LOCUS       sequence-199861-        4492 bp    DNA     circular SYN 15-DEC-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 4492)
  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 4492)
  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"; 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..4492
                     /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"
     primer_bind     complement(1816..1835)
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        complement(1817..1835)
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     primer_bind     complement(1840..1856)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(1840..1856)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     CDS             2287..3078
                     /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      3171..3759
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     3660..3679
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     3913..3930
                     /label=L4440
                     /note="L4440 vector, forward primer"
     terminator      complement(4089..4116)
                     /label=rrnB T2 terminator
                     /note="transcription terminator T2 from the E. coli rrnB 
                     gene"
     terminator      complement(4208..4294)
                     /gene="Escherichia coli rrnB"
                     /label=rrnB T1 terminator
                     /note="transcription terminator T1 from the E. coli rrnB 
                     gene"
     primer_bind     4343..4365
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     primer_bind     4357..4374
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     4358..4374
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     protein_bind    4389..4484
                     /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 tcatagctgt ttcctggcag
     1861 ctctggcccg tgtctcaaaa tctctgatgt tacattgcac aagataaaaa tatatcatca
     1921 tgcctcctct ggaccagcca ggacagaaat gcctcgactt cgctgctgcc caaggttgcc
     1981 gggtgacgca caccgtggaa acggatgaag gcacgaaccc agtggacata agcctgttcg
     2041 gttcgtaagc tgtaatgcaa gtagcgtatg cgctcacgca actggtccag aaccttgacc
     2101 gaacgcagcg gtggtaacgg cgcagtggcg gttttcatgg cttgttatga ctgttttttt
     2161 ggggtacagt ctatgcctcg ggcatccaag cagcaagcgc gttacgccgt gggtcgatgt
     2221 ttgatgttat ggagcagcaa cgatgttacg cagcagggca gtcgccctaa aacaaagtta
     2281 aacatcatga gggaagcggt gatcgccgaa gtatcgactc aactatcaga ggtagttggc
     2341 gtcatcgagc gccatctcga accgacgttg ctggccgtac atttgtacgg ctccgcagtg
     2401 gatggcggcc tgaagccaca cagtgatatt gatttgctgg ttacggtgac cgtaaggctt
     2461 gatgaaacaa cgcggcgagc tttgatcaac gaccttttgg aaacttcggc ttcccctgga
     2521 gagagcgaga ttctccgcgc tgtagaagtc accattgttg tgcacgacga catcattccg
     2581 tggcgttatc cagctaagcg cgaactgcaa tttggagaat ggcagcgcaa tgacattctt
     2641 gcaggtatct tcgagccagc cacgatcgac attgatctgg ctatcttgct gacaaaagca
     2701 agagaacata gcgttgcctt ggtaggtcca gcggcggagg aactctttga tccggttcct
     2761 gaacaggatc tatttgaggc gctaaatgaa accttaacgc tatggaactc gccgcccgac
     2821 tgggctggcg atgagcgaaa tgtagtgctt acgttgtccc gcatttggta cagcgcagta
     2881 accggcaaaa tcgcgccgaa ggatgtcgct gccgactggg caatggagcg cctgccggcc
     2941 cagtatcagc ccgtcatact tgaagctaga caggcttatc ttggacaaga agaagatcgc
     3001 ttggcctcgc gcgcagatca gttggaagaa tttgtccact acgtgaaagg cgagatcacc
     3061 aaggtagtcg gcaaataacc ctcgagccac ccatgaccaa aatcccttaa cgtgagttac
     3121 gcgtcgttcc actgagcgtc agaccccgta gaaaagatca aaggatcttc ttgagatcct
     3181 ttttttctgc gcgtaatctg ctgcttgcaa acaaaaaaac caccgctacc agcggtggtt
     3241 tgtttgccgg atcaagagct accaactctt tttccgaagg taactggctt cagcagagcg
     3301 cagataccaa atactgtcct tctagtgtag ccgtagttag gccaccactt caagaactct
     3361 gtagcaccgc ctacatacct cgctctgcta atcctgttac cagtggctgc tgccagtggc
     3421 gataagtcgt gtcttaccgg gttggactca agacgatagt taccggataa ggcgcagcgg
     3481 tcgggctgaa cggggggttc gtgcacacag cccagcttgg agcgaacgac ctacaccgaa
     3541 ctgagatacc tacagcgtga gcattgagaa agcgccacgc ttcccgaagg gagaaaggcg
     3601 gacaggtatc cggtaagcgg cagggtcgga acaggagagc gcacgaggga gcttccaggg
     3661 ggaaacgcct ggtatcttta tagtcctgtc gggtttcgcc acctctgact tgagcgtcga
     3721 tttttgtgat gctcgtcagg ggggcggagc ctatggaaaa acgccagcaa cgcggccttt
     3781 ttacggttcc tggccttttg ctggcctttt gctcacatgt tctttcctgc gttatcccct
     3841 gattctgtgg ataaccgtat taccgccttt gagtgagctg ataccgctcg ccgcagccga
     3901 acgaccgagc gcagcgagtc agtgagcgag gaagcggaag agcgcccaat acgcaaaccg
     3961 cctctccccg cgcgttggcc gattcattaa tgcagctggc acgacaggtt tcccgactgg
     4021 aaagcgggca gtgagcgcaa cgcaattaat acgcgtaccg ctcgccagga agagtttgta
     4081 gaaacgcaaa aaggccatcc gtcaggatgg ccttctgctt agtttgatgc ctggcagttt
     4141 atggcgggcg tcctgcccgc caccctccgg gccgttgctt cacaacgttc aaatccgctc
     4201 ccggcggatt tgtcctactc aggagagcgt tcaccgacaa acaacagata aaacgaaagg
     4261 cccagtcttc cgactgagcc tttcgtttta tttgatgcct ggcagttccc tactctcgcg
     4321 ttaacgcttg catggatgtt ttcccagtca cgacgttgta aaacgacggc cagtcttaag
     4381 ctcgggccca ataatgattt tattttgact gatagtgacc tgttcgttgc aacaaattga
     4441 tgagcaatgc ttttttataa tgccaacttt gtatacaaaa gttgccccat gg
//
