LOCUS       sequence-321995-        6740 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 6740)
  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 6740)
  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..6740
                     /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="MKVIAVLNQKGGSGKTTIATHLARALQLAGADVLLVDSDPQGSAR
                     DWAAVREDQPLTVVGIDRPTIDRDVKAIGRRDFVVIDGAPQAADLAVSAIKAADFVLIP
                     VQPSPYDIWATADLVELVKQRIEVTDGRLQAAFVVSRAIKGTRIGGEVAEALAGYELPI
                     LESRITQRVSYPGTAAAGTTVLESEPEGDAAREVQALAAEIKSKLI"
     CDS             2772..3845
                     /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      3911..4105
                     /label=pVS1 oriV
                     /note="origin of replication for the Pseudomonas plasmid 
                     pVS1 (Heeb et al., 2000)"
     primer_bind     complement(4341..4363)
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     misc_feature    4449..4589
                     /label=bom
                     /note="basis of mobility region from pBR322"
     primer_bind     4504..4523
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     complement(4604..4621)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(4775..5363)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(4855..4874)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(5451..6266)
                     /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(5451..6266)
                     /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     6174..6193
                     /label=Kan-R
                     /note="Kanamycin resistance gene, reverse primer"
     misc_feature    6699..6723
                     /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 atatgatgcg
     1681 atgagtaaat aaacaaatac gcaaggggaa cgcatgaagg ttatcgctgt acttaaccag
     1741 aaaggcgggt caggcaagac gaccatcgca 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 ctgaatacat cgcgcagcta ccagagtaaa
     2581 tgagcaaatg aataaatgag tagatgaatt ttagcggcta aaggaggcgg catggaaaat
     2641 caagaacaac caggcaccga cgccgtggaa tgccccatgt gtggaggaac gggcggttgg
     2701 ccaggcgtaa gcggctgggt tgcctgccgg ccctgcaatg gcactggaac ccccaagccc
     2761 gaggaatcgg cgtgagcggt cgcaaaccat ccggcccggt acaaatcggc gcggcgctgg
     2821 gtgatgacct ggtggagaag ttgaaggccg cgcaggccgc ccagcggcaa cgcatcgagg
     2881 cagaagcacg ccccggtgaa tcgtggcaag cggccgctga tcgaatccgc aaagaatccc
     2941 ggcaaccgcc ggcagccggt gcgccgtcga ttaggaagcc gcccaagggc gacgagcaac
     3001 cagatttttt cgttccgatg ctctatgacg tgggcacccg cgatagtcgc agcatcatgg
     3061 acgtggccgt tttccgtctg tcgaagcgtg accgacgagc tggcgaggtg atccgctacg
     3121 agcttccaga cgggcacgta gaggtttccg cagggccggc cggcatggcc agtgtgtggg
     3181 attacgacct ggtactgatg gcggtttccc atctaaccga atccatgaac cgataccggg
     3241 aagggaaggg agacaagccc ggccgcgtgt tccgtccaca cgttgcggac gtactcaagt
     3301 tctgccggcg agccgatggc ggaaagcaga aagacgacct ggtagaaacc tgcattcggt
     3361 taaacaccac gcacgttgcc atgcagcgta cgaagaaggc caagaacggc cgcctggtga
     3421 cggtatccga gggtgaagcc ttgattagcc gctacaagat cgtaaagagc gaaaccgggc
     3481 ggccggagta catcgagatc gagctagctg attggatgta ccgcgagatc acagaaggca
     3541 agaacccgga cgtgctgacg gttcaccccg attacttttt gatcgatccc ggcatcggcc
     3601 gttttctcta ccgcctggca cgccgcgccg caggcaaggc agaagccaga tggttgttca
     3661 agacgatcta cgaacgcagt ggcagcgccg gagagttcaa gaagttctgt ttcaccgtgc
     3721 gcaagctgat cgggtcaaat gacctgccgg agtacgattt gaaggaggag gcggggcagg
     3781 ctggcccgat cctagtcatg cgctaccgca acctgatcga gggcgaagca tccgccggtt
     3841 cctaatgtac ggagcagatg ctagggcaaa ttgccctagc aggggaaaaa ggtcgaaaag
     3901 gactctttcc tgtggatagc acgtacattg ggaacccaaa gccgtacatt gggaaccgga
     3961 acccgtacat tgggaaccca aagccgtaca ttgggaaccg gtcacacatg taagtgactg
     4021 atataaaaga gaaaaaaggc gatttttccg cctaaaactc tttaaaactt attaaaactc
     4081 ttaaaacccg cctggcctgt gcataactgt ctggccagcg cacagccgaa gagctgcaaa
     4141 aagcgcctac ccttcggtcg ctgcgctccc tacgccccgc cgcttcgcgt cggcctatcg
     4201 cggccgctgg ccgctcaaaa atggctggcc tacggccagg caatctacca gggcgcggac
     4261 aagccgcgcc gtcgccactc gaccgccggc gcccacatca aggcaccctg cctcgcgcgt
     4321 ttcggtgatg acggtgaaaa cctctgacac atgcagctcc cggtgacggt cacagcttgt
     4381 ctgtaagcgg atgccgggag cagacaagcc cgtcagggcg cgtcagcggg tgttggcggg
     4441 tgtcggggcg cagccatgac ccagtcacgt agcgatagcg gagtgtatac tggcttaact
     4501 atgcggcatc agagcagatt gtactgagag tgcaccatat gcggtgtgaa ataccgcaca
     4561 gatgcgtaag gagaaaatac cgcatcaggc gctcttccgc ttcctcgctc actgactcgc
     4621 tgcgctcggt cgttcggctg cggcgagcgg tatcagctca ctcaaaggcg gtaatacggt
     4681 tatccacaga atcaggggat aacgcaggaa agaacatgtg agcaaaaggc cagcaaaagg
     4741 ccaggaaccg taaaaaggcc gcgttgctgg cgtttttcca taggctccgc ccccctgacg
     4801 agcatcacaa aaatcgacgc tcaagtcaga ggtggcgaaa cccgacagga ctataaagat
     4861 accaggcgtt tccccctgga agctccctcg tgcgctctcc tgttccgacc ctgccgctta
     4921 ccggatacct gtccgccttt ctcccttcgg gaagcgtggc gctttctcat agctcacgct
     4981 gtaggtatct cagttcggtg taggtcgttc gctccaagct gggctgtgtg cacgaacccc
     5041 ccgttcagcc cgaccgctgc gccttatccg gtaactatcg tcttgagtcc aacccggtaa
     5101 gacacgactt atcgccactg gcagcagcca ctggtaacag gattagcaga gcgaggtatg
     5161 taggcggtgc tacagagttc ttgaagtggt ggcctaacta cggctacact agaaggacag
     5221 tatttggtat ctgcgctctg ctgaagccag ttaccttcgg aaaaagagtt ggtagctctt
     5281 gatccggcaa acaaaccacc gctggtagcg gtggtttttt tgtttgcaag cagcagatta
     5341 cgcgcagaaa aaaaggatct caagaagatc ctttgatctt ttctacgggg tctgacgctc
     5401 agtggaacga aaactcacgt taagggattt tggtcatgca ttctaggtga ttagaaaaac
     5461 tcatcgagca tcaaatgaaa ctgcaattta ttcatatcag gattatcaat accatatttt
     5521 tgaaaaagcc gtttctgtaa tgaaggagaa aactcaccga ggcagttcca taggatggca
     5581 agatcctggt atcggtctgc gattccgact cgtccaacat caatacaacc tattaatttc
     5641 ccctcgtcaa aaataaggtt atcaagtgag aaatcaccat gagtgacgac tgaatccggt
     5701 gagaatggca aaagtttatg catttctttc cagacttgtt caacaggcca gccattacgc
     5761 tcgtcatcaa aatcactcgc atcaaccaaa ccgttattca ttcgtgattg cgcctgagcg
     5821 agtcgaaata cgcgatcgct gttaaaagga caattacaaa caggaatcga atgcaaccgg
     5881 cgcaggaaca ctgccagcgc atcaacaata ttttcacctg aatcaggata ttcttctaat
     5941 acctggaatg ctgttttccc tgggatcgca gtggtgagta accatgcatc atcaggagta
     6001 cggataaaat gcttgatggt cggaagaggc ataaattccg tcagccagtt tagtctgacc
     6061 atctcatctg taacatcatt ggcaacgcta cctttgccat gtttcagaaa caactctggc
     6121 gcatcgggct tcccatacaa tcggtagatt gtcgcacctg attgcccgac attatcgcga
     6181 gcccatttat acccatataa atcagcatcc atgttggaat ttaatcgcgg ccttgagcaa
     6241 gacgtttccc gttgaatatg gctcataaca gaacttatta tttccttcct cttttctaca
     6301 gtatttaaag ataccccaag aagctaatta taacaagacg aactccaatt cactgttcct
     6361 tgcattctaa aaccttaaat accagaaaac agctttttca aagttgtttt caaagttggc
     6421 gtataacata gtatcgacgg agccgatttt gaaaccgcgg tgatcacagg cagcaacgct
     6481 ctgtcatcgt tacaatcaac atgctaccct ccgcgagatc atccgtgttt caaacccggc
     6541 agcttagttg ccgttcttcc gaatagcatc ggtaacatga gcaaagtctg ccgccttaca
     6601 acggctctcc cgctgacgcc gtcccggact gatgggctgc ctgtatcgag tggtgatttt
     6661 gtgccgagct gccggtcggg gagctgttgg ctggctggtg gcaggatata ttgtggtgta
     6721 aacataacga attcgtctca
//
