LOCUS       sequence-254115-        7357 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  CasA constitutive expression cassette for Zea mays.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 7357)
  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 7357)
  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: PV247
FEATURES             Location/Qualifiers
     source          1..7357
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     4..21
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     5..21
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     28..47
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        28..46
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     promoter        81..2076
                     /label=Ubi promoter
                     /note="maize polyubiquitin gene promoter"
     CDS             2101..2121
                     /codon_start=1
                     /product="nuclear localization signal of SV40 (simian virus
                     40) large T antigen"
                     /label=SV40 NLS
                     /translation="PKKKRKV"
     promoter        complement(4175..4193)
                     /label=SP6 promoter
                     /note="promoter for bacteriophage SP6 RNA polymerase"
     primer_bind     complement(4176..4193)
                     /label=SP6
                     /note="SP6 promoter, forward primer"
     primer_bind     complement(4211..4227)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(4211..4227)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(4224..4246)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    4235..4251
                     /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)."
     protein_bind    4311..4332
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(4449..4466)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(4620..5208)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(4700..4719)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     complement(5382..5401)
                     /label=pENTR-R
                     /note="pENTR vectors, reverse primer"
     CDS             5393..6208
                     /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             5393..6208
                     /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(5466..5485)
                     /label=Kan-R
                     /note="Kanamycin resistance gene, reverse primer"
     primer_bind     6502..6520
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     primer_bind     complement(6558..6580)
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     primer_bind     6680..6699
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     rep_origin      complement(6766..7221)
                     /direction=LEFT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(6903..6924)
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     7115..7134
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     join(7347..7357,1..12)
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
ORIGIN
        1 cgttgtaaaa cgacggccag tgaattgtaa tacgactcac tatagggcga attgggttac
       61 ccggaccgaa gcttgcatgc ctgcagtgca gcgtgacccg gtcgtgcccc tctctagaga
      121 taatgagcat tgcatgtcta agttataaaa aattaccaca tatttttttt gtcacacttg
      181 tttgaagtgc agtttatcta tctttataca tatatttaaa ctttactcta cgaataatat
      241 aatctatagt actacaataa tatcagtgtt ttagagaatc atataaatga acagttagac
      301 atggtctaaa ggacaattga gtattttgac aacaggactc tacagtttta tctttttagt
      361 gtgcatgtgt tctccttttt ttttgcaaat agcttcacct atataatact tcatccattt
      421 tattagtaca tccatttagg gtttagggtt aatggttttt atagactaat ttttttagta
      481 catctatttt attctatttt agcctctaaa ttaagaaaac taaaactcta ttttagtttt
      541 tttatttaat aatttagata taaaatagaa taaaataaag tgactaaaaa ttaaacaaat
      601 accctttaag aaattaaaaa aactaaggaa acatttttct tgtttcgagt agataatgcc
      661 agcctgttaa acgccgtcga cgagtctaac ggacaccaac cagcgaacca gcagcgtcgc
      721 gtcgggccaa gcgaagcaga cggcacggca tctctgtcgc tgcctctgga cccctctcga
      781 gagttccgct ccaccgttgg acttgctccg ctgtcggcat ccagaaattg cgtggcggag
      841 cggcagacgt gagccggcac ggcaggcggc ctcctcctcc tctcacggca ccggcagcta
      901 cgggggattc ctttcccacc gctccttcgc tttcccttcc tcgcccgccg taataaatag
      961 acaccccctc cacaccctct ttccccaacc tcgtgttgtt cggagcgcac acacacacaa
     1021 ccagatctcc cccaaatcca cccgtcggca cctccgcttc aaggtacgcc gctcgtcctc
     1081 cccccccccc ctctctacct tctctagatc ggcgttccgg tccatgcatg gttagggccc
     1141 ggtagttcta cttctgttca tgtttgtgtt agatccgtgt ttgtgttaga tccgtgctgc
     1201 tagcgttcgt acacggatgc gacctgtacg tcagacacgt tctgattgct aacttgccag
     1261 tgtttctctt tggggaatcc tgggatggct ctagccgttc cgcagacggg atcgatttca
     1321 tgattttttt tgtttcgttg catagggttt ggtttgccct tttcctttat ttcaatatat
     1381 gccgtgcact tgtttgtcgg gtcatctttt catgcttttt tttgtcttgg ttgtgatgat
     1441 gtggtctggt tgggcggtcg ttctagatcg gagtagaatt ctgtttcaaa ctacctggtg
     1501 gatttattaa ttttggatct gtatgtgtgt gccatacata ttcatagtta cgaattgaag
     1561 atgatggatg gaaatatcga tctaggatag gtatacatgt tgatgcgggt tttactgatg
     1621 catatacaga gatgcttttt gttcgcttgg ttgtgatgat gtggtgtggt tgggcggtcg
     1681 ttcattcgtt ctagatcgga gtagaatact gtttcaaact acctggtgta tttattaatt
     1741 ttggaactgt atgtgtgtgt catacatctt catagttacg agtttaagat ggatggaaat
     1801 atcgatctag gataggtata catgttgatg tgggttttac tgatgcatat acatgatggc
     1861 atatgcagca tctattcata tgctctaacc ttgagtacct atctattata ataaacaagt
     1921 atgttttata attattttga tcttgatata cttggatgat ggcatatgca gcagctatat
     1981 gtggattttt ttagccctgc cttcatacgc tatttatttg cttggtactg tttcttttgt
     2041 cgatgctcac cctgttgttt ggtgttactt ctgcaggtcg actctagagg atccatggcc
     2101 cccaagaaga agcgcaaggt catgagccgc ttcaacctcc tcgacgagcc ctggatcagc
     2161 gtcatctacg acgagaaggg cagcaccaag gacgtcagcc tccaggacct cttcaccaac
     2221 gcccaccagt acaaggagct cgccggcgac accaagaccc aggacttcgc cgtcctcagg
     2281 gtcctcctcg ccgtcctcca caccgtgttc agccgcttcg acatcgacgg caacgcctac
     2341 gagtacctca ccatcgacga gggctggaac cagctcgagc ccgtcgacga gatggacatc
     2401 gagaactacg aggaggccct ctatgaaaca tgggagaagc tctggaccaa taaaagattc
     2461 cccaacatcg tcaatcaata cctcgagaag tggagggacc gcttctacct cttcgaccag
     2521 aagtacccct tcttccaggt caccaaggag gacatcgccg gcgataaaat tagcaaggcc
     2581 aagggcacca gcatcctcgg caaaaatata aatcgcatca tcagcgagag cggcaacaag
     2641 atcgccctct tcagccccaa ggacgaggaa aataaaaata ctctcaccgc cgccgagctc
     2701 gccaggtggc tcatcacctt ccagggctac agcggcgtca gcgacaaggt catcttcggc
     2761 aatgaaaaat atacccccag caagggctgg ctcttcgaca tcggcgccat ctacattaaa
     2821 ggcaacaccc tcttcgaaac cctcctcctc aactacatca gcccctacaa cgagtgcggc
     2881 aatgtggaaa atatacagag gccctgctgg gagaggaaga gcagcgacat cattaaaagc
     2941 tacctcgatg aaaaagacat caccaacatc gccagcctct acaccgtctg gagcaagggc
     3001 atctacatcg accccgactt caacctcaat aaacccttca gcttcgacat cgtcaagctc
     3061 cccgacatca accaccgcga caacttcctc gagcccatga ccctctggaa gtacaacgtc
     3121 agcggcaaaa atagggacag ctacaccccc aagaagcacc tcctcaacca gagcctctgg
     3181 aggagcttcg gcctcctgag cataaaagac accgacctcc agcacaggaa gcccggcgtc
     3241 atcgagtggc tcacctacat cgacgatata ataggcaaca ggctcagcaa catcgtcgcc
     3301 atcagcatgc aggacgacgg caacccccag agcaggctgc ccaccgacga ggtcatcgac
     3361 agcatcttca tcaacgacct cgtcctcacc gacctcgacg agggcggctg ggtccccagg
     3421 atcaacgagg tcgtcgagga aaccaagaag atcatcagca ggacatataa aacgtacatt
     3481 aatgatataa aggagatccg caacatcagc aacggcagct acacccagca gatcgtcgag
     3541 agcctctact tcaagatcga ccagcccttc aggcagtggc tcgccagcat ccagcccgag
     3601 gacgacaagg acagcaagat ccaggagtgg cgcgtcctcc tcaagaaaat agtcaaggcc
     3661 gaggccgagg tcatcctcca ccagggcggc gccagggact acctcggcat ccagaaggac
     3721 ggccgaatta agaacatcgc caccgcctac aacagcttcg acttctggct ccgccagcag
     3781 ttaaaatagt cttcgtaagt taacctagac ttgtccatct tctggattgg ccaacttaat
     3841 taatgtatga aataaaagga tgcacacata gtgacatgct aatcactata atgtgggcat
     3901 caaagttgtg tgttatgtgt aattactagt tatctgaata aaagagaaag agatcatcca
     3961 tatttcttat cctaaatgaa tgtcacgtgt ctttataatt ctttgatgaa ccagatgcat
     4021 ttcattaacc aaatccatat acatataaat attaatcata tataattaat atcaattggg
     4081 ttagcaaaac aaatctagtc taggtgtgtt ttgcgaatgc ggccgccacc gcggtggagc
     4141 tcgaattccg gtccgggtca cccagcttga gtattctata gtgtcaccta aatagcttgg
     4201 cgtaatcatg gtcatagctg tttcctgtgt gaaattgtta tccgctcaca attccacaca
     4261 acatacgagc cggaagcata aagcataaag tgtaaagcct ggggtgccta atgagtgagc
     4321 taactcacat taattgcgtt gcgctcactg cccgctttcc agtcgggaaa cctgtcgtgc
     4381 cagctgcatt aatgaatcgg ccaacgcgcg gggagaggcg gtttgcgtat tgggcgctct
     4441 tccgcttcct cgctcactga ctcgctgcgc tcggtcgttc ggctgcggcg agcggtatca
     4501 gctcactcaa aggcggtaat acggttatcc acagaatcag gggataacgc aggaaagaac
     4561 atgtgagcaa aaggccagca aaaggccagg aaccgtaaaa aggccgcgtt gctggcgttt
     4621 ttccataggc tccgcccccc tgacgagcat cacaaaaatc gacgctcaag tcagaggtgg
     4681 cgaaacccga caggactata aagataccag gcgtttcccc ctggaagctc cctcgtgcgc
     4741 tctcctgttc cgaccctgcc gcttaccgga tacctgtccg cctttctccc ttcgggaagc
     4801 gtggcgcttt ctcatagctc acgctgtagg tatctcagtt cggtgtaggt cgttcgctcc
     4861 aagctgggct gtgtgcacga accccccgtt cagcccgacc gctgcgcctt atccggtaac
     4921 tatcgtcttg agtccaaccc ggtaagacac gacttatcgc cactggcagc agccactggt
     4981 aacaggatta gcagagcgag gtatgtaggc ggtgctacag agttcttgaa gtggtggcct
     5041 aactacggct acactagaag aacagtattt ggtatctgcg ctctgctgaa gccagttacc
     5101 ttcggaaaaa gagttggtag ctcttgatcc ggcaaacaaa ccaccgctgg tagcggtggt
     5161 ttttttgttt gcaagcagca gattacgcgc agaaaaaaag gatctcaaga agatcctttg
     5221 atcttttcta cggggtctga cgctcagtgg aacgaaaact cacgttaagg gattttggtc
     5281 atggagccac gttgtgtctc aaaatctctg atgttacatt gcacaagata aaaatatatc
     5341 atcatgaaca ataaaactgt ctgcttacat aaacagtaat acaaggggtg ttatgagcca
     5401 tattcaacgg gaaacgtctt gctcgaggcc gcgattaaat tccaacatgg atgctgattt
     5461 atatgggtat aaatgggctc gcgataatgt cgggcaatca ggtgcgacaa tctatcgatt
     5521 gtatgggaag cccgatgcgc cagagttgtt tctgaaacat ggcaaaggta gcgttgccaa
     5581 tgatgttaca gatgagatgg tcagactaaa ctggctgacg gaatttatgc ctcttccgac
     5641 catcaagcat tttatccgta ctcctgatga tgcatggtta ctcaccactg cgatccccgg
     5701 aaaaacagca ttccaggtat tagaagaata tcctgattca ggtgaaaata ttgttgatgc
     5761 gctggcagtg ttcctgcgcc ggttgcattc gattcctgtt tgtaattgtc cttttaacag
     5821 cgatcgcgta tttcgtctcg ctcaggcgca atcacgaatg aataacggtt tggttgatgc
     5881 gagtgatttt gatgacgagc gtaatggctg gcctgttgaa caagtctgga aagaaatgca
     5941 taaacttttg ccattctcac cggattcagt cgtcactcat ggtgatttct cacttgataa
     6001 ccttattttt gacgagggga aattaatagg ttgtattgat gttggacgag tcggaatcgc
     6061 agaccgatac caggatcttg ccatcctatg gaactgcctc ggtgagtttt ctccttcatt
     6121 acagaaacgg ctttttcaga aatatggtat tgataatcct gatatgaata aattgcagtt
     6181 tcatttgatg ctcgatgagt ttttctaatc agaattggtt aattggttgt aacactggca
     6241 gagcattacg ctgacttgac gggacggcgg ctttgttgaa taaatcgaac ttttgctgag
     6301 ttgaaggatc agatcacgca tcttcccgac aacgcagacc gttccgtggc aaagcaaaag
     6361 ttcaaaatca ccaactggtc cacctacaac aaagctctca tcaaccgtgg ctccctcact
     6421 ttctggctgg atgatggggc gattcaggcc tggtatgagt cagcaacacc ttcttcacga
     6481 gccatgacat taacctataa aaataggcgt atcacgaggc cctttcgtct cgcgcgtttc
     6541 ggtgatgacg gtgaaaacct ctgacacatg cagctcccgg agacggtcac agcttgtctg
     6601 taagcggatg ccgggagcag acaagcccgt cagggcgcgt cagcgggtgt tggcgggtgt
     6661 cggggctggc ttaactatgc ggcatcagag cagattgtac tgagagtgca ccatatgcgg
     6721 tgtgaaatac cgcacagatg cgtaaggaga aaataccgca tcaggaaatt gtaagcgtta
     6781 atattttgtt aaaattcgcg ttaaattttt gttaaatcag ctcatttttt aaccaatagg
     6841 ccgaaatcgg caaaatccct tataaatcaa aagaatagac cgagataggg ttgagtgttg
     6901 ttccagtttg gaacaagagt ccactattaa agaacgtgga ctccaacgtc aaagggcgaa
     6961 aaaccgtcta tcagggcgat ggcccactac gtgaaccatc accctaatca agttttttgg
     7021 ggtcgaggtg ccgtaaagca ctaaatcgga accctaaagg gagcccccga tttagagctt
     7081 gacggggaaa gccggcgaac gtggcgagaa aggaagggaa gaaagcgaaa ggagcgggcg
     7141 ctagggcgct ggcaagtgta gcggtcacgc tgcgcgtaac caccacaccc gccgcgctta
     7201 atgcgccgct acagggcgcg tccattcgcc attcaggctg cgcaactgtt gggaagggcg
     7261 atcggtgcgg gcctcttcgc tattacgcca gctggcgaaa gggggatgtg ctgcaaggcg
     7321 attaagttgg gtaacgccag ggttttccca gtcacga
//
