LOCUS       sequence-203133-        4527 bp    DNA     circular SYN 15-APR-2025
DEFINITION  Express two TALE-VP64 activator linked by in-frame T2A (encoding 
            Thosea asigna 'self-cleaving' 2A peptide) sequence under 2x35S 
            promoter.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 4527)
  AUTHORS   Lowder LG, Zhou J, Zhang Y, Malzahn A, Zhong Z, Hsieh TF, Voytas DF,
            Zhang Y, Qi Y
  TITLE     Robust transcriptional activation in plants using multiplexed 
            CRISPR-Act2.0 and mTALE-Act systems.
  JOURNAL   Mol Plant. 2017 Nov 29. pii: S1674-2052(17)30344-1. doi: 
            10.1016/j.molp.2017.11.010.
  PUBMED    29197638
REFERENCE   2  (bases 1 to 4527)
  AUTHORS   .
  TITLE     Direct Submission
  JOURNAL   Exported Apr 15, 2025 from SnapGene Server 8.0.1
            https://www.snapgene.com
COMMENT     SGRef: number: 1; type: "Journal Article"; journalName: "Mol Plant. 
            2017 Nov 29. pii: S1674-2052(17)30344-1. doi: 
            10.1016/j.molp.2017.11.010."
COMMENT     Sequence Label: pYPQ127B
FEATURES             Location/Qualifiers
     source          1..4527
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     promoter        427..771
                     /label=CaMV 35S promoter
                     /note="strong constitutive promoter from cauliflower mosaic
                     virus"
     primer_bind     715..735
                     /label=35S promoter
                     /note="CaMV 35S promoter, forward primer"
     CDS             793..816
                     /codon_start=1
                     /product="FLAG(R) epitope tag, followed by an enterokinase 
                     cleavage site"
                     /label=FLAG
                     /translation="DYKDDDDK"
     CDS             820..840
                     /codon_start=1
                     /product="nuclear localization signal of SV40 (simian virus
                     40) large T antigen"
                     /label=SV40 NLS
                     /translation="PKKKRKV"
     CDS             948..968
                     /codon_start=1
                     /product="nuclear localization signal of SV40 (simian virus
                     40) large T antigen"
                     /label=SV40 NLS
                     /translation="PKKKRKV"
     CDS             1101..1154
                     /codon_start=1
                     /product="2A peptide from Thosea asigna virus capsid 
                     protein"
                     /label=T2A
                     /note="Eukaryotic ribosomes fail to insert a peptide bond 
                     between the Gly and Pro residues, yielding separate 
                     polypeptides."
                     /translation="EGRGSLLTCGDVEENPGP"
     CDS             1161..1187
                     /codon_start=1
                     /product="HA (human influenza hemagglutinin) epitope tag"
                     /label=HA
                     /translation="YPYDVPDYA"
     CDS             1188..1208
                     /codon_start=1
                     /product="nuclear localization signal of SV40 (simian virus
                     40) large T antigen"
                     /label=SV40 NLS
                     /translation="PKKKRKV"
     CDS             1249..1269
                     /codon_start=1
                     /product="nuclear localization signal of SV40 (simian virus
                     40) large T antigen"
                     /label=SV40 NLS
                     /translation="PKKKRKV"
     terminator      1415..1667
                     /label=NOS terminator
                     /note="nopaline synthase terminator and poly(A) signal"
     protein_bind    complement(1700..1799)
                     /gene="mutant version of attL"
                     /label=attL2
                     /bound_moiety="LR Clonase(TM)"
                     /note="recombination site for the Gateway(R) LR reaction"
     promoter        complement(1817..1835)
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     promoter        complement(1852..1870)
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     primer_bind     complement(1875..1891)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(1875..1891)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     CDS             2322..3113
                     /codon_start=1
                     /gene="aadA"
                     /product="aminoglycoside adenylyltransferase (Murphy, 
                     1985)"
                     /label=SmR
                     /note="confers resistance to spectinomycin and 
                     streptomycin"
                     /translation="MREAVIAEVSTQLSEVVGVIERHLEPTLLAVHLYGSAVDGGLKPH
                     SDIDLLVTVTVRLDETTRRALINDLLETSASPGESEILRAVEVTIVVHDDIIPWRYPAK
                     RELQFGEWQRNDILAGIFEPATIDIDLAILLTKAREHSVALVGPAAEELFDPVPEQDLF
                     EALNETLTLWNSPPDWAGDERNVVLTLSRIWYSAVTGKIAPKDVAADWAMERLPAQYQP
                     VILEARQAYLGQEEDRLASRADQLEEFVHYVKGEITKVVGK"
     rep_origin      3206..3794
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     3695..3714
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     3948..3965
                     /label=L4440
                     /note="L4440 vector, forward primer"
     terminator      complement(4124..4151)
                     /label=rrnB T2 terminator
                     /note="transcription terminator T2 from the E. coli rrnB 
                     gene"
     terminator      complement(4243..4329)
                     /gene="Escherichia coli rrnB"
                     /label=rrnB T1 terminator
                     /note="transcription terminator T1 from the E. coli rrnB 
                     gene"
     primer_bind     4378..4400
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     primer_bind     4392..4409
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     4393..4409
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     protein_bind    4424..4519
                     /gene="mutant version of attL"
                     /label=attL5
                     /bound_moiety="LR Clonase(TM)"
                     /note="recombination site for the Gateway(R) LR reaction"
ORIGIN
        1 gcgaagcttg gcgtgcctgc aggtcaacat ggtggagcac gacacacttg tctactccaa
       61 aaatatcaaa gatacagtct cagaagacca aagggcaatt gagacttttc aacaaagggt
      121 aatatccgga aacctcctcg gattccattg cccagctatc tgtcacttta ttgtgaagat
      181 agtggaaaag gaaggtggct cctacaaatg ccatcattgc gataaaggaa aggccatcgt
      241 tgaagatgcc tctgccgaca gtggtcccaa agatggaccc ccacccacga ggagcatcgt
      301 ggaaaaagaa gacgttccaa ccacgtcttc aaagcaagtg gattgatgtg ataacatggt
      361 ggagcacgac acacttgtct actccaaaaa tatcaaagat acagtctcag aagaccaaag
      421 ggcaattgag acttttcaac aaagggtaat atccggaaac ctcctcggat tccattgccc
      481 agctatctgt cactttattg tgaagatagt ggaaaaggaa ggtggctcct acaaatgcca
      541 tcattgcgat aaaggaaagg ccatcgttga agatgcctct gccgacagtg gtcccaaaga
      601 tggaccccca cccacgagga gcatcgtgga aaaagaagac gttccaacca cgtcttcaaa
      661 gcaagtggat tgatgtgata tctccactga cgtaagggat gacgcacaat cccactatcc
      721 ttcgcaagac ccttcctcta tataaggaag ttcatttcat ttggagagga cctcgactct
      781 ggtaccgata tggactacaa agacgatgac gataaagctc ctaagaaaaa gcgcaaagtc
      841 ccctctagat aacgcaggat ccgtaagttt atcagttaaa tataataaat aaagaagaaa
      901 accaaaaaaa tggctaacta aaacgatggt cttatgattt tatgcagcca aaaaagaaac
      961 gtaaggttga tgctcttgac gattttgacc tcgatatgct cgacgctctt gatgattttg
     1021 atctcgacat gctcgatgca cttgatgact ttgaccttga catgctcgac gcactcgatg
     1081 acttcgacct cgacatgctt gagggcagag gaagtcttct aacatgcggt gacgtggagg
     1141 agaatcccgg ccctatcgat tacccttatg atgtgccaga ctacgctcct aaaaagaagc
     1201 gcaaagttgg cattcacggg gtgccggcta gctaataggc ttagatctcc aaaaaagaaa
     1261 cgtaaggttg atgctcttga cgattttgac ctcgatatgc tcgacgctct tgatgatttt
     1321 gatctcgaca tgctcgatgc acttgatgac tttgaccttg acatgctcga cgcactcgat
     1381 gacttcgacc tcgacatgct ttaataggac gtccgatcgt tcaaacattt ggcaataaag
     1441 tttcttaaga ttgaatcctg ttgccggtct tgcgatgatt atcatataat ttctgttgaa
     1501 ttacgttaag catgtaataa ttaacatgta atgcatgacg ttatttatga gatgggtttt
     1561 tatgattaga gtcccgcaat tatacattta atacgcgata gaaaacaaaa tatagcgcgc
     1621 aaactaggat aaattatcga aagcggtgtc atctatgtta ctagatcggg aattgatccc
     1681 actagtaagg gcgaattcga cccagctttc ttgtacaaag ttggcattat aaaaaataat
     1741 tgctcatcaa tttgttgcaa cgaacaggtc actatcagtc aaaataaaat cattatttgc
     1801 catccagctg atatccccta tagtgagtcg tattacatgg tcatagcatc ccctatagtg
     1861 agtcgtatta catggtcata gctgtttcct ggcagctctg gcccgtgtct caaaatctct
     1921 gatgttacat tgcacaagat aaaaatatat catcatgcct cctctggacc agccaggaca
     1981 gaaatgcctc gacttcgctg ctacccaagg ttgccgggtg acgcacaccg tggaaacgga
     2041 tgaaggcacg aacccagtgg acataagcct gttcggttcg taagctgtaa tgcaagtagc
     2101 gtatgcgctc acgcaactgg tccagaacct tgaccgaacg cagcggtggt aacggcgcag
     2161 tggcggtttt catggcttgt tatgactgtt tttttggggt acagtctatg cctcgggcat
     2221 ccaagcagca agcgcgttac gccgtgggtc gatgtttgat gttatggagc agcaacgatg
     2281 ttacgcagca gggcagtcgc cctaaaacaa agttaaacat tatgagggaa gcggtgatcg
     2341 ccgaagtatc gactcaacta tcagaggtag ttggcgtcat cgagcgccat ctcgaaccga
     2401 cgttgctggc cgtacatttg tacggctccg cagtggatgg cggcctgaag ccacacagtg
     2461 atattgattt gctggttacg gtgaccgtaa ggcttgatga aacaacgcgg cgagctttga
     2521 tcaacgacct tttggaaact tcggcttccc ctggagagag cgagattctc cgcgctgtag
     2581 aagtcaccat tgttgtgcac gacgacatca ttccgtggcg ttatccagct aagcgcgaac
     2641 tgcaatttgg agaatggcag cgcaatgaca ttcttgcagg tatcttcgag ccagccacga
     2701 tcgacattga tctggctatc ttgctgacaa aagcaagaga acatagcgtt gccttggtag
     2761 gtccagcggc ggaggaactc tttgatccgg ttcctgaaca ggatctattt gaggcgctaa
     2821 atgaaacctt aacgctatgg aactcgccgc ccgactgggc tggcgatgag cgaaatgtag
     2881 tgcttacgtt gtcccgcatt tggtacagcg cagtaaccgg caaaatcgcg ccgaaggatg
     2941 tcgctgccga ctgggcaatg gagcgcctgc cggcccagta tcagcccgtc atacttgaag
     3001 ctagacaggc ttatcttgga caagaagaag atcgcttggc ctcgcgcgca gatcagttgg
     3061 aagaatttgt ccactacgtg aaaggcgaga tcaccaaggt agtcggcaaa taaccctcga
     3121 gccacccatg accaaaatcc cttaacgtga gttacgcgtc gttccactga gcgtcagacc
     3181 ccgtagaaaa gatcaaagga tcttcttgag atcctttttt tctgcgcgta atctgctgct
     3241 tgcaaacaaa aaaaccaccg ctaccagcgg tggtttgttt gccggatcaa gagctaccaa
     3301 ctctttttcc gaaggtaact ggcttcagca gagcgcagat accaaatact gttcttctag
     3361 tgtagccgta gttaggccac cacttcaaga actctgtagc accgcctaca tacctcgctc
     3421 tgctaatcct gttaccagtg gctgctgcca gtggcgataa gtcgtgtctt accgggttgg
     3481 actcaagacg atagttaccg gataaggcgc agcggtcggg ctgaacgggg ggttcgtgca
     3541 cacagcccag cttggagcga acgacctaca ccgaactgag atacctacag cgtgagctat
     3601 gagaaagcgc cacgcttccc gaagggagaa aggcggacag gtatccggta agcggcaggg
     3661 tcggaacagg agagcgcacg agggagcttc cagggggaaa cgcctggtat ctttatagtc
     3721 ctgtcgggtt tcgccacctc tgacttgagc gtcgattttt gtgatgctcg tcaggggggc
     3781 ggagcctatg gaaaaacgcc agcaacgcgg cctttttacg gttcctggcc ttttgctggc
     3841 cttttgctca catgttcttt cctgcgttat cccctgattc tgtggataac cgtattaccg
     3901 cctttgagtg agctgatacc gctcgccgca gccgaacgac cgagcgcagc gagtcagtga
     3961 gcgaggaagc ggaagagcgc ccaatacgca aaccgcctct ccccgcgcgt tggccgattc
     4021 attaatgcag ctggcacgac aggtttcccg actggaaagc gggcagtgag cgcaacgcaa
     4081 ttaatacgcg taccgcgagc caggaagagt ttgtagaaac gcaaaaaggc catccgtcag
     4141 gatggccttc tgcttagttt gatgcctggc agtttatggc gggcgtcctg cccgccaccc
     4201 tccgggccgt tgcttcacaa cgttcaaatc cgctcccggc ggatttgtcc tactcaggag
     4261 agcgttcacc gacaaacaac agataaaacg aaaggcccag tcttccgact gagcctttcg
     4321 ttttatttga tgcctggcag ttccctactc tcgcgttaac gcttgcatgg atgttttccc
     4381 agtcacgacg ttgtaaaacg acggccagtc ttaagctcgg gcccaataat gattttattt
     4441 tgactgatag tgacctgttc gttgcaacaa attgatgagc aatgcttttt tataatgcca
     4501 actttgtata caaaagttgc cccatgg
//
