagcctaa cttcgatcat
     1321 tggaccgctg atcgtcacgg cgatttatgc cgcctcggcg agcacatgga acgggttggc
     1381 atggattgta ggcgccgccc tataccttgt ctgcctcccc gcgttgcgtc gcggtgcatg
     1441 gagccgggcc acctcgacct gaatggaagc cggcggcacc tcgctaacgg attcaccact
     1501 ccaagaattg gagccaatca attcttgcgg agaactgtga atgcgcaaac caacccttga
     1561 tcggggaaga acagtatgtc gagctatttt ttgacttact ggggatcaag cctgattggg
     1621 agaaaataaa atatccccta tagtgagtcg tattacatgg tcatagctgt ttcctggcag
     1681 ctctggcccg tgtctcaaaa tctctgatgt tacattgcac aagataaaaa tatatcatca
     1741 tgcctcctct agaggtctca ggacaacaaa gcaccagtgg tctagtggta gaatagtacc
     1801 ctgccacggt acagacccgg gttcgattcc cggctggtgc aagagacgag atctagtctg
     1861 agtcgaccgt ctctgtttta gagctaggcc aacatgagga tcacccatgt ctgcagggcc
     1921 tagcaagtta aaataaggct agtccgttat caacttggcc aacatgagga tcacccatgt
     1981 ctgcagggcc aagtggcacc gagtcggtgc ccagggagac cctcgagcca cccatgacca
     2041 aaatccctta acgtgagtta cgcgtcgttc cactgagcgt cagaccccgt agaaaagatc
     2101 aaaggatctt cttgagatcc tttttttctg cgcgtaatct gctgcttgca aacaaaaaaa
     2161 ccaccgctac cagcggtggt ttgtttgccg gatcaagagc taccaactct ttttccgaag
     2221 gtaactggct tcagcagagc gcagatacca aatactgttc ttctagtgta gccgtagtta
     2281 ggccaccact tcaagaactc tgtagcaccg cctacatacc tcgctctgct aatcctgtta
     2341 ccagtggctg ctgccagtgg cgataagtcg tgtcttaccg ggttggactc aagacgatag
     2401 ttaccggata aggcgcagcg gtcgggctga acggggggtt cgtgcacaca gcccagcttg
     2461 gagcgaacga cctacaccga actgagatac ctacagcgtg agctatgaga aagcgccacg
     2521 cttcccgaag ggagaaaggc ggacaggtat ccggtaagcg gcagggtcgg aacaggagag
     2581 cgcacgaggg agcttccagg gggaaacgcc tggtatcttt atagtcctgt cgggtttcgc
     2641 cacctctgac ttgagcgtcg atttttgtga tgctcgtcag gggggcggag cctatggaaa
     2701 aacgccagca acgcggcctt tttacggttc ctggcctttt gctggccttt tgctcacatg
     2761 ttctttcctg cgttatcccc tgattctgtg gataaccgta ttaccgcctt tgagtgagct
     2821 gataccgctc gccgcagccg aacgaccgag cgcagcgagt cagtgagcga ggaagcggaa
     2881 gagcgcccaa tacgcaaacc gcctctcccc gcgcgttggc cgattcatta atgcagctgg
     2941 cacgacaggt ttcccgactg gaaagcgggc agtgagcgca acgcaattaa tacgcgtacc
     3001 gctagccagg aagagtttgt agaaacgcaa aaaggccatc cgtcaggatg gccttctgct
     3061 tagtttgatg cctggcagtt tatggcgggc gtcctgcccg ccaccctccg ggccgttgct
     3121 tcacaacgtt caaatccgct cccggcggat ttgtcctact caggagagcg ttcaccgaca
     3181 aacaacagat aaaacgaaag gcccagtctt ccgactgagc ctttcgtttt atttgatgcc
     3241 tggcagttcc ctactctcgc gtt
//
