LOCUS       sequence-258453-        6726 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  CasD plant transcriptional activator (AT-CBF1) linked constitutive 
            expression cassette for Zea mays.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 6726)
  AUTHORS   Young JK, Gasior SL, Jones S, Wang L, Navarro P, Vickroy B, 
            Barrangou R
  TITLE     The repurposing of type I-E CRISPR-Cascade for gene activation in 
            plants.
  JOURNAL   Commun Biol. 2019 Oct 18;2:383. doi: 10.1038/s42003-019-0637-6. 
            eCollection 2019.
  PUBMED    31646186
REFERENCE   2  (bases 1 to 6726)
  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: "Commun 
            Biol."; date: "2019-10-18"; volume: "2:383. doi"; pages: " 
            10.1038/s42003-019-0637-6. eCollection 2019"
COMMENT     Sequence Label: PV259
FEATURES             Location/Qualifiers
     source          1..6726
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     1..20
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        1..19
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     promoter        54..2049
                     /label=Ubi promoter
                     /note="maize polyubiquitin gene promoter"
     CDS             2812..2832
                     /codon_start=1
                     /product="nuclear localization signal of SV40 (simian virus
                     40) large T antigen"
                     /label=SV40 NLS
                     /translation="PKKKRKV"
     promoter        complement(3524..3542)
                     /label=SP6 promoter
                     /note="promoter for bacteriophage SP6 RNA polymerase"
     primer_bind     complement(3525..3542)
                     /label=SP6
                     /note="SP6 promoter, forward primer"
     primer_bind     complement(3560..3576)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(3560..3576)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(3573..3595)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    3584..3600
                     /label=lac operator
                     /bound_moiety="lac repressor encoded by lacI"
                     /note="The lac repressor binds to the lac operator to 
                     inhibit transcription in E. coli. This inhibition can be 
                     relieved by adding lactose or 
                     isopropyl-beta-D-thiogalactopyranoside (IPTG)."
     promoter        complement(join(3608..3614,3615..3632,3633..3638))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    3653..3674
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(3791..3808)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(3962..4550)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(4042..4061)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     complement(4724..4743)
                     /label=pENTR-R
                     /note="pENTR vectors, reverse primer"
     CDS             4735..5550
                     /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             4735..5550
                     /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     complement(4808..4827)
                     /label=Kan-R
                     /note="Kanamycin resistance gene, reverse primer"
     primer_bind     5844..5862
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     primer_bind     complement(5900..5922)
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     primer_bind     6022..6041
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     rep_origin      complement(6108..6563)
                     /direction=LEFT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(6245..6266)
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     6457..6476
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     6689..6711
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     primer_bind     6703..6720
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     6704..6720
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
ORIGIN
        1 taatacgact cactataggg cgaattgggt tacccggacc gaagcttgca tgcctgcagt
       61 gcagcgtgac ccggtcgtgc ccctctctag agataatgag cattgcatgt ctaagttata
      121 aaaaattacc acatattttt tttgtcacac ttgtttgaag tgcagtttat ctatctttat
      181 acatatattt aaactttact ctacgaataa tataatctat agtactacaa taatatcagt
      241 gttttagaga atcatataaa tgaacagtta gacatggtct aaaggacaat tgagtatttt
      301 gacaacagga ctctacagtt ttatcttttt agtgtgcatg tgttctcctt tttttttgca
      361 aatagcttca cctatataat acttcatcca ttttattagt acatccattt agggtttagg
      421 gttaatggtt tttatagact aattttttta gtacatctat tttattctat tttagcctct
      481 aaattaagaa aactaaaact ctattttagt ttttttattt aataatttag atataaaata
      541 gaataaaata aagtgactaa aaattaaaca aatacccttt aagaaattaa aaaaactaag
      601 gaaacatttt tcttgtttcg agtagataat gccagcctgt taaacgccgt cgacgagtct
      661 aacggacacc aaccagcgaa ccagcagcgt cgcgtcgggc caagcgaagc agacggcacg
      721 gcatctctgt cgctgcctct ggacccctct cgagagttcc gctccaccgt tggacttgct
      781 ccgctgtcgg catccagaaa ttgcgtggcg gagcggcaga cgtgagccgg cacggcaggc
      841 ggcctcctcc tcctctcacg gcaccggcag ctacggggga ttcctttccc accgctcctt
      901 cgctttccct tcctcgcccg ccgtaataaa tagacacccc ctccacaccc tctttcccca
      961 acctcgtgtt gttcggagcg cacacacaca caaccagatc tcccccaaat ccacccgtcg
     1021 gcacctccgc ttcaaggtac gccgctcgtc ctcccccccc ccnctctcta ccttctctag
     1081 atcggcgttc cggtccatgc atggttaggg cccggtagtt ctacttctgt tcatgtttgt
     1141 gttagatccg tgtttgtgtt agatccgtgc tgctagcgtt cgtacacgga tgcgacctgt
     1201 acgtcagaca cgttctgatt gctaacttgc cagtgtttct ctttggggaa tcctgggatg
     1261 gctctagccg ttccgcagac gggatcgatt tcatgatttt ttttgtttcg ttgcataggg
     1321 tttggtttgc ccttttcctt tatttcaata tatgccgtgc acttgtttgt cgggtcatct
     1381 tttcatgctt ttttttgtct tggttgtgat gatgtggtct ggttgggcgg tcgttctaga
     1441 tcggagtaga attctgtttc aaactacctg gtggatttat taattttgga tctgtatgtg
     1501 tgtgccatac atattcatag ttacgaattg aagatgatgg atggaaatat cgatctagga
     1561 taggtataca tgttgatgcg ggttttactg atgcatatac agagatgctt tttgttcgct
     1621 tggttgtgat gatgtggtgt ggttgggcgg tcgttcattc gttctagatc ggagtagaat
     1681 actgtttcaa actacctggt gtatttatta attttggaac tgtatgtgtg tgtcatacat
     1741 cttcatagtt acgagtttaa gatggatgga aatatcgatc taggataggt atacatgttg
     1801 atgtgggttt tactgatgca tatacatgat ggcatatgca gcatctattc atatgctcta
     1861 accttgagta cctatctatt ataataaaca agtatgtttt ataattattt tgatcttgat
     1921 atacttggat gatggcatat gcagcagcta tatgtggatt tttttagccc tgccttcata
     1981 cgctatttat ttgcttggta ctgtttcttt tgtcgatgct caccctgttg tttggtgtta
     2041 cttctgcagg tcgactctag aggatccatg aacgccatga aaaccatcct cctcaagttc
     2101 gccggccccc tccagagctg gggcaccagc agccacttcg aaacccgcca caccgacttc
     2161 taccccagca agagcgcggt catcggcctc ctcgccgcca gcctcgggta caggcgcgac
     2221 gaggatgaaa aaatccagaa attaaatgag ctcgacttcg ccgtcagggt cgaccagcag
     2281 ggcaacctcc tcagggactt ccacgtcgcc cagaagtaca ccgagaaggg caagttcgac
     2341 cgcaactacg tcaccaaccg ctactacctc gaggacgccg tgttcgtcgt cgccctcagc
     2401 cacaccgacg acagcttcat cgacatcatt aaaggcgccc tcaagaaccc ctacttccag
     2461 cccttcatgg gcaggagggc cctccccatc accgccgact tcatcctcgg cgaaaccgac
     2521 gtcaaccccc tcgagagcct gaagagcctc aagtggcagg ccagcctctg gttcatgaaa
     2581 aagagccacg acaaggacgc catcagcctc gaggtctacc tcgacagcca gctcgtcaac
     2641 aaggacgtct accagctccg ccaggactac gtccgcagct tcagccagaa gggcaggagg
     2701 ttcggcttca ggtacgaggt ccgcgaatta atcagcgtcc agaaccccca ctacctcaac
     2761 aagaatacag agcacaacat cttcgagagc atcggcgaca gcagggccga ccccaagaag
     2821 aagaggaagg tgggtcgggc gacatgtgcc aaggacatcc agaaggccgc tgccgaagcg
     2881 gcgctcgctt ttcaggacga aacctgcgac accaccacca ccaaccacgg cctcgacatg
     2941 gaggaaacaa tggtggaggc catctacacg cctgaacaga gcgagggggc tttctacatg
     3001 gacgaggaaa ccatgttcgg catgcccaca ctcctcgaca acatggccga gggcatgctc
     3061 ctcccccccc cgagcgtcca gtggaaccac aactacgacg gcgaaggcga cggcgacgtc
     3121 agcctctggt cctactagtc ttcgtaagtt aacctagact tgtccatctt ctggattggc
     3181 caacttaatt aatgtatgaa ataaaaggat gcacacatag tgacatgcta atcactataa
     3241 tgtgggcatc aaagttgtgt gttatgtgta attactagtt atctgaataa aagagaaaga
     3301 gatcatccat atttcttatc ctaaatgaat gtcacgtgtc tttataattc tttgatgaac
     3361 cagatgcatt tcattaacca aatccatata catataaata ttaatcatat ataattaata
     3421 tcaattgggt tagcaaaaca aatctagtct aggtgtgttt tgcgaatgcg gccgccaccg
     3481 cggtggagct cgaattccgg tccgggtcac ccagcttgag tattctatag tgtcacctaa
     3541 atagcttggc gtaatcatgg tcatagctgt ttcctgtgtg aaattgttat ccgctcacaa
     3601 ttccacacaa catacgagcc ggaagcataa agtgtaaagc ctggggtgcc taatgagtga
     3661 gctaactcac attaattgcg ttgcgctcac tgcccgcttt ccagtcggga aacctgtcgt
     3721 gccagctgca ttaatgaatc ggccaacgcg cggggagagg cggtttgcgt attgggcgct
     3781 cttccgcttc ctcgctcact gactcgctgc gctcggtcgt tcggctgcgg cgagcggtat
     3841 cagctcactc aaaggcggta atacggttat ccacagaatc aggggataac gcaggaaaga
     3901 acatgtgagc aaaaggccag caaaaggcca ggaaccgtaa aaaggccgcg ttgctggcgt
     3961 ttttccatag gctccgcccc cctgacgagc atcacaaaaa tcgacgctca agtcagaggt
     4021 ggcgaaaccc gacaggacta taaagatacc aggcgtttcc ccctggaagc tccctcgtgc
     4081 gctctcctgt tccgaccctg ccgcttaccg gatacctgtc cgcctttctc ccttcgggaa
     4141 gcgtggcgct ttctcatagc tcacgctgta ggtatctcag ttcggtgtag gtcgttcgct
     4201 ccaagctggg ctgtgtgcac gaaccccccg ttcagcccga ccgctgcgcc ttatccggta
     4261 actatcgtct tgagtccaac ccggtaagac acgacttatc gccactggca gcagccactg
     4321 gtaacaggat tagcagagcg aggtatgtag gcggtgctac agagttcttg aagtggtggc
     4381 ctaactacgg ctacactaga agaacagtat ttggtatctg cgctctgctg aagccagtta
     4441 ccttcggaaa aagagttggt agctcttgat ccggcaaaca aaccaccgct ggtagcggtg
     4501 gtttttttgt ttgcaagcag cagattacgc gcagaaaaaa aggatctcaa gaagatcctt
     4561 tgatcttttc tacggggtct gacgctcagt ggaacgaaaa ctcacgttaa gggattttgg
     4621 tcatggagcc acgttgtgtc tcaaaatctc tgatgttaca ttgcacaaga taaaaatata
     4681 tcatcatgaa caataaaact gtctgcttac ataaacagta atacaagggg tgttatgagc
     4741 catattcaac gggaaacgtc ttgctcgagg ccgcgattaa attccaacat ggatgctgat
     4801 ttatatgggt ataaatgggc tcgcgataat gtcgggcaat caggtgcgac aatctatcga
     4861 ttgtatggga agcccgatgc gccagagttg tttctgaaac atggcaaagg tagcgttgcc
     4921 aatgatgtta cagatgagat ggtcagacta aactggctga cggaatttat gcctcttccg
     4981 accatcaagc attttatccg tactcctgat gatgcatggt tactcaccac tgcgatcccc
     5041 ggaaaaacag cattccaggt attagaagaa tatcctgatt caggtgaaaa tattgttgat
     5101 gcgctggcag tgttcctgcg ccggttgcat tcgattcctg tttgtaattg tccttttaac
     5161 agcgatcgcg tatttcgtct cgctcaggcg caatcacgaa tgaataacgg tttggttgat
     5221 gcgagtgatt ttgatgacga gcgtaatggc tggcctgttg aacaagtctg gaaagaaatg
     5281 cataaacttt tgccattctc accggattca gtcgtcactc atggtgattt ctcacttgat
     5341 aaccttattt ttgacgaggg gaaattaata ggttgtattg atgttggacg agtcggaatc
     5401 gcagaccgat accaggatct tgccatccta tggaactgcc tcggtgagtt ttctccttca
     5461 ttacagaaac ggctttttca gaaatatggt attgataatc ctgatatgaa taaattgcag
     5521 tttcatttga tgctcgatga gtttttctaa tcagaattgg ttaattggtt gtaacactgg
     5581 cagagcatta cgctgacttg acgggacggc ggctttgttg aataaatcga acttttgctg
     5641 agttgaagga tcagatcacg catcttcccg acaacgcaga ccgttccgtg gcaaagcaaa
     5701 agttcaaaat caccaactgg tccacctaca acaaagctct catcaaccgt ggctccctca
     5761 ctttctggct ggatgatggg gcgattcagg cctggtatga gtcagcaaca ccttcttcac
     5821 gagccatgac attaacctat aaaaataggc gtatcacgag gccctttcgt ctcgcgcgtt
     5881 tcggtgatga cggtgaaaac ctctgacaca tgcagctccc ggagacggtc acagcttgtc
     5941 tgtaagcgga tgccgggagc agacaagccc gtcagggcgc gtcagcgggt gttggcgggt
     6001 gtcggggctg gcttaactat gcggcatcag agcagattgt actgagagtg caccatatgc
     6061 ggtgtgaaat accgcacaga tgcgtaagga gaaaataccg catcaggaaa ttgtaagcgt
     6121 taatattttg ttaaaattcg cgttaaattt ttgttaaatc agctcatttt ttaaccaata
     6181 ggccgaaatc ggcaaaatcc cttataaatc aaaagaatag accgagatag ggttgagtgt
     6241 tgttccagtt tggaacaaga gtccactatt aaagaacgtg gactccaacg tcaaagggcg
     6301 aaaaaccgtc tatcagggcg atggcccact acgtgaacca tcaccctaat caagtttttt
     6361 ggggtcgagg tgccgtaaag cactaaatcg gaaccctaaa gggagccccc gatttagagc
     6421 ttgacgggga aagccggcga acgtggcgag aaaggaaggg aagaaagcga aaggagcggg
     6481 cgctagggcg ctggcaagtg tagcggtcac gctgcgcgta accaccacac ccgccgcgct
     6541 taatgcgccg ctacagggcg cgtccattcg ccattcaggc tgcgcaactg ttgggaaggg
     6601 cgatcggtgc gggcctcttc gctattacgc cagctggcga aagggggatg tgctgcaagg
     6661 cgattaagtt gggtaacgcc agggttttcc cagtcacgac gttgtaaaac gacggccagt
     6721 gaattg
//
