LOCUS       sequence-310986-        6739 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  GB-cassette for the expression of a guide RNA targeting the DFR 
            promoter with 2.1 Ms2 aptamer in the 3' of the scaffold.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 6739)
  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 6739)
  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: "10.1111/pbi.13138";
            journalName: "Plant Biotechnol J"; date: "2019-09"; volume: "17"; 
            issue: "9"; pages: "1703-1705"
COMMENT     Sequence Label: pDGB3 alpha1 U6-26-1gRNA-DFR 2.1 (GB1838)
FEATURES             Location/Qualifiers
     source          1..6739
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     misc_RNA        230..305
                     /label=gRNA scaffold
                     /note="guide RNA scaffold for the Streptococcus pyogenes 
                     CRISPR/Cas9 system"
     misc_feature    389..413
                     /label=RB T-DNA repeat
                     /note="right border repeat from nopaline C58 T-DNA"
     CDS             1714..2343
                     /codon_start=1
                     /product="stability protein from the Pseudomonas plasmid 
                     pVS1 (Heeb et al., 2000)"
                     /label=pVS1 StaA
                     /translation="MKVIAVLNQKGGSGKTTIGTHLARALQLAGADVLLVDSDPQGSAR
                     DWAAVREDQPLTVVGIDRPTIDRDVKAIGRRDFVVIDGAPQAADLAVSAIKAADFVLIP
                     VQPSPYDIWATADLVELVKQRIEVTDGRLQAAFVVSRAIKGTRIGGEVAEALAGYELPI
                     LESRITQRVSYPGTAAAGTTVLESEPEGDAAREVQALAAEIKSKLI"
     CDS             2777..3844
                     /codon_start=1
                     /product="replication protein from the Pseudomonas plasmid 
                     pVS1 (Heeb et al., 2000)"
                     /label=pVS1 RepA
                     /translation="GRKPSGPVQIGAALGDDLVEKLKAAQAAQRQRIEAEARPGESWQA
                     AADRIRKESRQPPAAGAPSIRKPPKGDEQPDFFVPMLYDVGTRDSRSIMDVAVFRLSKR
                     DRRAGEVIRYELPDGHVEVSAGPAGMASVWDYDLVLMAVSHLTESMNRYREGKGDKPGR
                     VFRPHVADVLKFCRRADGGKQKDDLVETCIRLNTTHVAMQRTKKAKNGRLVTVSEGEAL
                     ISRYKIVKSETGRPEYIEIELADWMYREITEGKNPDVLTVHPDYFLIDPGIGRFLYRLA
                     RRAAGKAEARWLFKTIYERSGSAGEFKKFCFTVRKLIGSNDLPEYDLKEEAGQAGPILV
                     MRYRNLIEGEASAGS"
     rep_origin      3910..4104
                     /label=pVS1 oriV
                     /note="origin of replication for the Pseudomonas plasmid 
                     pVS1 (Heeb et al., 2000)"
     primer_bind     complement(4340..4362)
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     misc_feature    4448..4588
                     /label=bom
                     /note="basis of mobility region from pBR322"
     primer_bind     4503..4522
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     complement(4603..4620)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(4774..5362)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(4854..4873)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(5450..6265)
                     /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(5450..6265)
                     /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     6173..6192
                     /label=Kan-R
                     /note="Kanamycin resistance gene, reverse primer"
     misc_feature    6698..6722
                     /label=LB T-DNA repeat
                     /note="left border repeat from nopaline C58 T-DNA"
ORIGIN
        1 ggagcatctt cattcttaag atatgaagat aatcttcaaa aggcccctgg gaatctgaaa
       61 gaagagaagc aggcccattt atatgggaaa gaacaatagt atttcttata taggcccatt
      121 taagttgaaa acaatcttca aaagtcccac atcgcttaga taagaaaacg aagctgagtt
      181 tatatacagc tagagtcgaa gtagtgattg actggttggt gagagaagag ttttagagct
      241 agaaatagca agttaaaata aggctagtcc gttatcaact tgaaaaagtg gcaccgagtc
      301 ggtgcgggag cacatgagga atcacccatg tgcgactccc acagtcactg gggagtcttc
      361 ccttttttcg ctgtcatgag acgaattctg acaggatata ttggcgggta aacctaagag
      421 aaaagagcgt ttattagaat aatcggatat ttaaaagggc gtgaaaaggt ttatccgttc
      481 gtccatttgt atgtgcatgc caaccacagg gttcccctcg ggatcaaagt actttgatcc
      541 aacccctccg ctgctatagt gcagtcggct tctgacgttc agtgcagccg tcatctgaaa
      601 acgacatgtc gcacaagtcc taagttacgc gacaggctgc cgccctgccc ttttcctggc
      661 gttttcttgt cgcgtgtttt agtcgcataa agtagaatac ttgcgactag aaccggagac
      721 attacgccat gaacaagagc gccgccgctg gcctgctggg ctatgcccgc gtcagcaccg
      781 acgaccagga cttgaccaac caacgggccg aactgcacgc ggccggctgc accaagctgt
      841 tttccgagaa gatcaccggc accaggcgcg accgcccgga gctggccagg atgcttgacc
      901 acctacgccc tggcgacgtt gtgacagtga ccaggctaga ccgcctggcc cgcagcaccc
      961 gcgacctact ggacattgcc gagcgcatcc aggaggccgg cgcgggcctg cgtagcctgg
     1021 cagagccgtg ggccgacacc accacgccgg ccggccgcat ggtgttgacc gtgttcgccg
     1081 gcattgccga gttcgagcgt tccctaatca tcgaccgcac ccggagcggg cgcgaggccg
     1141 ccaaggcccg aggcgtgaag tttggccccc gccctaccct caccccggca cagatcgcgc
     1201 acgcccgcga gctgatcgac caggaaggcc gcaccgtgaa agaggcggct gcactgcttg
     1261 gcgtgcatcg ctcgaccctg taccgcgcac ttgagcgcag cgaggaagtg acgcccaccg
     1321 aggccaggcg gcgcggtgcc ttccgtgagg acgcattgac cgaggccgac gccctggcgg
     1381 ccgccgagaa tgaacgccaa gaggaacaag catgaaaccg caccaggacg gccaggacga
     1441 accgtttttc attaccgaag agatcgaggc ggagatgatc gcggccgggt acgtgttcga
     1501 gccgcccgcg cacctctcaa ccgtgcggct gcatgaaatc ctggccggtt tgtctgatgc
     1561 caagctggcg gcctggccgg ccagcttggc cgctgaagaa accgagcgcc gccgtctaaa
     1621 aaggtgatgt gtatttgagt aaaacagctt gcgtcatgcg gtcgctgcgt atatgatccg
     1681 atgagtaaat aaacaaatac gcaaggggaa cgcatgaagg ttatcgctgt acttaaccag
     1741 aaaggcgggt caggcaagac gaccatcgga acccatctag cccgcgccct gcaactcgcc
     1801 ggggccgatg ttctgttagt cgattccgat ccccagggca gtgcccgcga ttgggcggcc
     1861 gtgcgggaag atcaaccgct aaccgttgtc ggcatcgacc gcccgacgat tgaccgcgac
     1921 gtgaaggcca tcggccggcg cgacttcgta gtgatcgacg gagcgcccca ggcggcggac
     1981 ttggctgtgt ccgcgatcaa ggcagccgac ttcgtgctga ttccggtgca gccaagccct
     2041 tacgacatat gggccaccgc cgacctggtg gagctggtta agcagcgcat tgaggtcacg
     2101 gatggaaggc tacaagcggc ctttgtcgtg tcgcgggcga tcaaaggcac gcgcatcggc
     2161 ggtgaggttg ccgaggcgct ggccgggtac gagctgccca ttcttgagtc ccgtatcacg
     2221 cagcgcgtga gctacccagg cactgccgcc gccggcacaa ccgttcttga atcagaaccc
     2281 gagggcgacg ctgcccgcga ggtccaggcg ctggccgctg aaattaaatc aaaactcatt
     2341 tgagttaatg aggtaaagag aaaatgagca aaagcacaaa cacgctaagt gccggccgtc
     2401 cgagcgcacg cagcagcaag gctgcaacgt tggccagcct ggcagacacg ccagccatga
     2461 agcgggtcaa ctttcagttg ccggcggagg atcacaccaa gctgaagatg tacgcggtac
     2521 gccaaggcaa gaccattacc gagctgctat ctgaatagat cgcgcagcta ccagagtaaa
     2581 tgagcaaatg aataaatgag tagatgaatt ttagcggcta aaggaggcgg catggaaaat
     2641 caagaacaac caggcaccga cgccgtggaa tgccccatgt gtggaggaac gggcggttgg
     2701 ccaggcgtaa gcggctgggt tgtctgccgg ccctgcaatg gcactggaac ccccaagccc
     2761 gaggaatcgg cgtgacggtc gcaaaccatc cggcccggta caaatcggcg cggcgctggg
     2821 tgatgacctg gtggagaagt tgaaggccgc gcaggccgcc cagcggcaac gcatcgaggc
     2881 agaagcacgc cccggtgaat cgtggcaagc ggccgctgat cgaatccgca aagaatcccg
     2941 gcaaccgccg gcagccggtg cgccgtcgat taggaagccg cccaagggcg acgagcaacc
     3001 agattttttc gttccgatgc tctatgacgt gggcacccgc gatagtcgca gcatcatgga
     3061 cgtggccgtt ttccgtctgt cgaagcgtga ccgacgagct ggcgaggtga tccgctacga
     3121 gcttccagac gggcacgtag aggtttccgc agggccggcc ggcatggcca gtgtgtggga
     3181 ttacgacctg gtactgatgg cggtttccca tctaaccgaa tccatgaacc gataccggga
     3241 agggaaggga gacaagcccg gccgcgtgtt ccgtccacac gttgcggacg tactcaagtt
     3301 ctgccggcga gccgatggcg gaaagcagaa agacgacctg gtagaaacct gcattcggtt
     3361 aaacaccacg cacgttgcca tgcagcgtac gaagaaggcc aagaacggcc gcctggtgac
     3421 ggtatccgag ggtgaagcct tgattagccg ctacaagatc gtaaagagcg aaaccgggcg
     3481 gccggagtac atcgagatcg agctagctga ttggatgtac cgcgagatca cagaaggcaa
     3541 gaacccggac gtgctgacgg ttcaccccga ttactttttg atcgatcccg gcatcggccg
     3601 ttttctctac cgcctggcac gccgcgccgc aggcaaggca gaagccagat ggttgttcaa
     3661 gacgatctac gaacgcagtg gcagcgccgg agagttcaag aagttctgtt tcaccgtgcg
     3721 caagctgatc gggtcaaatg acctgccgga gtacgatttg aaggaggagg cggggcaggc
     3781 tggcccgatc ctagtcatgc gctaccgcaa cctgatcgag ggcgaagcat ccgccggttc
     3841 ctaatgtacg gagcagatgc tagggcaaat tgccctagca ggggaaaaag gtcgaaaagg
     3901 actctttcct gtggatagca cgtacattgg gaacccaaag ccgtacattg ggaaccggaa
     3961 cccgtacatt gggaacccaa agccgtacat tgggaaccgg tcacacatgt aagtgactga
     4021 tataaaagag aaaaaaggcg atttttccgc ctaaaactct ttaaaactta ttaaaactct
     4081 taaaacccgc ctggcctgtg cataactgtc tggccagcgc acagccgaag agctgcaaaa
     4141 agcgcctacc cttcggtcgc tgcgctccct acgccccgcc gcttcgcgtc ggcctatcgc
     4201 ggccgctggc cgctcaaaaa tggctggcct acggccaggc aatctaccag ggcgcggaca
     4261 agccgcgccg tcgccactcg accgccggcg cccacatcaa ggcaccctgc ctcgcgcgtt
     4321 tcggtgatga cggtgaaaac ctctgacaca tgcagctccc ggtgacggtc acagcttgtc
     4381 tgtaagcgga tgccgggagc agacaagccc gtcagggcgc gtcagcgggt gttggcgggt
     4441 gtcggggcgc agccatgacc cagtcacgta gcgatagcgg agtgtatact ggcttaacta
     4501 tgcggcatca gagcagattg tactgagagt gcaccatatg cggtgtgaaa taccgcacag
     4561 atgcgtaagg agaaaatacc gcatcaggcg ctcttccgct tcctcgctca ctgactcgct
     4621 gcgctcggtc gttcggctgc ggcgagcggt atcagctcac tcaaaggcgg taatacggtt
     4681 atccacagaa tcaggggata acgcaggaaa gaacatgtga gcaaaaggcc agcaaaaggc
     4741 caggaaccgt aaaaaggccg cgttgctggc gtttttccat aggctccgcc cccctgacga
     4801 gcatcacaaa aatcgacgct caagtcagag gtggcgaaac ccgacaggac tataaagata
     4861 ccaggcgttt ccccctggaa gctccctcgt gcgctctcct gttccgaccc tgccgcttac
     4921 cggatacctg tccgcctttc tcccttcggg aagcgtggcg ctttctcata gctcacgctg
     4981 taggtatctc agttcggtgt aggtcgttcg ctccaagctg ggctgtgtgc acgaaccccc
     5041 cgttcagccc gaccgctgcg ccttatccgg taactatcgt cttgagtcca acccggtaag
     5101 acacgactta tcgccactgg cagcagccac tggtaacagg attagcagag cgaggtatgt
     5161 aggcggtgct acagagttct tgaagtggtg gcctaactac ggctacacta gaaggacagt
     5221 atttggtatc tgcgctctgc tgaagccagt taccttcgga aaaagagttg gtagctcttg
     5281 atccggcaaa caaaccaccg ctggtagcgg tggttttttt gtttgcaagc agcagattac
     5341 gcgcagaaaa aaaggatctc aagaagatcc tttgatcttt tctacggggt ctgacgctca
     5401 gtggaacgaa aactcacgtt aagggatttt ggtcatgcat tctaggtgat tagaaaaact
     5461 catcgagcat caaatgaaac tgcaatttat tcatatcagg attatcaata ccatattttt
     5521 gaaaaagccg tttctgtaat gaaggagaaa actcaccgag gcagttccat aggatggcaa
     5581 gatcctggta tcggtctgcg attccgactc gtccaacatc aatacaacct attaatttcc
     5641 cctcgtcaaa aataaggtta tcaagtgaga aatcaccatg agtgacgact gaatccggtg
     5701 agaatggcaa aagtttatgc atttctttcc agacttgttc aacaggccag ccattacgct
     5761 cgtcatcaaa atcactcgca tcaaccaaac cgttattcat tcgtgattgc gcctgagcga
     5821 gtcgaaatac gcgatcgctg ttaaaaggac aattacaaac aggaatcgaa tgcaaccggc
     5881 gcaggaacac tgccagcgca tcaacaatat tttcacctga atcaggatat tcttctaata
     5941 cctggaatgc tgttttccct gggatcgcag tggtgagtaa ccatgcatca tcaggagtac
     6001 ggataaaatg cttgatggtc ggaagaggca taaattccgt cagccagttt agtctgacca
     6061 tctcatctgt aacatcattg gcaacgctac ctttgccatg tttcagaaac aactctggcg
     6121 catcgggctt cccatacaat cggtagattg tcgcacctga ttgcccgaca ttatcgcgag
     6181 cccatttata cccatataaa tcagcatcca tgttggaatt taatcgcggc cttgagcaag
     6241 acgtttcccg ttgaatatgg ctcataacag aacttattat ttccttcctc ttttctacag
     6301 tatttaaaga taccccaaga agctaattat aacaagacga actccaattc actgttcctt
     6361 gcattctaaa accttaaata ccagaaaaca gctttttcaa agttgttttc aaagttggcg
     6421 tataacatag tatcgacgga gccgattttg aaaccgcggt gatcacaggc agcaacgctc
     6481 tgtcatcgtt acaatcaaca tgctaccctc cgcgagatca tccgtgtttc aaacccggca
     6541 gcttagttgc cgttcttccg aatagcatcg gtaacatgag caaagtctgc cgccttacaa
     6601 cggctctccc gctgacgccg tcccggactg atgggctgcc tgtatcgagt ggtgattttg
     6661 tgccgagctg ccggtcgggg agctgttggc tggctggtgg caggatatat tgtggtgtaa
     6721 acataacgaa ttcgtctca
//
