LOCUS       sequence-258477-        6416 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  CasD constitutive expression cassette for Zea mays.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 6416)
  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 6416)
  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: PV248
FEATURES             Location/Qualifiers
     source          1..6416
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     1..17
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     24..43
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        24..42
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     promoter        77..2071
                     /label=Ubi promoter
                     /note="maize polyubiquitin gene promoter"
     CDS             2096..2116
                     /codon_start=1
                     /product="nuclear localization signal of SV40 (simian virus
                     40) large T antigen"
                     /label=SV40 NLS
                     /translation="PKKKRKV"
     promoter        complement(3237..3255)
                     /label=SP6 promoter
                     /note="promoter for bacteriophage SP6 RNA polymerase"
     primer_bind     complement(3238..3255)
                     /label=SP6
                     /note="SP6 promoter, forward primer"
     primer_bind     complement(3273..3289)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(3273..3289)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(3286..3308)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    3297..3313
                     /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(3321..3327,3328..3345,3346..3351))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    3366..3387
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(3504..3521)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(3675..4263)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(3755..3774)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     complement(4437..4456)
                     /label=pENTR-R
                     /note="pENTR vectors, reverse primer"
     CDS             4448..5263
                     /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             4448..5263
                     /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(4521..4540)
                     /label=Kan-R
                     /note="Kanamycin resistance gene, reverse primer"
     primer_bind     5557..5575
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     primer_bind     complement(5613..5635)
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     primer_bind     5735..5754
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     rep_origin      complement(5821..6276)
                     /direction=LEFT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(5958..5979)
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     6170..6189
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     join(6402..6416,1..8)
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     primer_bind     join(6416,1..17)
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
ORIGIN
        1 gtaaaacgac ggccagtgaa ttgtaatacg actcactata gggcgaattg ggttacccgg
       61 accgaagctt gcatgcctgc agtgcagcgt gacccggtcg tgcccctctc tagagataat
      121 gagcattgca tgtctaagtt ataaaaaatt accacatatt ttttttgtca cacttgtttg
      181 aagtgcagtt tatctatctt tatacatata tttaaacttt actctacgaa taatataatc
      241 tatagtacta caataatatc agtgttttag agaatcatat aaatgaacag ttagacatgg
      301 tctaaaggac aattgagtat tttgacaaca ggactctaca gttttatctt tttagtgtgc
      361 atgtgttctc cttttttttt gcaaatagct tcacctatat aatacttcat ccattttatt
      421 agtacatcca tttagggttt agggttaatg gtttttatag actaattttt ttagtacatc
      481 tattttattc tattttagcc tctaaattaa gaaaactaaa actctatttt agttttttta
      541 tttaataatt tagatataaa atagaataaa ataaagtgac taaaaattaa acaaataccc
      601 tttaagaaat taaaaaaact aaggaaacat ttttcttgtt tcgagtagat aatgccagcc
      661 tgttaaacgc cgtcgacgag tctaacggac accaaccagc gaaccagcag cgtcgcgtcg
      721 ggccaagcga agcagacggc acggcatctc tgtcgctgcc tctggacccc tctcgagagt
      781 tccgctccac cgttggactt gctccgctgt cggcatccag aaattgcgtg gcggagcggc
      841 agacgtgagc cggcacggca ggcggcctcc tcctcctctc acggcaccgg cagctacggg
      901 ggattccttt cccaccgctc cttcgctttc ccttcctcgc ccgccgtaat aaatagacac
      961 cccctccaca ccctctttcc ccaacctcgt gttgttcgga gcgcacacac acacaaccag
     1021 atctccccca aatccacccg tcggcacctc cgcttcaagg tacgccgctc gtcctccccc
     1081 cccccctctc taccttctct agatcggcgt tccggtccat gcatggttag ggcccggtag
     1141 ttctacttct gttcatgttt gtgttagatc cgtgtttgtg ttagatccgt gctgctagcg
     1201 ttcgtacacg gatgcgacct gtacgtcaga cacgttctga ttgctaactt gccagtgttt
     1261 ctctttgggg aatcctggga tggctctagc cgttccgcag acgggatcga tttcatgatt
     1321 ttttttgttt cgttgcatag ggtttggttt gcccttttcc tttatttcaa tatatgccgt
     1381 gcacttgttt gtcgggtcat cttttcatgc ttttttttgt cttggttgtg atgatgtggt
     1441 ctggttgggc ggtcgttcta gatcggagta gaattctgtt tcaaactacc tggtggattt
     1501 attaattttg gatctgtatg tgtgtgccat acatattcat agttacgaat tgaagatgat
     1561 ggatggaaat atcgatctag gataggtata catgttgatg cgggttttac tgatgcatat
     1621 acagagatgc tttttgttcg cttggttgtg atgatgtggt gtggttgggc ggtcgttcat
     1681 tcgttctaga tcggagtaga atactgtttc aaactacctg gtgtatttat taattttgga
     1741 actgtatgtg tgtgtcatac atcttcatag ttacgagttt aagatggatg gaaatatcga
     1801 tctaggatag gtatacatgt tgatgtgggt tttactgatg catatacatg atggcatatg
     1861 cagcatctat tcatatgctc taaccttgag tacctatcta ttataataaa caagtatgtt
     1921 ttataattat tttgatcttg atatacttgg atgatggcat atgcagcagc tatatgtgga
     1981 tttttttagc cctgccttca tacgctattt atttgcttgg tactgtttct tttgtcgatg
     2041 ctcaccctgt tgtttggtgt tacttctgca ggtcgactct agaggatcca tggcccccaa
     2101 gaagaagcgc aaggtcatga acgccatgaa aaccatcctc ctcaagttcg ccggccccct
     2161 ccagagctgg ggcaccagca gccacttcga aacccgccac accgacttct accccagcaa
     2221 gagcgcggtc atcggcctcc tcgccgccag cctcgggtac aggcgcgacg aggatgaaaa
     2281 aatccagaaa ttaaatgagc tcgacttcgc cgtcagggtc gaccagcagg gcaacctcct
     2341 cagggacttc cacgtcgccc agaagtacac cgagaagggc aagttcgacc gcaactacgt
     2401 caccaaccgc tactacctcg aggacgccgt gttcgtcgtc gccctcagcc acaccgacga
     2461 cagcttcatc gacatcatta aaggcgccct caagaacccc tacttccagc ccttcatggg
     2521 caggagggcc ctccccatca ccgccgactt catcctcggc gaaaccgacg tcaaccccct
     2581 cgagagcctg aagagcctca agtggcaggc cagcctctgg ttcatgaaaa agagccacga
     2641 caaggacgcc atcagcctcg aggtctacct cgacagccag ctcgtcaaca aggacgtcta
     2701 ccagctccgc caggactacg tccgcagctt cagccagaag ggcaggaggt tcggcttcag
     2761 gtacgaggtc cgcgaattaa tcagcgtcca gaacccccac tacctcaaca agaatacaga
     2821 gcacaacatc ttcgagagca tcggcgacta gtcttcgtaa gttaacctag acttgtccat
     2881 cttctggatt ggccaactta attaatgtat gaaataaaag gatgcacaca tagtgacatg
     2941 ctaatcacta taatgtgggc atcaaagttg tgtgttatgt gtaattacta gttatctgaa
     3001 taaaagagaa agagatcatc catatttctt atcctaaatg aatgtcacgt gtctttataa
     3061 ttctttgatg aaccagatgc atttcattaa ccaaatccat atacatataa atattaatca
     3121 tatataatta atatcaattg ggttagcaaa acaaatctag tctaggtgtg ttttgcgaat
     3181 gcggccgcca ccgcggtgga gctcgaattc cggtccgggt cacccagctt gagtattcta
     3241 tagtgtcacc taaatagctt ggcgtaatca tggtcatagc tgtttcctgt gtgaaattgt
     3301 tatccgctca caattccaca caacatacga gccggaagca taaagtgtaa agcctggggt
     3361 gcctaatgag tgagctaact cacattaatt gcgttgcgct cactgcccgc tttccagtcg
     3421 ggaaacctgt cgtgccagct gcattaatga atcggccaac gcgcggggag aggcggtttg
     3481 cgtattgggc gctcttccgc ttcctcgctc actgactcgc tgcgctcggt cgttcggctg
     3541 cggcgagcgg tatcagctca ctcaaaggcg gtaatacggt tatccacaga atcaggggat
     3601 aacgcaggaa agaacatgtg agcaaaaggc cagcaaaagg ccaggaaccg taaaaaggcc
     3661 gcgttgctgg cgtttttcca taggctccgc ccccctgacg agcatcacaa aaatcgacgc
     3721 tcaagtcaga ggtggcgaaa cccgacagga ctataaagat accaggcgtt tccccctgga
     3781 agctccctcg tgcgctctcc tgttccgacc ctgccgctta ccggatacct gtccgccttt
     3841 ctcccttcgg gaagcgtggc gctttctcat agctcacgct gtaggtatct cagttcggtg
     3901 taggtcgttc gctccaagct gggctgtgtg cacgaacccc ccgttcagcc cgaccgctgc
     3961 gccttatccg gtaactatcg tcttgagtcc aacccggtaa gacacgactt atcgccactg
     4021 gcagcagcca ctggtaacag gattagcaga gcgaggtatg taggcggtgc tacagagttc
     4081 ttgaagtggt ggcctaacta cggctacact agaagaacag tatttggtat ctgcgctctg
     4141 ctgaagccag ttaccttcgg aaaaagagtt ggtagctctt gatccggcaa acaaaccacc
     4201 gctggtagcg gtggtttttt tgtttgcaag cagcagatta cgcgcagaaa aaaaggatct
     4261 caagaagatc ctttgatctt ttctacgggg tctgacgctc agtggaacga aaactcacgt
     4321 taagggattt tggtcatgga gccacgttgt gtctcaaaat ctctgatgtt acattgcaca
     4381 agataaaaat atatcatcat gaacaataaa actgtctgct tacataaaca gtaatacaag
     4441 gggtgttatg agccatattc aacgggaaac gtcttgctcg aggccgcgat taaattccaa
     4501 catggatgct gatttatatg ggtataaatg ggctcgcgat aatgtcgggc aatcaggtgc
     4561 gacaatctat cgattgtatg ggaagcccga tgcgccagag ttgtttctga aacatggcaa
     4621 aggtagcgtt gccaatgatg ttacagatga gatggtcaga ctaaactggc tgacggaatt
     4681 tatgcctctt ccgaccatca agcattttat ccgtactcct gatgatgcat ggttactcac
     4741 cactgcgatc cccggaaaaa cagcattcca ggtattagaa gaatatcctg attcaggtga
     4801 aaatattgtt gatgcgctgg cagtgttcct gcgccggttg cattcgattc ctgtttgtaa
     4861 ttgtcctttt aacagcgatc gcgtatttcg tctcgctcag gcgcaatcac gaatgaataa
     4921 cggtttggtt gatgcgagtg attttgatga cgagcgtaat ggctggcctg ttgaacaagt
     4981 ctggaaagaa atgcataaac ttttgccatt ctcaccggat tcagtcgtca ctcatggtga
     5041 tttctcactt gataacctta tttttgacga ggggaaatta ataggttgta ttgatgttgg
     5101 acgagtcgga atcgcagacc gataccagga tcttgccatc ctatggaact gcctcggtga
     5161 gttttctcct tcattacaga aacggctttt tcagaaatat ggtattgata atcctgatat
     5221 gaataaattg cagtttcatt tgatgctcga tgagtttttc taatcagaat tggttaattg
     5281 gttgtaacac tggcagagca ttacgctgac ttgacgggac ggcggctttg ttgaataaat
     5341 cgaacttttg ctgagttgaa ggatcagatc acgcatcttc ccgacaacgc agaccgttcc
     5401 gtggcaaagc aaaagttcaa aatcaccaac tggtccacct acaacaaagc tctcatcaac
     5461 cgtggctccc tcactttctg gctggatgat ggggcgattc aggcctggta tgagtcagca
     5521 acaccttctt cacgagccat gacattaacc tataaaaata ggcgtatcac gaggcccttt
     5581 cgtctcgcgc gtttcggtga tgacggtgaa aacctctgac acatgcagct cccggagacg
     5641 gtcacagctt gtctgtaagc ggatgccggg agcagacaag cccgtcaggg cgcgtcagcg
     5701 ggtgttggcg ggtgtcgggg ctggcttaac tatgcggcat cagagcagat tgtactgaga
     5761 gtgcaccata tgcggtgtga aataccgcac agatgcgtaa ggagaaaata ccgcatcagg
     5821 aaattgtaag cgttaatatt ttgttaaaat tcgcgttaaa tttttgttaa atcagctcat
     5881 tttttaacca ataggccgaa atcggcaaaa tcccttataa atcaaaagaa tagaccgaga
     5941 tagggttgag tgttgttcca gtttggaaca agagtccact attaaagaac gtggactcca
     6001 acgtcaaagg gcgaaaaacc gtctatcagg gcgatggccc actacgtgaa ccatcaccct
     6061 aatcaagttt tttggggtcg aggtgccgta aagcactaaa tcggaaccct aaagggagcc
     6121 cccgatttag agcttgacgg ggaaagccgg cgaacgtggc gagaaaggaa gggaagaaag
     6181 cgaaaggagc gggcgctagg gcgctggcaa gtgtagcggt cacgctgcgc gtaaccacca
     6241 cacccgccgc gcttaatgcg ccgctacagg gcgcgtccat tcgccattca ggctgcgcaa
     6301 ctgttgggaa gggcgatcgg tgcgggcctc ttcgctatta cgccagctgg cgaaaggggg
     6361 atgtgctgca aggcgattaa gttgggtaac gccagggttt tcccagtcac gacgtt
//
