LOCUS       sequence-258478-        7350 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 7350)
  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 7350)
  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..7350
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     7..26
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        7..25
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     promoter        60..2055
                     /label=Ubi promoter
                     /note="maize polyubiquitin gene promoter"
     CDS             2080..2100
                     /codon_start=1
                     /product="nuclear localization signal of SV40 (simian virus
                     40) large T antigen"
                     /label=SV40 NLS
                     /translation="PKKKRKV"
     promoter        complement(4154..4172)
                     /label=SP6 promoter
                     /note="promoter for bacteriophage SP6 RNA polymerase"
     primer_bind     complement(4155..4172)
                     /label=SP6
                     /note="SP6 promoter, forward primer"
     primer_bind     complement(4190..4206)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(4190..4206)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(4203..4225)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    4214..4230
                     /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(4238..4244,4245..4262,4263..4268))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    4283..4304
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(4421..4438)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(4592..5180)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(4672..4691)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     complement(5354..5373)
                     /label=pENTR-R
                     /note="pENTR vectors, reverse primer"
     CDS             5365..6180
                     /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             5365..6180
                     /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(5438..5457)
                     /label=Kan-R
                     /note="Kanamycin resistance gene, reverse primer"
     primer_bind     6474..6492
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     primer_bind     complement(6530..6552)
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     primer_bind     6652..6671
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     rep_origin      complement(6738..7193)
                     /direction=LEFT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(6875..6896)
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     7087..7106
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     7319..7341
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     primer_bind     7333..7350
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     7334..7350
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
ORIGIN
        1 gaattgtaat acgactcact atagggcgaa ttgggttacc cggaccgaag cttgcatgcc
       61 tgcagtgcag cgtgacccgg tcgtgcccct ctctagagat aatgagcatt gcatgtctaa
      121 gttataaaaa attaccacat attttttttg tcacacttgt ttgaagtgca gtttatctat
      181 ctttatacat atatttaaac tttactctac gaataatata atctatagta ctacaataat
      241 atcagtgttt tagagaatca tataaatgaa cagttagaca tggtctaaag gacaattgag
      301 tattttgaca acaggactct acagttttat ctttttagtg tgcatgtgtt ctcctttttt
      361 tttgcaaata gcttcaccta tataatactt catccatttt attagtacat ccatttaggg
      421 tttagggtta atggttttta tagactaatt tttttagtac atctatttta ttctatttta
      481 gcctctaaat taagaaaact aaaactctat tttagttttt ttatttaata atttagatat
      541 aaaatagaat aaaataaagt gactaaaaat taaacaaata ccctttaaga aattaaaaaa
      601 actaaggaaa catttttctt gtttcgagta gataatgcca gcctgttaaa cgccgtcgac
      661 gagtctaacg gacaccaacc agcgaaccag cagcgtcgcg tcgggccaag cgaagcagac
      721 ggcacggcat ctctgtcgct gcctctggac ccctctcgag agttccgctc caccgttgga
      781 cttgctccgc tgtcggcatc cagaaattgc gtggcggagc ggcagacgtg agccggcacg
      841 gcaggcggcc tcctcctcct ctcacggcac cggcagctac gggggattcc tttcccaccg
      901 ctccttcgct ttcccttcct cgcccgccgt aataaataga caccccctcc acaccctctt
      961 tccccaacct cgtgttgttc ggagcgcaca cacacacaac cagatctccc ccaaatccac
     1021 ccgtcggcac ctccgcttca aggtacgccg ctcgtcctcc cccccccccn tctctacctt
     1081 ctctagatcg gcgttccggt ccatgcatgg ttagggcccg gtagttctac ttctgttcat
     1141 gtttgtgtta gatccgtgtt tgtgttagat ccgtgctgct agcgttcgta cacggatgcg
     1201 acctgtacgt cagacacgtt ctgattgcta acttgccagt gtttctcttt ggggaatcct
     1261 gggatggctc tagccgttcc gcagacggga tcgatttcat gatttttttt gtttcgttgc
     1321 atagggtttg gtttgccctt ttcctttatt tcaatatatg ccgtgcactt gtttgtcggg
     1381 tcatcttttc atgctttttt ttgtcttggt tgtgatgatg tggtctggtt gggcggtcgt
     1441 tctagatcgg agtagaattc tgtttcaaac tacctggtgg atttattaat tttggatctg
     1501 tatgtgtgtg ccatacatat tcatagttac gaattgaaga tgatggatgg aaatatcgat
     1561 ctaggatagg tatacatgtt gatgcgggtt ttactgatgc atatacagag atgctttttg
     1621 ttcgcttggt tgtgatgatg tggtgtggtt gggcggtcgt tcattcgttc tagatcggag
     1681 tagaatactg tttcaaacta cctggtgtat ttattaattt tggaactgta tgtgtgtgtc
     1741 atacatcttc atagttacga gtttaagatg gatggaaata tcgatctagg ataggtatac
     1801 atgttgatgt gggttttact gatgcatata catgatggca tatgcagcat ctattcatat
     1861 gctctaacct tgagtaccta tctattataa taaacaagta tgttttataa ttattttgat
     1921 cttgatatac ttggatgatg gcatatgcag cagctatatg tggatttttt tagccctgcc
     1981 ttcatacgct atttatttgc ttggtactgt ttcttttgtc gatgctcacc ctgttgtttg
     2041 gtgttacttc tgcaggtcga ctctagagga tccatggccc ccaagaagaa gcgcaaggtc
     2101 atgagccgct tcaacctcct cgacgagccc tggatcagcg tcatctacga cgagaagggc
     2161 agcaccaagg acgtcagcct ccaggacctc ttcaccaacg cccaccagta caaggagctc
     2221 gccggcgaca ccaagaccca ggacttcgcc gtcctcaggg tcctcctcgc cgtcctccac
     2281 accgtgttca gccgcttcga catcgacggc aacgcctacg agtacctcac catcgacgag
     2341 ggctggaacc agctcgagcc cgtcgacgag atggacatcg agaactacga ggaggccctc
     2401 tatgaaacat gggagaagct ctggaccaat aaaagattcc ccaacatcgt caatcaatac
     2461 ctcgagaagt ggagggaccg cttctacctc ttcgaccaga agtacccctt cttccaggtc
     2521 accaaggagg acatcgccgg cgataaaatt agcaaggcca agggcaccag catcctcggc
     2581 aaaaatataa atcgcatcat cagcgagagc ggcaacaaga tcgccctctt cagccccaag
     2641 gacgaggaaa ataaaaatac tctcaccgcc gccgagctcg ccaggtggct catcaccttc
     2701 cagggctaca gcggcgtcag cgacaaggtc atcttcggca atgaaaaata tacccccagc
     2761 aagggctggc tcttcgacat cggcgccatc tacattaaag gcaacaccct cttcgaaacc
     2821 ctcctcctca actacatcag cccctacaac gagtgcggca atgtggaaaa tatacagagg
     2881 ccctgctggg agaggaagag cagcgacatc attaaaagct acctcgatga aaaagacatc
     2941 accaacatcg ccagcctcta caccgtctgg agcaagggca tctacatcga ccccgacttc
     3001 aacctcaata aacccttcag cttcgacatc gtcaagctcc ccgacatcaa ccaccgcgac
     3061 aacttcctcg agcccatgac cctctggaag tacaacgtca gcggcaaaaa tagggacagc
     3121 tacaccccca agaagcacct cctcaaccag agcctctgga ggagcttcgg cctcctgagc
     3181 ataaaagaca ccgacctcca gcacaggaag cccggcgtca tcgagtggct cacctacatc
     3241 gacgatataa taggcaacag gctcagcaac atcgtcgcca tcagcatgca ggacgacggc
     3301 aacccccaga gcaggctgcc caccgacgag gtcatcgaca gcatcttcat caacgacctc
     3361 gtcctcaccg acctcgacga gggcggctgg gtccccagga tcaacgaggt cgtcgaggaa
     3421 accaagaaga tcatcagcag gacatataaa acgtacatta atgatataaa ggagatccgc
     3481 aacatcagca acggcagcta cacccagcag atcgtcgaga gcctctactt caagatcgac
     3541 cagcccttca ggcagtggct cgccagcatc cagcccgagg acgacaagga cagcaagatc
     3601 caggagtggc gcgtcctcct caagaaaata gtcaaggccg aggccgaggt catcctccac
     3661 cagggcggcg ccagggacta cctcggcatc cagaaggacg gccgaattaa gaacatcgcc
     3721 accgcctaca acagcttcga cttctggctc cgccagcagt taaaatagtc ttcgtaagtt
     3781 aacctagact tgtccatctt ctggattggc caacttaatt aatgtatgaa ataaaaggat
     3841 gcacacatag tgacatgcta atcactataa tgtgggcatc aaagttgtgt gttatgtgta
     3901 attactagtt atctgaataa aagagaaaga gatcatccat atttcttatc ctaaatgaat
     3961 gtcacgtgtc tttataattc tttgatgaac cagatgcatt tcattaacca aatccatata
     4021 catataaata ttaatcatat ataattaata tcaattgggt tagcaaaaca aatctagtct
     4081 aggtgtgttt tgcgaatgcg gccgccaccg cggtggagct cgaattccgg tccgggtcac
     4141 ccagcttgag tattctatag tgtcacctaa atagcttggc gtaatcatgg tcatagctgt
     4201 ttcctgtgtg aaattgttat ccgctcacaa ttccacacaa catacgagcc ggaagcataa
     4261 agtgtaaagc ctggggtgcc taatgagtga gctaactcac attaattgcg ttgcgctcac
     4321 tgcccgcttt ccagtcggga aacctgtcgt gccagctgca ttaatgaatc ggccaacgcg
     4381 cggggagagg cggtttgcgt attgggcgct cttccgcttc ctcgctcact gactcgctgc
     4441 gctcggtcgt tcggctgcgg cgagcggtat cagctcactc aaaggcggta atacggttat
     4501 ccacagaatc aggggataac gcaggaaaga acatgtgagc aaaaggccag caaaaggcca
     4561 ggaaccgtaa aaaggccgcg ttgctggcgt ttttccatag gctccgcccc cctgacgagc
     4621 atcacaaaaa tcgacgctca agtcagaggt ggcgaaaccc gacaggacta taaagatacc
     4681 aggcgtttcc ccctggaagc tccctcgtgc gctctcctgt tccgaccctg ccgcttaccg
     4741 gatacctgtc cgcctttctc ccttcgggaa gcgtggcgct ttctcatagc tcacgctgta
     4801 ggtatctcag ttcggtgtag gtcgttcgct ccaagctggg ctgtgtgcac gaaccccccg
     4861 ttcagcccga ccgctgcgcc ttatccggta actatcgtct tgagtccaac ccggtaagac
     4921 acgacttatc gccactggca gcagccactg gtaacaggat tagcagagcg aggtatgtag
     4981 gcggtgctac agagttcttg aagtggtggc ctaactacgg ctacactaga agaacagtat
     5041 ttggtatctg cgctctgctg aagccagtta ccttcggaaa aagagttggt agctcttgat
     5101 ccggcaaaca aaccaccgct ggtagcggtg gtttttttgt ttgcaagcag cagattacgc
     5161 gcagaaaaaa aggatctcaa gaagatcctt tgatcttttc tacggggtct gacgctcagt
     5221 ggaacgaaaa ctcacgttaa gggattttgg tcatggagcc acgttgtgtc tcaaaatctc
     5281 tgatgttaca ttgcacaaga taaaaatata tcatcatgaa caataaaact gtctgcttac
     5341 ataaacagta atacaagggg tgttatgagc catattcaac gggaaacgtc ttgctcgagg
     5401 ccgcgattaa attccaacat ggatgctgat ttatatgggt ataaatgggc tcgcgataat
     5461 gtcgggcaat caggtgcgac aatctatcga ttgtatggga agcccgatgc gccagagttg
     5521 tttctgaaac atggcaaagg tagcgttgcc aatgatgtta cagatgagat ggtcagacta
     5581 aactggctga cggaatttat gcctcttccg accatcaagc attttatccg tactcctgat
     5641 gatgcatggt tactcaccac tgcgatcccc ggaaaaacag cattccaggt attagaagaa
     5701 tatcctgatt caggtgaaaa tattgttgat gcgctggcag tgttcctgcg ccggttgcat
     5761 tcgattcctg tttgtaattg tccttttaac agcgatcgcg tatttcgtct cgctcaggcg
     5821 caatcacgaa tgaataacgg tttggttgat gcgagtgatt ttgatgacga gcgtaatggc
     5881 tggcctgttg aacaagtctg gaaagaaatg cataaacttt tgccattctc accggattca
     5941 gtcgtcactc atggtgattt ctcacttgat aaccttattt ttgacgaggg gaaattaata
     6001 ggttgtattg atgttggacg agtcggaatc gcagaccgat accaggatct tgccatccta
     6061 tggaactgcc tcggtgagtt ttctccttca ttacagaaac ggctttttca gaaatatggt
     6121 attgataatc ctgatatgaa taaattgcag tttcatttga tgctcgatga gtttttctaa
     6181 tcagaattgg ttaattggtt gtaacactgg cagagcatta cgctgacttg acgggacggc
     6241 ggctttgttg aataaatcga acttttgctg agttgaagga tcagatcacg catcttcccg
     6301 acaacgcaga ccgttccgtg gcaaagcaaa agttcaaaat caccaactgg tccacctaca
     6361 acaaagctct catcaaccgt ggctccctca ctttctggct ggatgatggg gcgattcagg
     6421 cctggtatga gtcagcaaca ccttcttcac gagccatgac attaacctat aaaaataggc
     6481 gtatcacgag gccctttcgt ctcgcgcgtt tcggtgatga cggtgaaaac ctctgacaca
     6541 tgcagctccc ggagacggtc acagcttgtc tgtaagcgga tgccgggagc agacaagccc
     6601 gtcagggcgc gtcagcgggt gttggcgggt gtcggggctg gcttaactat gcggcatcag
     6661 agcagattgt actgagagtg caccatatgc ggtgtgaaat accgcacaga tgcgtaagga
     6721 gaaaataccg catcaggaaa ttgtaagcgt taatattttg ttaaaattcg cgttaaattt
     6781 ttgttaaatc agctcatttt ttaaccaata ggccgaaatc ggcaaaatcc cttataaatc
     6841 aaaagaatag accgagatag ggttgagtgt tgttccagtt tggaacaaga gtccactatt
     6901 aaagaacgtg gactccaacg tcaaagggcg aaaaaccgtc tatcagggcg atggcccact
     6961 acgtgaacca tcaccctaat caagtttttt ggggtcgagg tgccgtaaag cactaaatcg
     7021 gaaccctaaa gggagccccc gatttagagc ttgacgggga aagccggcga acgtggcgag
     7081 aaaggaaggg aagaaagcga aaggagcggg cgctagggcg ctggcaagtg tagcggtcac
     7141 gctgcgcgta accaccacac ccgccgcgct taatgcgccg ctacagggcg cgtccattcg
     7201 ccattcaggc tgcgcaactg ttgggaaggg cgatcggtgc gggcctcttc gctattacgc
     7261 cagctggcga aagggggatg tgctgcaagg cgattaagtt gggtaacgcc agggttttcc
     7321 cagtcacgac gttgtaaaac gacggccagt
//
