LOCUS       sequence-330365-        3263 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Golden Gate entry vector to express the 1st 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: pYPQ131-tRNA2.0
FEATURES             Location/Qualifiers
     source          1..3263
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     promoter        join(185..190,191..207,208..213)
                     /label=tet promoter
                     /note="E. coli promoter for tetracycline efflux protein 
                     gene"
     CDS             261..1451
                     /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(613..632)
                     /label=pBRrevBam
                     /note="pBR322 vectors, tet region, downstream of BamHI, 
                     reverse primer"
     primer_bind     complement(1625..1644)
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        complement(1626..1644)
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     primer_bind     complement(1649..1665)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(1649..1665)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     misc_RNA        1881..1899
                     /label=MS2 stem loop
                     /note="stem loop that binds the bacteriophage MS2 coat 
                     protein"
     misc_RNA        1951..1969
                     /label=MS2 stem loop
                     /note="stem loop that binds the bacteriophage MS2 coat 
                     protein"
     rep_origin      2101..2689
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     2590..2609
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     terminator      complement(3019..3046)
                     /label=rrnB T2 terminator
                     /note="transcription terminator T2 from the E. coli rrnB 
                     gene"
     terminator      complement(3138..3224)
                     /gene="Escherichia coli rrnB"
                     /label=rrnB T1 terminator
                     /note="transcription terminator T1 from the E. coli rrnB 
                     gene"
ORIGIN
        1 aatcggccaa cgcgcgggga gaggcggttt gcgtattggg cgctcttccg cttcctcgct
       61 cactgactcg ctgcgctcgg tcgttcggct gcggcgagcg gtatcagctc actcaaaggc
      121 ggtaatacgg ttatccacag aatcagggga taacgcagga aagaacatga attaattctc
      181 atgtttgaca gcttatcatc gattagcttt aatgcggtag tttatcacag ttaaattgct
      241 aacgcagtca ggcaccgtgt atgaaatcta acaatgcgct catcgtcatc ctcggcaccg
      301 tcaccctgga tgctgtaggc ataggcttgg ttatgccggt actgccgggc ctcttgcggg
      361 atatcgtcca ttccgacagc atcgccagtc actatggcgt gctgctagcg ctatatgcgt
      421 tgatgcaatt tctatgcgca cccgttctcg gagcactgtc cgaccgcttt ggccgccgcc
      481 cagtcctgct cgcttcgcta cttggagcca ctatcgacta cgcgatcatg gcgaccacac
      541 ccgtcctgtg gattctctac gccggacgca tcgtggccgg catcaccggc gccacaggtg
      601 cggttgctgg cgcctatatc gccgacatca ccgatgggga agatcgggct cgccacttcg
      661 ggctcatgag cgcttgtttc ggcgtgggta tggtggcagg ccccgtggcc gggggactgt
      721 tgggcgccat ctccttacat gcaccattcc ttgcggcggc ggtgctcaac ggcctcaacc
      781 tactactggg ctgcttccta atgcaggagt cgcataaggg agagcgccga cccatgccct
      841 tgagagcctt caacccagtc agctccttcc ggtgggcgcg gggcatgact atcgtcgccg
      901 cacttatgac tgtcttcttt atcatgcaac tcgtaggaca ggtgccggca gcgctctggg
      961 tcattttcgg cgaggaccgc tttcgctgga gcgcgacgat gatcggcctg tcgcttgcgg
     1021 tattcggaat cttgcacgcc ctcgctcaag ccttcgtcac tggtcccgcc accaaacgtt
     1081 tcggcgagaa gcaggccatt atcgccggca tggcggccga cgcgctgggc tacgtcttgc
     1141 tggcgttcgc gacgcgaggc tggatggcct tccccattat gattcttctc gcttccggcg
     1201 gcatcgggat gcccgcgttg caggccatgc tgtccaggca ggtagatgac gaccatcagg
     1261 gacagcttca aggatcgctc gcggctctta ccagcctaac ttcgatcatt ggaccgctga
     1321 tcgtcacggc gatttatgcc gcctcggcga gcacatggaa cgggttggca tggattgtag
     1381 gcgccgccct ataccttgtc tgcctccccg cgttgcgtcg cggtgcatgg agccgggcca
     1441 cctcgacctg aatggaagcc ggcggcacct cgctaacgga ttcaccactc caagaattgg
     1501 agccaatcaa ttcttgcgga gaactgtgaa tgcgcaaacc aacccttgat cggggaagaa
     1561 cagtatgtcg agctattttt tgacttactg gggatcaagc ctgattggga gaaaataaaa
     1621 tatcccctat agtgagtcgt attacatggt catagctgtt tcctggcagc tctggcccgt
     1681 gtctcaaaat ctctgatgtt acattgcaca agataaaaat atatcatcat gcctcctcta
     1741 gaggtctcgc tataacaaag caccagtggt ctagtggtag aatagtaccc tgccacggta
     1801 cagacccggg ttcgattccc ggctggtgca agagacgaga tctagtctga gtcgaccgtc
     1861 tctgttttag agctaggcca acatgaggat cacccatgtc tgcagggcct agcaagttaa
     1921 aataaggcta gtccgttatc aacttggcca acatgaggat cacccatgtc tgcagggcca
     1981 agtggcaccg agtcggtgcc atgggagacc ctcgagccac ccatgaccaa aatcccttaa
     2041 cgtgagttac gcgtcgttcc actgagcgtc agaccccgta gaaaagatca aaggatcttc
     2101 ttgagatcct ttttttctgc gcgtaatctg ctgcttgcaa acaaaaaaac caccgctacc
     2161 agcggtggtt tgtttgccgg atcaagagct accaactctt tttccgaagg taactggctt
     2221 cagcagagcg cagataccaa atactgttct tctagtgtag ccgtagttag gccaccactt
     2281 caagaactct gtagcaccgc ctacatacct cgctctgcta atcctgttac cagtggctgc
     2341 tgccagtggc gataagtcgt gtcttaccgg gttggactca agacgatagt taccggataa
     2401 ggcgcagcgg tcgggctgaa cggggggttc gtgcacacag cccagcttgg agcgaacgac
     2461 ctacaccgaa ctgagatacc tacagcgtga gctatgagaa agcgccacgc ttcccgaagg
     2521 gagaaaggcg gacaggtatc cggtaagcgg cagggtcgga acaggagagc gcacgaggga
     2581 gcttccaggg ggaaacgcct ggtatcttta tagtcctgtc gggtttcgcc acctctgact
     2641 tgagcgtcga tttttgtgat gctcgtcagg ggggcggagc ctatggaaaa acgccagcaa
     2701 cgcggccttt ttacggttcc tggccttttg ctggcctttt gctcacatgt tctttcctgc
     2761 gttatcccct gattctgtgg ataaccgtat taccgccttt gagtgagctg ataccgctcg
     2821 ccgcagccga acgaccgagc gcagcgagtc agtgagcgag gaagcggaag agcgcccaat
     2881 acgcaaaccg cctctccccg cgcgttggcc gattcattaa tgcagctggc acgacaggtt
     2941 tcccgactgg aaagcgggca gtgagcgcaa cgcaattaat acgcgtaccg ctagccagga
     3001 agagtttgta gaaacgcaaa aaggccatcc gtcaggatgg ccttctgctt agtttgatgc
     3061 ctggcagttt atggcgggcg tcctgcccgc caccctccgg gccgttgctt cacaacgttc
     3121 aaatccgctc ccggcggatt tgtcctactc aggagagcgt tcaccgacaa acaacagata
     3181 aaacgaaagg cccagtcttc cgactgagcc tttcgtttta tttgatgcct ggcagttccc
     3241 tactctcgcg ttctgcatta atg
//
