LOCUS       sequence-322041-        8523 bp    DNA     circular SYN 15-APR-2025
DEFINITION  TU for the constitutive expresion of the viral coat protein MS2 
            fused on C-terminal with the VP64.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 8523)
  AUTHORS   Selma S, Bernabe-Orts JM, Vazquez-Vilar M, Diego-Martin B, Ajenjo M,
            Garcia-Carpintero V, Granell A, Orzaez D
  TITLE     Strong gene activation in plants with genome-wide specificity using 
            a new orthogonal CRISPR/Cas9-based programmable transcriptional 
            activator.
  JOURNAL   Plant Biotechnol J. 2019 Sep;17(9):1703-1705. doi: 
            10.1111/pbi.13138. Epub 2019 May 23.
  PUBMED    31034138
REFERENCE   2  (bases 1 to 8523)
  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"; doi: "10.1111/pbi.13138";
            journalName: "Plant Biotechnol J"; date: "2019-09"; volume: "17"; 
            issue: "9"; pages: "1703-1705"
COMMENT     Sequence Label: pDGB3 alpha2 P35S:MS2-VP64:Tnos (GB1395)
FEATURES             Location/Qualifiers
     source          1..8523
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     promoter        616..960
                     /label=CaMV 35S promoter
                     /note="strong constitutive promoter from cauliflower mosaic
                     virus"
     primer_bind     904..924
                     /label=35S promoter
                     /note="CaMV 35S promoter, forward primer"
     CDS             1040..1426
                     /codon_start=1
                     /product="bacteriophage MS2 coat protein"
                     /label=MS2
                     /note="binds to a specific stem-loop structure in the viral
                     RNA (Peabody, 1993)"
                     /translation="ASNFTQFVLVDNGGTGDVTVAPSNFANGVAEWISSNSRSQAYKVT
                     CSVRQSSAQNRKYTIKVEVPKVATQTVGGVELPVAAWRSYLNMELTIPIFATNSDCELI
                     VKAMQGLLKDGNPIPSAIAANSGIY"
     CDS             1433..1453
                     /codon_start=1
                     /product="nuclear localization signal of SV40 (simian virus
                     40) large T antigen"
                     /label=SV40 NLS
                     /translation="PKKKRKV"
     CDS             1457..1477
                     /codon_start=1
                     /product="nuclear localization signal of SV40 (simian virus
                     40) large T antigen"
                     /label=SV40 NLS
                     /translation="PKKKRKV"
     CDS             1481..1501
                     /codon_start=1
                     /product="nuclear localization signal of SV40 (simian virus
                     40) large T antigen"
                     /label=SV40 NLS
                     /translation="PKKKRKV"
     CDS             1502..1651
                     /codon_start=1
                     /product="tetrameric repeat of the minimal activation 
                     domain of herpes simplex virus VP16 (Beerli et al., 1998)"
                     /label=VP64
                     /translation="DALDDFDLDMLGSDALDDFDLDMLGSDALDDFDLDMLGSDALDDF
                     DLDML"
     terminator      1678..1930
                     /label=NOS terminator
                     /note="nopaline synthase terminator and poly(A) signal"
     primer_bind     complement(1965..1983)
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     misc_feature    2167..2191
                     /label=RB T-DNA repeat
                     /note="right border repeat from nopaline C58 T-DNA"
     CDS             3492..4121
                     /codon_start=1
                     /product="stability protein from the Pseudomonas plasmid 
                     pVS1 (Heeb et al., 2000)"
                     /label=pVS1 StaA
                     /translation="MKVIAVLNQKGGSGKTTIATHLARALQLAGADVLLVDSDPQGSAR
                     DWAAVREDQPLTVVGIDRPTIDRDVKAIGRRDFVVIDGAPQAADLAVSAIKAADFVLIP
                     VQPSPYDIWATADLVELVKQRIEVTDGRLQAAFVVSRAIKGTRIGGEVAEALAGYELPI
                     LESRITQRVSYPGTAAAGTTVLESEPEGDAAREVQALAAEIKSKLI"
     CDS             4550..5623
                     /codon_start=1
                     /product="replication protein from the Pseudomonas plasmid 
                     pVS1 (Heeb et al., 2000)"
                     /label=pVS1 RepA
                     /translation="MSGRKPSGPVQIGAALGDDLVEKLKAAQAAQRQRIEAEARPGESW
                     QAAADRIRKESRQPPAAGAPSIRKPPKGDEQPDFFVPMLYDVGTRDSRSIMDVAVFRLS
                     KRDRRAGEVIRYELPDGHVEVSAGPAGMASVWDYDLVLMAVSHLTESMNRYREGKGDKP
                     GRVFRPHVADVLKFCRRADGGKQKDDLVETCIRLNTTHVAMQRTKKAKNGRLVTVSEGE
                     ALISRYKIVKSETGRPEYIEIELADWMYREITEGKNPDVLTVHPDYFLIDPGIGRFLYR
                     LARRAAGKAEARWLFKTIYERSGSAGEFKKFCFTVRKLIGSNDLPEYDLKEEAGQAGPI
                     LVMRYRNLIEGEASAGS"
     rep_origin      5689..5883
                     /label=pVS1 oriV
                     /note="origin of replication for the Pseudomonas plasmid 
                     pVS1 (Heeb et al., 2000)"
     primer_bind     complement(6119..6141)
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     misc_feature    6227..6367
                     /label=bom
                     /note="basis of mobility region from pBR322"
     primer_bind     6282..6301
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     complement(6382..6399)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(6553..7141)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(6633..6652)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(7229..8044)
                     /codon_start=1
                     /product="aminoglycoside phosphotransferase"
                     /label=KanR
                     /note="confers resistance to kanamycin in bacteria or G418 
                     (Geneticin(R)) in eukaryotes"
                     /translation="MSHIQRETSCSRPRLNSNMDADLYGYKWARDNVGQSGATIYRLYG
                     KPDAPELFLKHGKGSVANDVTDEMVRLNWLTEFMPLPTIKHFIRTPDDAWLLTTAIPGK
                     TAFQVLEEYPDSGENIVDALAVFLRRLHSIPVCNCPFNSDRVFRLAQAQSRMNNGLVDA
                     SDFDDERNGWPVEQVWKEMHKLLPFSPDSVVTHGDFSLDNLIFDEGKLIGCIDVGRVGI
                     ADRYQDLAILWNCLGEFSPSLQKRLFQKYGIDNPDMNKLQFHLMLDEFF"
     CDS             complement(7229..8044)
                     /codon_start=1
                     /gene="aph(3')-Ia"
                     /product="aminoglycoside phosphotransferase"
                     /label=KanR
                     /note="confers resistance to kanamycin in bacteria or G418 
                     (Geneticin(R)) in eukaryotes"
                     /translation="MSHIQRETSCSRPRLNSNMDADLYGYKWARDNVGQSGATIYRLYG
                     KPDAPELFLKHGKGSVANDVTDEMVRLNWLTEFMPLPTIKHFIRTPDDAWLLTTAIPGK
                     TAFQVLEEYPDSGENIVDALAVFLRRLHSIPVCNCPFNSDRVFRLAQAQSRMNNGLVDA
                     SDFDDERNGWPVEQVWKEMHKLLPFSPDSVVTHGDFSLDNLIFDEGKLIGCIDVGRVGI
                     ADRYQDLAILWNCLGEFSPSLQKRLFQKYGIDNPDMNKLQFHLMLDEFF"
     primer_bind     7952..7971
                     /label=Kan-R
                     /note="Kanamycin resistance gene, reverse primer"
     misc_feature    8477..8501
                     /label=LB T-DNA repeat
                     /note="left border repeat from nopaline C58 T-DNA"
ORIGIN
        1 ggagactaga gccaagctga tctcctttgc cccggagatc accatggacg actttctcta
       61 tctctacgat ctaggaagaa agttcgacgg agaaggtgac gataccatgt tcaccaccga
      121 taatgagaag attagcctct tcaatttcag aaagaatgct gacccacaga tggttagaga
      181 ggcctacgcg gcaggtctga tcaagacgat ctacccgagt aataatctcc aggagatcaa
      241 ataccttccc aagaaggtta aagatgcagt caaaagattc aggactaact gcatcaagaa
      301 cacagagaaa gatatatttc tcaagatcag aagtactatt ccagtatgga cgattcaagg
      361 cttgcttcat aaaccaaggc aagtaataga gattggagtc tctaagaaag tagttcctac
      421 tgaatcaaag gccatggagt caaaaattca gatcgaggat ctaacagaac tcgccgtgaa
      481 gactggcgaa cagttcatac agagtctttt acgactcaat gacaagaaga aaatcttcgt
      541 caacatggtg gagcacgaca ctctcgtcta ctccaagaat atcaaagata cagtctcaga
      601 agaccaaagg gctattgaga cttttcaaca aagggtaata tcgggaaacc tcctcggatt
      661 ccattgccca gctatctgtc acttcatcaa aaggacagta gaaaaggaag gtggcaccta
      721 caaatgccat cattgcgata aaggaaaggc tatcgttcaa gatgcccctg ccgacagtgg
      781 tcccaaagat ggacccccac ccacgaggag catcgtggaa aaagaagacg ttccaaccac
      841 gtcttcaaag caagtggatt gatgtgatat ctccactgac gtaagggatg acgcacaatc
      901 ccactatcct tcgcaagacc cttcctctat ataaggaagt tcatttcatt tggagaggac
      961 tccggtattt ttacaacaat taccacaaca aaacaaacaa caaacaacat tacaatttac
     1021 tattctagtc gaaatgatgg cttctaattt tactcaattt gttcttgttg ataatggtgg
     1081 tactggtgat gttactgttg ctccttctaa ttttgctaat ggtgttgctg aatggatttc
     1141 ttctaattct agatctcaag cttataaggt tacttgttct gttagacaat cttctgctca
     1201 aaatagaaag tatactatta aggttgaagt tcctaaggtt gctactcaaa ctgttggtgg
     1261 tgttgaactt cctgttgctg cttggagatc ttatcttaat atggaactta ctattcctat
     1321 ttttgctact aattctgatt gtgaacttat tgttaaggct atgcaaggtc ttcttaagga
     1381 tggtaatcct attccttctg ctattgctgc taattctggt atttattcgg acccaaaaaa
     1441 gaagcgtaag gtcgatccca agaagaagag aaaggtagat cctaagaaga agagaaaggt
     1501 agacgcattg gatgacttcg acttagacat gcttgggtca gatgctttgg acgactttga
     1561 cctcgacatg ttgggttctg acgcgctaga tgacttcgac ttggacatgt tgggctccga
     1621 cgctttggac gactttgatc tggacatgtt atgagcttgg aatggatctt cgatcccgat
     1681 cgttcaaaca tttggcaata aagtttctta agattgaatc ctgttgccgg tcttgcgacg
     1741 attatcatat aatttctgtt gaattacgtt aagcatgtaa taattaacat gtaatgcatg
     1801 acgttattta tgagatgggt ttttatgatt agagtcccgc aattatacat ttaatacgcg
     1861 atagaaaaca aaatatagcg cgcaaactag gataaattat cgcgcgcggt gtcatctatg
     1921 ttactagatc gggaattgcc aagctaattc ttgaagacga aagggcctcg tgatacgcct
     1981 atttttatag gttaatgtca tgataataat ggtttcttag acgtcaggtg gcacttttcg
     2041 gggaaatgtg cgcggaaccc ctatttgttt atttttctaa atacattcaa atatgtatcc
     2101 gctcatgaga caataaccct gataaatgct tcaataatgg gaccgactcg cgcttgagac
     2161 gaagcttgac aggatatatt ggcgggtaaa cctaagagaa aagagcgttt attagaataa
     2221 tcggatattt aaaagggcgt gaaaaggttt atccgttcgt ccatttgtat gtgcatgcca
     2281 accacagggt tcccctcggg atcaaagtac tttgatccaa cccctccgct gctatagtgc
     2341 agtcggcttc tgacgttcag tgcagccgtc atctgaaaac gacatgtcgc acaagtccta
     2401 agttacgcga caggctgccg ccctgccctt ttcctggcgt tttcttgtcg cgtgttttag
     2461 tcgcataaag tagaatactt gcgactagaa ccggagacat tacgccatga acaagagcgc
     2521 cgccgctggc ctgctgggct atgcccgcgt cagcaccgac gaccaggact tgaccaacca
     2581 acgggccgaa ctgcacgcgg ccggctgcac caagctgttt tccgagaaga tcaccggcac
     2641 caggcgcgac cgcccggagc tggccaggat gcttgaccac ctacgccctg gcgacgttgt
     2701 gacagtgacc aggctagacc gcctggcccg cagcacccgc gacctactgg acattgccga
     2761 gcgcatccag gaggccggcg cgggcctgcg tagcctggca gagccgtggg ccgacaccac
     2821 cacgccggcc ggccgcatgg tgttgaccgt gttcgccggc attgccgagt tcgagcgttc
     2881 cctaatcatc gaccgcaccc ggagcgggcg cgaggccgcc aaggcccgag gcgtgaagtt
     2941 tggcccccgc cctaccctca ccccggcaca gatcgcgcac gcccgcgagc tgatcgacca
     3001 ggaaggccgc accgtgaaag aggcggctgc actgcttggc gtgcatcgct cgaccctgta
     3061 ccgcgcactt gagcgcagcg aggaagtgac gcccaccgag gccaggcggc gcggtgcctt
     3121 ccgtgaggac gcattgaccg aggccgacgc cctggcggcc gccgagaatg aacgccaaga
     3181 ggaacaagca tgaaaccgca ccaggacggc caggacgaac cgtttttcat taccgaagag
     3241 atcgaggcgg agatgatcgc ggccgggtac gtgttcgagc cgcccgcgca cctctcaacc
     3301 gtgcggctgc atgaaatcct ggccggtttg tctgatgcca agctggcggc ctggccggcc
     3361 agcttggccg ctgaagaaac cgagcgccgc cgtctaaaaa ggtgatgtgt atttgagtaa
     3421 aacagcttgc gtcatgcggt cgctgcgtat atgatgcgat gagtaaataa acaaatacgc
     3481 aaggggaacg catgaaggtt atcgctgtac ttaaccagaa aggcgggtca ggcaagacga
     3541 ccatcgcaac ccatctagcc cgcgccctgc aactcgccgg ggccgatgtt ctgttagtcg
     3601 attccgatcc ccagggcagt gcccgcgatt gggcggccgt gcgggaagat caaccgctaa
     3661 ccgttgtcgg catcgaccgc ccgacgattg accgcgacgt gaaggccatc ggccggcgcg
     3721 acttcgtagt gatcgacgga gcgccccagg cggcggactt ggctgtgtcc gcgatcaagg
     3781 cagccgactt cgtgctgatt ccggtgcagc caagccctta cgacatatgg gccaccgccg
     3841 acctggtgga gctggttaag cagcgcattg aggtcacgga tggaaggcta caagcggcct
     3901 ttgtcgtgtc gcgggcgatc aaaggcacgc gcatcggcgg tgaggttgcc gaggcgctgg
     3961 ccgggtacga gctgcccatt cttgagtccc gtatcacgca gcgcgtgagc tacccaggca
     4021 ctgccgccgc cggcacaacc gttcttgaat cagaacccga gggcgacgct gcccgcgagg
     4081 tccaggcgct ggccgctgaa attaaatcaa aactcatttg agttaatgag gtaaagagaa
     4141 aatgagcaaa agcacaaaca cgctaagtgc cggccgtccg agcgcacgca gcagcaaggc
     4201 tgcaacgttg gccagcctgg cagacacgcc agccatgaag cgggtcaact ttcagttgcc
     4261 ggcggaggat cacaccaagc tgaagatgta cgcggtacgc caaggcaaga ccattaccga
     4321 gctgctatct gaatacatcg cgcagctacc agagtaaatg agcaaatgaa taaatgagta
     4381 gatgaatttt agcggctaaa ggaggcggca tggaaaatca agaacaacca ggcaccgacg
     4441 ccgtggaatg ccccatgtgt ggaggaacgg gcggttggcc aggcgtaagc ggctgggttg
     4501 cctgccggcc ctgcaatggc actggaaccc ccaagcccga ggaatcggcg tgagcggtcg
     4561 caaaccatcc ggcccggtac aaatcggcgc ggcgctgggt gatgacctgg tggagaagtt
     4621 gaaggccgcg caggccgccc agcggcaacg catcgaggca gaagcacgcc ccggtgaatc
     4681 gtggcaagcg gccgctgatc gaatccgcaa agaatcccgg caaccgccgg cagccggtgc
     4741 gccgtcgatt aggaagccgc ccaagggcga cgagcaacca gattttttcg ttccgatgct
     4801 ctatgacgtg ggcacccgcg atagtcgcag catcatggac gtggccgttt tccgtctgtc
     4861 gaagcgtgac cgacgagctg gcgaggtgat ccgctacgag cttccagacg ggcacgtaga
     4921 ggtttccgca gggccggccg gcatggccag tgtgtgggat tacgacctgg tactgatggc
     4981 ggtttcccat ctaaccgaat ccatgaaccg ataccgggaa gggaagggag acaagcccgg
     5041 ccgcgtgttc cgtccacacg ttgcggacgt actcaagttc tgccggcgag ccgatggcgg
     5101 aaagcagaaa gacgacctgg tagaaacctg cattcggtta aacaccacgc acgttgccat
     5161 gcagcgtacg aagaaggcca agaacggccg cctggtgacg gtatccgagg gtgaagcctt
     5221 gattagccgc tacaagatcg taaagagcga aaccgggcgg ccggagtaca tcgagatcga
     5281 gctagctgat tggatgtacc gcgagatcac agaaggcaag aacccggacg tgctgacggt
     5341 tcaccccgat tactttttga tcgatcccgg catcggccgt tttctctacc gcctggcacg
     5401 ccgcgccgca ggcaaggcag aagccagatg gttgttcaag acgatctacg aacgcagtgg
     5461 cagcgccgga gagttcaaga agttctgttt caccgtgcgc aagctgatcg ggtcaaatga
     5521 cctgccggag tacgatttga aggaggaggc ggggcaggct ggcccgatcc tagtcatgcg
     5581 ctaccgcaac ctgatcgagg gcgaagcatc cgccggttcc taatgtacgg agcagatgct
     5641 agggcaaatt gccctagcag gggaaaaagg tcgaaaagga ctctttcctg tggatagcac
     5701 gtacattggg aacccaaagc cgtacattgg gaaccggaac ccgtacattg ggaacccaaa
     5761 gccgtacatt gggaaccggt cacacatgta agtgactgat ataaaagaga aaaaaggcga
     5821 tttttccgcc taaaactctt taaaacttat taaaactctt aaaacccgcc tggcctgtgc
     5881 ataactgtct ggccagcgca cagccgaaga gctgcaaaaa gcgcctaccc ttcggtcgct
     5941 gcgctcccta cgccccgccg cttcgcgtcg gcctatcgcg gccgctggcc gctcaaaaat
     6001 ggctggccta cggccaggca atctaccagg gcgcggacaa gccgcgccgt cgccactcga
     6061 ccgccggcgc ccacatcaag gcaccctgcc tcgcgcgttt cggtgatgac ggtgaaaacc
     6121 tctgacacat gcagctcccg gtgacggtca cagcttgtct gtaagcggat gccgggagca
     6181 gacaagcccg tcagggcgcg tcagcgggtg ttggcgggtg tcggggcgca gccatgaccc
     6241 agtcacgtag cgatagcgga gtgtatactg gcttaactat gcggcatcag agcagattgt
     6301 actgagagtg caccatatgc ggtgtgaaat accgcacaga tgcgtaagga gaaaataccg
     6361 catcaggcgc tcttccgctt cctcgctcac tgactcgctg cgctcggtcg ttcggctgcg
     6421 gcgagcggta tcagctcact caaaggcggt aatacggtta tccacagaat caggggataa
     6481 cgcaggaaag aacatgtgag caaaaggcca gcaaaaggcc aggaaccgta aaaaggccgc
     6541 gttgctggcg tttttccata ggctccgccc ccctgacgag catcacaaaa atcgacgctc
     6601 aagtcagagg tggcgaaacc cgacaggact ataaagatac caggcgtttc cccctggaag
     6661 ctccctcgtg cgctctcctg ttccgaccct gccgcttacc ggatacctgt ccgcctttct
     6721 cccttcggga agcgtggcgc tttctcatag ctcacgctgt aggtatctca gttcggtgta
     6781 ggtcgttcgc tccaagctgg gctgtgtgca cgaacccccc gttcagcccg accgctgcgc
     6841 cttatccggt aactatcgtc ttgagtccaa cccggtaaga cacgacttat cgccactggc
     6901 agcagccact ggtaacagga ttagcagagc gaggtatgta ggcggtgcta cagagttctt
     6961 gaagtggtgg cctaactacg gctacactag aaggacagta tttggtatct gcgctctgct
     7021 gaagccagtt accttcggaa aaagagttgg tagctcttga tccggcaaac aaaccaccgc
     7081 tggtagcggt ggtttttttg tttgcaagca gcagattacg cgcagaaaaa aaggatctca
     7141 agaagatcct ttgatctttt ctacggggtc tgacgctcag tggaacgaaa actcacgtta
     7201 agggattttg gtcatgcatt ctaggtgatt agaaaaactc atcgagcatc aaatgaaact
     7261 gcaatttatt catatcagga ttatcaatac catatttttg aaaaagccgt ttctgtaatg
     7321 aaggagaaaa ctcaccgagg cagttccata ggatggcaag atcctggtat cggtctgcga
     7381 ttccgactcg tccaacatca atacaaccta ttaatttccc ctcgtcaaaa ataaggttat
     7441 caagtgagaa atcaccatga gtgacgactg aatccggtga gaatggcaaa agtttatgca
     7501 tttctttcca gacttgttca acaggccagc cattacgctc gtcatcaaaa tcactcgcat
     7561 caaccaaacc gttattcatt cgtgattgcg cctgagcgag tcgaaatacg cgatcgctgt
     7621 taaaaggaca attacaaaca ggaatcgaat gcaaccggcg caggaacact gccagcgcat
     7681 caacaatatt ttcacctgaa tcaggatatt cttctaatac ctggaatgct gttttccctg
     7741 ggatcgcagt ggtgagtaac catgcatcat caggagtacg gataaaatgc ttgatggtcg
     7801 gaagaggcat aaattccgtc agccagttta gtctgaccat ctcatctgta acatcattgg
     7861 caacgctacc tttgccatgt ttcagaaaca actctggcgc atcgggcttc ccatacaatc
     7921 ggtagattgt cgcacctgat tgcccgacat tatcgcgagc ccatttatac ccatataaat
     7981 cagcatccat gttggaattt aatcgcggcc ttgagcaaga cgtttcccgt tgaatatggc
     8041 tcataacaga acttattatt tccttcctct tttctacagt atttaaagat accccaagaa
     8101 gctaattata acaagacgaa ctccaattca ctgttccttg cattctaaaa ccttaaatac
     8161 cagaaaacag ctttttcaaa gttgttttca aagttggcgt ataacatagt atcgacggag
     8221 ccgattttga aaccgcggtg atcacaggca gcaacgctct gtcatcgtta caatcaacat
     8281 gctaccctcc gcgagatcat ccgtgtttca aacccggcag cttagttgcc gttcttccga
     8341 atagcatcgg taacatgagc aaagtctgcc gccttacaac ggctctcccg ctgacgccgt
     8401 cccggactga tgggctgcct gtatcgagtg gtgattttgt gccgagctgc cggtcgggga
     8461 gctgttggct ggctggtggc aggatatatt gtggtgtaaa cataacaagc ttcgtctcag
     8521 tca
//
