LOCUS       sequence-331282-        3699 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Golden Gate entry vector to express the 6th gRNA with gRNA2.0 
            scaffold (with four MS2 binding sites) under AtU3 promoter.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 3699)
  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 3699)
  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: pYPQ136B2.0
FEATURES             Location/Qualifiers
     source          1..3699
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     promoter        join(206..211,212..228,229..234)
                     /label=tet promoter
                     /note="E. coli promoter for tetracycline efflux protein 
                     gene"
     CDS             282..1472
                     /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(634..653)
                     /label=pBRrevBam
                     /note="pBR322 vectors, tet region, downstream of BamHI, 
                     reverse primer"
     primer_bind     complement(1646..1665)
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        complement(1647..1665)
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     primer_bind     complement(1670..1686)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(1670..1686)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     misc_RNA        2153..2171
                     /label=MS2 stem loop
                     /note="stem loop that binds the bacteriophage MS2 coat 
                     protein"
     misc_RNA        2223..2241
                     /label=MS2 stem loop
                     /note="stem loop that binds the bacteriophage MS2 coat 
                     protein"
     rep_origin      2558..3146
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     3047..3066
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     terminator      complement(3476..3503)
                     /label=rrnB T2 terminator
                     /note="transcription terminator T2 from the E. coli rrnB 
                     gene"
     terminator      complement(3595..3681)
                     /gene="Escherichia coli rrnB"
                     /label=rrnB T1 terminator
                     /note="transcription terminator T1 from the E. coli rrnB 
                     gene"
ORIGIN
        1 ctctcgcgtt ctgcattaat gaatcggcca acgcgcgggg agaggcggtt tgcgtattgg
       61 gcgctcttcc gcttcctcgc tcactgactc gctgcgctcg gtcgttcggc tgcggcgagc
      121 ggtatcagct cactcaaagg cggtaatacg gttatccaca gaatcagggg ataacgcagg
      181 aaagaacatg aattaattct catgtttgac agcttatcat cgattagctt taatgcggta
      241 gtttatcaca gttaaattgc taacgcagtc aggcaccgtg tatgaaatct aacaatgcgc
      301 tcatcgtcat cctcggcacc gtcaccctgg atgctgtagg cataggcttg gttatgccgg
      361 tactgccggg cctcttgcgg gatatcgtcc attccgacag catcgccagt cactatggcg
      421 tgctgctagc gctatatgcg ttgatgcaat ttctatgcgc acccgttctc ggagcactgt
      481 ccgaccgctt tggccgccgc ccagtcctgc tcgcttcgct acttggagcc actatcgact
      541 acgcgatcat ggcgaccaca cccgtcctgt ggattctcta cgccggacgc atcgtggccg
      601 gcatcaccgg cgccacaggt gcggttgctg gcgcctatat cgccgacatc accgatgggg
      661 aagatcgggc tcgccacttc gggctcatga gcgcttgttt cggcgtgggt atggtggcag
      721 gccccgtggc cgggggactg ttgggcgcca tctccttaca tgcaccattc cttgcggcgg
      781 cggtgctcaa cggcctcaac ctactactgg gctgcttcct aatgcaggag tcgcataagg
      841 gagagcgccg acccatgccc ttgagagcct tcaacccagt cagctccttc cggtgggcgc
      901 ggggcatgac tatcgtcgcc gcacttatga ctgtcttctt tatcatgcaa ctcgtaggac
      961 aggtgccggc agcgctctgg gtcattttcg gcgaggaccg ctttcgctgg agcgcgacga
     1021 tgatcggcct gtcgcttgcg gtattcggaa tcttgcacgc cctcgctcaa gccttcgtca
     1081 ctggtcccgc caccaaacgt ttcggcgaga agcaggccat tatcgccggc atggcggccg
     1141 acgcgctggg ctacgtcttg ctggcgttcg cgacgcgagg ctggatggcc ttccccatta
     1201 tgattcttct cgcttccggc ggcatcggga tgcccgcgtt gcaggccatg ctgtccaggc
     1261 aggtagatga cgaccatcag ggacagcttc aaggatcgct cgcggctctt accagcctaa
     1321 cttcgatcat tggaccgctg atcgtcacgg cgatttatgc cgcctcggcg agcacatgga
     1381 acgggttggc atggattgta ggcgccgccc tataccttgt ctgcctcccc gcgttgcgtc
     1441 gcggtgcatg gagccgggcc acctcgacct gaatggaagc cggcggcacc tcgctaacgg
     1501 attcaccact ccaagaattg gagccaatca attcttgcgg agaactgtga atgcgcaaac
     1561 caacccttga tcggggaaga acagtatgtc gagctatttt ttgacttact ggggatcaag
     1621 cctgattggg agaaaataaa atatccccta tagtgagtcg tattacatgg tcatagctgt
     1681 ttcctggcag ctctggcccg tgtctcaaaa tctctgatgt tacattgcac aagataaaaa
     1741 tatatcatca tgcctcctct agaggtctcg tgcaagttta ctttaaattt ttcttatggc
     1801 tcagcctgtg atggataact gaatcaaaca aatggcgtct gggtttaaga acatctgttt
     1861 tggctatgtt ggacgaaaca agtgaacttt taggatcaac ttccgtttat atacggagct
     1921 tatatcgagc aataagataa gtgggctttt tatgtaattt aatgggctat cgtccatata
     1981 ttcactaata cccatgccca gtacccatgt atgcgtttca tataagctcc taatttctcc
     2041 cacatcgctc aaatctaaac aaatcttgtt gtatatataa cactgaggga gcaccattgg
     2101 tcagagacga gatctagtct gagtcgaccg tctctgtttt agagctaggc caacatgagg
     2161 atcacccatg tctgcagggc ctagcaagtt aaaataaggc tagtccgtta tcaacttggc
     2221 caacatgagg atcacccatg tctgcagggc caagtggcac cgagtcggtg cttttttttc
     2281 cctttccttt tttctttttt ttgccataaa cttaaatttg tatatcgatc attgtagata
     2341 ttgaaaacct agaacaaacc aacatccatg tgaatgtctt tcatgactga tttagagata
     2401 attcttgaat tttggaacta gaatctataa tgagcctaaa ttaaaacatt gtgagccggt
     2461 tgagaccctc gagccaccca tgaccaaaat cccttaacgt gagttacgcg tcgttccact
     2521 gagcgtcaga ccccgtagaa aagatcaaag gatcttcttg agatcctttt tttctgcgcg
     2581 taatctgctg cttgcaaaca aaaaaaccac cgctaccagc ggtggtttgt ttgccggatc
     2641 aagagctacc aactcttttt ccgaaggtaa ctggcttcag cagagcgcag ataccaaata
     2701 ctgttcttct agtgtagccg tagttaggcc accacttcaa gaactctgta gcaccgccta
     2761 catacctcgc tctgctaatc ctgttaccag tggctgctgc cagtggcgat aagtcgtgtc
     2821 ttaccgggtt ggactcaaga cgatagttac cggataaggc gcagcggtcg ggctgaacgg
     2881 ggggttcgtg cacacagccc agcttggagc gaacgaccta caccgaactg agatacctac
     2941 agcgtgagct atgagaaagc gccacgcttc ccgaagggag aaaggcggac aggtatccgg
     3001 taagcggcag ggtcggaaca ggagagcgca cgagggagct tccaggggga aacgcctggt
     3061 atctttatag tcctgtcggg tttcgccacc tctgacttga gcgtcgattt ttgtgatgct
     3121 cgtcaggggg gcggagccta tggaaaaacg ccagcaacgc ggccttttta cggttcctgg
     3181 ccttttgctg gccttttgct cacatgttct ttcctgcgtt atcccctgat tctgtggata
     3241 accgtattac cgcctttgag tgagctgata ccgctcgccg cagccgaacg accgagcgca
     3301 gcgagtcagt gagcgaggaa gcggaagagc gcccaatacg caaaccgcct ctccccgcgc
     3361 gttggccgat tcattaatgc agctggcacg acaggtttcc cgactggaaa gcgggcagtg
     3421 agcgcaacgc aattaatacg cgtaccgcta gccaggaaga gtttgtagaa acgcaaaaag
     3481 gccatccgtc aggatggcct tctgcttagt ttgatgcctg gcagtttatg gcgggcgtcc
     3541 tgcccgccac cctccgggcc gttgcttcac aacgttcaaa tccgctcccg gcggatttgt
     3601 cctactcagg agagcgttca ccgacaaaca acagataaaa cgaaaggccc agtcttccga
     3661 ctgagccttt cgttttattt gatgcctggc agttcccta
//
