LOCUS       sequence-331276-        3694 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Golden Gate entry vector to express the 4th 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 3694)
  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 3694)
  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: pYPQ134B2.0
FEATURES             Location/Qualifiers
     source          1..3694
                     /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        2105..2123
                     /label=MS2 stem loop
                     /note="stem loop that binds the bacteriophage MS2 coat 
                     protein"
     misc_RNA        2175..2193
                     /label=MS2 stem loop
                     /note="stem loop that binds the bacteriophage MS2 coat 
                     protein"
     rep_origin      2507..3095
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     2996..3015
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     terminator      complement(3425..3452)
                     /label=rrnB T2 terminator
                     /note="transcription terminator T2 from the E. coli rrnB 
                     gene"
     terminator      complement(3544..3630)
                     /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 ctcgccagtt tactttaaat
     1741 ttttcttatg gctcagcctg tgatggataa ctgaatcaaa caaatggcgt ctgggtttaa
     1801 gaacatctgt tttggctatg ttggacgaaa caagtgaact tttaggatca acttccgttt
     1861 atatacggag cttatatcga gcaataagat aagtgggctt tttatgtaat ttaatgggct
     1921 atcgtccata tattcactaa tacccatgcc cagtacccat gtatgcgttt catataagct
     1981 cctaatttct cccacatcgc tcaaatctaa acaaatcttg ttgtatatat aacactgagg
     2041 gagcaccatt ggtcagagac gagatctagt ctgagtcgac cgtctctgtt ttagagctag
     2101 gccaacatga ggatcaccca tgtctgcagg gcctagcaag ttaaaataag gctagtccgt
     2161 tatcaacttg gccaacatga ggatcaccca tgtctgcagg gccaagtggc accgagtcgg
     2221 tgcttttttt tccctttcct tttttctttt ttttgccata aacttaaatt tgtatatcga
     2281 tcattgtaga tattgaaaac ctagaacaaa ccaacatcca tgtgaatgtc tttcatgact
     2341 gatttagaga taattcttga attttggaac tagaatctat aatgagccta aattaaaaca
     2401 ttgtgtgttg gagaccctcg agccacccat gaccaaaatc ccttaacgtg agttacgcgt
     2461 cgttccactg agcgtcagac cccgtagaaa agatcaaagg atcttcttga gatccttttt
     2521 ttctgcgcgt aatctgctgc ttgcaaacaa aaaaaccacc gctaccagcg gtggtttgtt
     2581 tgccggatca agagctacca actctttttc cgaaggtaac tggcttcagc agagcgcaga
     2641 taccaaatac tgttcttcta gtgtagccgt agttaggcca ccacttcaag aactctgtag
     2701 caccgcctac atacctcgct ctgctaatcc tgttaccagt ggctgctgcc agtggcgata
     2761 agtcgtgtct taccgggttg gactcaagac gatagttacc ggataaggcg cagcggtcgg
     2821 gctgaacggg gggttcgtgc acacagccca gcttggagcg aacgacctac accgaactga
     2881 gatacctaca gcgtgagcta tgagaaagcg ccacgcttcc cgaagggaga aaggcggaca
     2941 ggtatccggt aagcggcagg gtcggaacag gagagcgcac gagggagctt ccagggggaa
     3001 acgcctggta tctttatagt cctgtcgggt ttcgccacct ctgacttgag cgtcgatttt
     3061 tgtgatgctc gtcagggggg cggagcctat ggaaaaacgc cagcaacgcg gcctttttac
     3121 ggttcctggc cttttgctgg ccttttgctc acatgttctt tcctgcgtta tcccctgatt
     3181 ctgtggataa ccgtattacc gcctttgagt gagctgatac cgctcgccgc agccgaacga
     3241 ccgagcgcag cgagtcagtg agcgaggaag cggaagagcg cccaatacgc aaaccgcctc
     3301 tccccgcgcg ttggccgatt cattaatgca gctggcacga caggtttccc gactggaaag
     3361 cgggcagtga gcgcaacgca attaatacgc gtaccgctag ccaggaagag tttgtagaaa
     3421 cgcaaaaagg ccatccgtca ggatggcctt ctgcttagtt tgatgcctgg cagtttatgg
     3481 cgggcgtcct gcccgccacc ctccgggccg ttgcttcaca acgttcaaat ccgctcccgg
     3541 cggatttgtc ctactcagga gagcgttcac cgacaaacaa cagataaaac gaaaggccca
     3601 gtcttccgac tgagcctttc gttttatttg atgcctggca gttccctact ctcgcgttct
     3661 gcattaatga atcggccaac gcgcggggag aggc
//
