LOCUS       sequence-331281-        3694 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Golden Gate entry vector to express the 5th 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: pYPQ135B2.0
FEATURES             Location/Qualifiers
     source          1..3694
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     terminator      complement(44..130)
                     /gene="Escherichia coli rrnB"
                     /label=rrnB T1 terminator
                     /note="transcription terminator T1 from the E. coli rrnB 
                     gene"
     promoter        join(354..359,360..376,377..382)
                     /label=tet promoter
                     /note="E. coli promoter for tetracycline efflux protein 
                     gene"
     CDS             430..1620
                     /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(782..801)
                     /label=pBRrevBam
                     /note="pBR322 vectors, tet region, downstream of BamHI, 
                     reverse primer"
     primer_bind     complement(1794..1813)
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        complement(1795..1813)
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     primer_bind     complement(1818..1834)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(1818..1834)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     misc_RNA        2299..2317
                     /label=MS2 stem loop
                     /note="stem loop that binds the bacteriophage MS2 coat 
                     protein"
     misc_RNA        2369..2387
                     /label=MS2 stem loop
                     /note="stem loop that binds the bacteriophage MS2 coat 
                     protein"
     rep_origin      2701..3289
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     3190..3209
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     terminator      complement(3619..3646)
                     /label=rrnB T2 terminator
                     /note="transcription terminator T2 from the E. coli rrnB 
                     gene"
ORIGIN
        1 ctccgggccg ttgcttcaca acgttcaaat ccgctcccgg cggatttgtc ctactcagga
       61 gagcgttcac cgacaaacaa cagataaaac gaaaggccca gtcttccgac tgagcctttc
      121 gttttatttg atgcctggca gttccctact ctcgcgttct gcattaatga atcggccaac
      181 gcgcggggag aggcggtttg cgtattgggc gctcttccgc ttcctcgctc actgactcgc
      241 tgcgctcggt cgttcggctg cggcgagcgg tatcagctca ctcaaaggcg gtaatacggt
      301 tatccacaga atcaggggat aacgcaggaa agaacatgaa ttaattctca tgtttgacag
      361 cttatcatcg attagcttta atgcggtagt ttatcacagt taaattgcta acgcagtcag
      421 gcaccgtgta tgaaatctaa caatgcgctc atcgtcatcc tcggcaccgt caccctggat
      481 gctgtaggca taggcttggt tatgccggta ctgccgggcc tcttgcggga tatcgtccat
      541 tccgacagca tcgccagtca ctatggcgtg ctgctagcgc tatatgcgtt gatgcaattt
      601 ctatgcgcac ccgttctcgg agcactgtcc gaccgctttg gccgccgccc agtcctgctc
      661 gcttcgctac ttggagccac tatcgactac gcgatcatgg cgaccacacc cgtcctgtgg
      721 attctctacg ccggacgcat cgtggccggc atcaccggcg ccacaggtgc ggttgctggc
      781 gcctatatcg ccgacatcac cgatggggaa gatcgggctc gccacttcgg gctcatgagc
      841 gcttgtttcg gcgtgggtat ggtggcaggc cccgtggccg ggggactgtt gggcgccatc
      901 tccttacatg caccattcct tgcggcggcg gtgctcaacg gcctcaacct actactgggc
      961 tgcttcctaa tgcaggagtc gcataaggga gagcgccgac ccatgccctt gagagccttc
     1021 aacccagtca gctccttccg gtgggcgcgg ggcatgacta tcgtcgccgc acttatgact
     1081 gtcttcttta tcatgcaact cgtaggacag gtgccggcag cgctctgggt cattttcggc
     1141 gaggaccgct ttcgctggag cgcgacgatg atcggcctgt cgcttgcggt attcggaatc
     1201 ttgcacgccc tcgctcaagc cttcgtcact ggtcccgcca ccaaacgttt cggcgagaag
     1261 caggccatta tcgccggcat ggcggccgac gcgctgggct acgtcttgct ggcgttcgcg
     1321 acgcgaggct ggatggcctt ccccattatg attcttctcg cttccggcgg catcgggatg
     1381 cccgcgttgc aggccatgct gtccaggcag gtagatgacg accatcaggg acagcttcaa
     1441 ggatcgctcg cggctcttac cagcctaact tcgatcattg gaccgctgat cgtcacggcg
     1501 atttatgccg cctcggcgag cacatggaac gggttggcat ggattgtagg cgccgcccta
     1561 taccttgtct gcctccccgc gttgcgtcgc ggtgcatgga gccgggccac ctcgacctga
     1621 atggaagccg gcggcacctc gctaacggat tcaccactcc aagaattgga gccaatcaat
     1681 tcttgcggag aactgtgaat gcgcaaacca acccttgatc ggggaagaac agtatgtcga
     1741 gctatttttt gacttactgg ggatcaagcc tgattgggag aaaataaaat atcccctata
     1801 gtgagtcgta ttacatggtc atagctgttt cctggcagct ctggcccgtg tctcaaaatc
     1861 tctgatgtta cattgcacaa gataaaaata tatcatcatg cctcctctag aggtctcctg
     1921 tttttacttt aaatttttct tatggctcag cctgtgatgg ataactgaat caaacaaatg
     1981 gcgtctgggt ttaagaacat ctgttttggc tatgttggac gaaacaagtg aacttttagg
     2041 atcaacttcc gtttatatac ggagcttata tcgagcaata agataagtgg gctttttatg
     2101 taatttaatg ggctatcgtc catatattca ctaataccca tgcccagtac ccatgtatgc
     2161 gtttcatata agctcctaat ttctcccaca tcgctcaaat ctaaacaaat cttgttgtat
     2221 atataacact gagggagcac cattggtcag agacgagatc tagtctgagt cgaccgtctc
     2281 tgttttagag ctaggccaac atgaggatca cccatgtctg cagggcctag caagttaaaa
     2341 taaggctagt ccgttatcaa cttggccaac atgaggatca cccatgtctg cagggccaag
     2401 tggcaccgag tcggtgcttt tttttccctt tccttttttc ttttttttgc cataaactta
     2461 aatttgtata tcgatcattg tagatattga aaacctagaa caaaccaaca tccatgtgaa
     2521 tgtctttcat gactgattta gagataattc ttgaattttg gaactagaat ctataatgag
     2581 cctaaattaa aacattgtgt gcaggagacc ctcgagccac ccatgaccaa aatcccttaa
     2641 cgtgagttac gcgtcgttcc actgagcgtc agaccccgta gaaaagatca aaggatcttc
     2701 ttgagatcct ttttttctgc gcgtaatctg ctgcttgcaa acaaaaaaac caccgctacc
     2761 agcggtggtt tgtttgccgg atcaagagct accaactctt tttccgaagg taactggctt
     2821 cagcagagcg cagataccaa atactgttct tctagtgtag ccgtagttag gccaccactt
     2881 caagaactct gtagcaccgc ctacatacct cgctctgcta atcctgttac cagtggctgc
     2941 tgccagtggc gataagtcgt gtcttaccgg gttggactca agacgatagt taccggataa
     3001 ggcgcagcgg tcgggctgaa cggggggttc gtgcacacag cccagcttgg agcgaacgac
     3061 ctacaccgaa ctgagatacc tacagcgtga gctatgagaa agcgccacgc ttcccgaagg
     3121 gagaaaggcg gacaggtatc cggtaagcgg cagggtcgga acaggagagc gcacgaggga
     3181 gcttccaggg ggaaacgcct ggtatcttta tagtcctgtc gggtttcgcc acctctgact
     3241 tgagcgtcga tttttgtgat gctcgtcagg ggggcggagc ctatggaaaa acgccagcaa
     3301 cgcggccttt ttacggttcc tggccttttg ctggcctttt gctcacatgt tctttcctgc
     3361 gttatcccct gattctgtgg ataaccgtat taccgccttt gagtgagctg ataccgctcg
     3421 ccgcagccga acgaccgagc gcagcgagtc agtgagcgag gaagcggaag agcgcccaat
     3481 acgcaaaccg cctctccccg cgcgttggcc gattcattaa tgcagctggc acgacaggtt
     3541 tcccgactgg aaagcgggca gtgagcgcaa cgcaattaat acgcgtaccg ctagccagga
     3601 agagtttgta gaaacgcaaa aaggccatcc gtcaggatgg ccttctgctt agtttgatgc
     3661 ctggcagttt atggcgggcg tcctgcccgc cacc
//
