LOCUS       sequence-330366-        3263 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Golden Gate entry vector to express the 2nd 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: pYPQ132-tRNA2.0
FEATURES             Location/Qualifiers
     source          1..3263
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     promoter        join(160..165,166..182,183..188)
                     /label=tet promoter
                     /note="E. coli promoter for tetracycline efflux protein 
                     gene"
     CDS             236..1426
                     /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(588..607)
                     /label=pBRrevBam
                     /note="pBR322 vectors, tet region, downstream of BamHI, 
                     reverse primer"
     primer_bind     complement(1600..1619)
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        complement(1601..1619)
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     primer_bind     complement(1624..1640)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(1624..1640)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     misc_RNA        1856..1874
                     /label=MS2 stem loop
                     /note="stem loop that binds the bacteriophage MS2 coat 
                     protein"
     misc_RNA        1926..1944
                     /label=MS2 stem loop
                     /note="stem loop that binds the bacteriophage MS2 coat 
                     protein"
     rep_origin      2076..2664
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     2565..2584
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     terminator      complement(2994..3021)
                     /label=rrnB T2 terminator
                     /note="transcription terminator T2 from the E. coli rrnB 
                     gene"
     terminator      complement(3113..3199)
                     /gene="Escherichia coli rrnB"
                     /label=rrnB T1 terminator
                     /note="transcription terminator T1 from the E. coli rrnB 
                     gene"
ORIGIN
        1 ggtttgcgta ttgggcgctc ttccgcttcc tcgctcactg actcgctgcg ctcggtcgtt
       61 cggctgcggc gagcggtatc agctcactca aaggcggtaa tacggttatc cacagaatca
      121 ggggataacg caggaaagaa catgaattaa ttctcatgtt tgacagctta tcatcgatta
      181 gctttaatgc ggtagtttat cacagttaaa ttgctaacgc agtcaggcac cgtgtatgaa
      241 atctaacaat gcgctcatcg tcatcctcgg caccgtcacc ctggatgctg taggcatagg
      301 cttggttatg ccggtactgc cgggcctctt gcgggatatc gtccattccg acagcatcgc
      361 cagtcactat ggcgtgctgc tagcgctata tgcgttgatg caatttctat gcgcacccgt
      421 tctcggagca ctgtccgacc gctttggccg ccgcccagtc ctgctcgctt cgctacttgg
      481 agccactatc gactacgcga tcatggcgac cacacccgtc ctgtggattc tctacgccgg
      541 acgcatcgtg gccggcatca ccggcgccac aggtgcggtt gctggcgcct atatcgccga
      601 catcaccgat ggggaagatc gggctcgcca cttcgggctc atgagcgctt gtttcggcgt
      661 gggtatggtg gcaggccccg tggccggggg actgttgggc gccatctcct tacatgcacc
      721 attccttgcg gcggcggtgc tcaacggcct caacctacta ctgggctgct tcctaatgca
      781 ggagtcgcat aagggagagc gccgacccat gcccttgaga gccttcaacc cagtcagctc
      841 cttccggtgg gcgcggggca tgactatcgt cgccgcactt atgactgtct tctttatcat
      901 gcaactcgta ggacaggtgc cggcagcgct ctgggtcatt ttcggcgagg accgctttcg
      961 ctggagcgcg acgatgatcg gcctgtcgct tgcggtattc ggaatcttgc acgccctcgc
     1021 tcaagccttc gtcactggtc ccgccaccaa acgtttcggc gagaagcagg ccattatcgc
     1081 cggcatggcg gccgacgcgc tgggctacgt cttgctggcg ttcgcgacgc gaggctggat
     1141 ggccttcccc attatgattc ttctcgcttc cggcggcatc gggatgcccg cgttgcaggc
     1201 catgctgtcc aggcaggtag atgacgacca tcagggacag cttcaaggat cgctcgcggc
     1261 tcttaccagc ctaacttcga tcattggacc gctgatcgtc acggcgattt atgccgcctc
     1321 ggcgagcaca tggaacgggt tggcatggat tgtaggcgcc gccctatacc ttgtctgcct
     1381 ccccgcgttg cgtcgcggtg catggagccg ggccacctcg acctgaatgg aagccggcgg
     1441 cacctcgcta acggattcac cactccaaga attggagcca atcaattctt gcggagaact
     1501 gtgaatgcgc aaaccaaccc ttgatcgggg aagaacagta tgtcgagcta ttttttgact
     1561 tactggggat caagcctgat tgggagaaaa taaaatatcc cctatagtga gtcgtattac
     1621 atggtcatag ctgtttcctg gcagctctgg cccgtgtctc aaaatctctg atgttacatt
     1681 gcacaagata aaaatatatc atcatgcctc ctctagaggt ctcccatgaa caaagcacca
     1741 gtggtctagt ggtagaatag taccctgcca cggtacagac ccgggttcga ttcccggctg
     1801 gtgcaagaga cgagatctag tctgagtcga ccgtctctgt tttagagcta ggccaacatg
     1861 aggatcaccc atgtctgcag ggcctagcaa gttaaaataa ggctagtccg ttatcaactt
     1921 ggccaacatg aggatcaccc atgtctgcag ggccaagtgg caccgagtcg gtgcggaccg
     1981 agaccctcga gccacccatg accaaaatcc cttaacgtga gttacgcgtc gttccactga
     2041 gcgtcagacc ccgtagaaaa gatcaaagga tcttcttgag atcctttttt tctgcgcgta
     2101 atctgctgct tgcaaacaaa aaaaccaccg ctaccagcgg tggtttgttt gccggatcaa
     2161 gagctaccaa ctctttttcc gaaggtaact ggcttcagca gagcgcagat accaaatact
     2221 gttcttctag tgtagccgta gttaggccac cacttcaaga actctgtagc accgcctaca
     2281 tacctcgctc tgctaatcct gttaccagtg gctgctgcca gtggcgataa gtcgtgtctt
     2341 accgggttgg actcaagacg atagttaccg gataaggcgc agcggtcggg ctgaacgggg
     2401 ggttcgtgca cacagcccag cttggagcga acgacctaca ccgaactgag atacctacag
     2461 cgtgagctat gagaaagcgc cacgcttccc gaagggagaa aggcggacag gtatccggta
     2521 agcggcaggg tcggaacagg agagcgcacg agggagcttc cagggggaaa cgcctggtat
     2581 ctttatagtc ctgtcgggtt tcgccacctc tgacttgagc gtcgattttt gtgatgctcg
     2641 tcaggggggc ggagcctatg gaaaaacgcc agcaacgcgg cctttttacg gttcctggcc
     2701 ttttgctggc cttttgctca catgttcttt cctgcgttat cccctgattc tgtggataac
     2761 cgtattaccg cctttgagtg agctgatacc gctcgccgca gccgaacgac cgagcgcagc
     2821 gagtcagtga gcgaggaagc ggaagagcgc ccaatacgca aaccgcctct ccccgcgcgt
     2881 tggccgattc attaatgcag ctggcacgac aggtttcccg actggaaagc gggcagtgag
     2941 cgcaacgcaa ttaatacgcg taccgctagc caggaagagt ttgtagaaac gcaaaaaggc
     3001 catccgtcag gatggccttc tgcttagttt gatgcctggc agtttatggc gggcgtcctg
     3061 cccgccaccc tccgggccgt tgcttcacaa cgttcaaatc cgctcccggc ggatttgtcc
     3121 tactcaggag agcgttcacc gacaaacaac agataaaacg aaaggcccag tcttccgact
     3181 gagcctttcg ttttatttga tgcctggcag ttccctactc tcgcgttctg cattaatgaa
     3241 tcggccaacg cgcggggaga ggc
//
