LOCUS       sequence-258182-        4440 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Cas9 R promoter targeting sgRNA2 expression cassette for Zea mays.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 4440)
  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 4440)
  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: PV523
FEATURES             Location/Qualifiers
     source          1..4440
                     /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"
     primer_bind     67..83
                     /label=pBluescriptKS
                     /note="For pBluescript vector"
     primer_bind     68..84
                     /label=KS primer
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     misc_RNA        1113..1188
                     /label=gRNA scaffold
                     /note="guide RNA scaffold for the Streptococcus pyogenes 
                     CRISPR/Cas9 system"
     promoter        complement(1653..1671)
                     /label=T3 promoter
                     /note="promoter for bacteriophage T3 RNA polymerase"
     primer_bind     complement(1692..1708)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(1692..1708)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(1705..1727)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    1716..1732
                     /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(1740..1746,1747..1764,1765..1770))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    1785..1806
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(1923..1940)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(2094..2682)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(2174..2193)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(2853..3644,3645..3713))
                     /codon_start=1
                     /gene="bla"
                     /product="beta-lactamase"
                     /label=AmpR
                     /note="confers resistance to ampicillin, carbenicillin, and
                     related antibiotics"
                     /translation="MSIQHFRVALIPFFAAFCLPVFAHPETLVKVKDAEDQLGARVGYI
                     ELDLNSGKILESFRPEERFPMMSTFKVLLCGAVLSRIDAGQEQLGRRIHYSQNDLVEYS
                     PVTEKHLTDGMTVRELCSAAITMSDNTAANLLLTTIGGPKELTAFLHNMGDHVTRLDRW
                     EPELNEAIPNDERDTTMPVAMATTLRKLLTGELLTLASRQQLIDWMEADKVAGPLLRSA
                     LPAGWFIADKSGAGERGSRGIIAALGPDGKPSRIVVIYTTGSQATMDERNRQIAEIGAS
                     LIKHW"
     primer_bind     3476..3495
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(3714..3818)
                     /gene="bla"
                     /label=AmpR promoter
     rep_origin      complement(3844..4299)
                     /direction=LEFT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(3981..4002)
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     4193..4212
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     join(4426..4440,1..8)
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     primer_bind     join(4440,1..17)
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
ORIGIN
        1 gtaaaacgac ggccagtgaa ttgtaatacg actcactata gggcgaattg ggtaccgggc
       61 cccccctcga ggtcgacggt atcgataagc tttgagagta caatgatgaa cctagattaa
      121 tcaatgccaa agtctgaaaa atgcaccctc agtctatgat ccagaaaatc aagattgctt
      181 gaggccctgt tcggttgttc cggattagag ccccggatta attcctagcc ggattacttc
      241 tctaatttat atagattttg atgagctgga atgaatcctg gcttattccg gtacaaccga
      301 acaggccctg aaggatacca gtaatcgctg agctaaattg gcatgctgtc agagtgtcag
      361 tattgcagca aggtagtgag ataaccggca tcatggtgcc agtttgatgg caccattagg
      421 gttagagatg gtggccatgg gcgcatgtcc tggccaactt tgtatgatat atggcagggt
      481 gaataggaaa gtaaaattgt attgtaaaaa gggatttctt ctgtttgtta gcgcatgtac
      541 aaggaatgca agttttgagc gagggggcat caaagatctg gctgtgtttc cagctgtttt
      601 tgttagcccc atcgaatcct tgacataatg atcccgctta aataagcaac ctcgcttgta
      661 tagttccttg tgctctaaca cacgatgatg ataagtcgta aaatagtggt gtccaaagaa
      721 tttccaggcc cagttgtaaa agctaaaatg ctattcgaat ttctactagc agtaagtcgt
      781 gtttagaaat tattttttta tatacctttt ttccttctat gtacagtagg acacagtgtc
      841 agcgccgcgt tgacggagaa tatttgcaaa aaagtaaaag agaaagtcat agcggcgtat
      901 gtgccaaaaa cttcgtcaca gagagggcca taagaaacat ggcccacggc ccaatacgaa
      961 gcaccgcgac gaagcccaaa cagcagtccg taggtggagc aaagcgctgg gtaatacgca
     1021 aacgttttgt cccaccttga ctaatcacaa gagtggagcg taccttataa accgagccgc
     1081 aagcaccgaa ttgagtgcta taatatgagt gggttttaga gctagaaata gcaagttaaa
     1141 ataaggctag tccgttatca acttgaaaaa gtggcaccga gtcggtgctt tttttttgcg
     1201 gccgcgaatt cctgcagggc cctcttgtcg gaccagttgc ccaccacgtt ggtgagctcg
     1261 gtgaggccct tcattgagag gaaggaggtc atgaggtgcc taccgatgtg ggacttgggg
     1321 ccgttcttga tggcgaagat ggagtagggg gcgttcttct tgagggcctt gttgtaggac
     1381 ctcacgaggt tgtccttgag gagctggtac tcctgcttgt tggaggagga gttgccggtc
     1441 ctgttcaccc tcttgagcac gggctctgag ttcctgagga actcgtcgag gtacacgagg
     1501 gggtcgatcc tgccgcgagc ggagaagaag tagatgtgcc tggacacgga ggtcttggtc
     1561 tcggtcacga ggcactggat gatcacgccg aggtacttgt tctgactagt tctagagcgg
     1621 ccgccaccgc ggtggagctc cagcttttgt tccctttagt gagggttaat ttcgagcttg
     1681 gcgtaatcat ggtcatagct gtttcctgtg tgaaattgtt atccgctcac aattccacac
     1741 aacatacgag ccggaagcat aaagtgtaaa gcctggggtg cctaatgagt gagctaactc
     1801 acattaattg cgttgcgctc actgcccgct ttccagtcgg gaaacctgtc gtgccagctg
     1861 cattaatgaa tcggccaacg cgcggggaga ggcggtttgc gtattgggcg ctcttccgct
     1921 tcctcgctca ctgactcgct gcgctcggtc gttcggctgc ggcgagcggt atcagctcac
     1981 tcaaaggcgg taatacggtt atccacagaa tcaggggata acgcaggaaa gaacatgtga
     2041 gcaaaaggcc agcaaaaggc caggaaccgt aaaaaggccg cgttgctggc gtttttccat
     2101 aggctccgcc cccctgacga gcatcacaaa aatcgacgct caagtcagag gtggcgaaac
     2161 ccgacaggac tataaagata ccaggcgttt ccccctggaa gctccctcgt gcgctctcct
     2221 gttccgaccc tgccgcttac cggatacctg tccgcctttc tcccttcggg aagcgtggcg
     2281 ctttctcata gctcacgctg taggtatctc agttcggtgt aggtcgttcg ctccaagctg
     2341 ggctgtgtgc acgaaccccc cgttcagccc gaccgctgcg ccttatccgg taactatcgt
     2401 cttgagtcca acccggtaag acacgactta tcgccactgg cagcagccac tggtaacagg
     2461 attagcagag cgaggtatgt aggcggtgct acagagttct tgaagtggtg gcctaactac
     2521 ggctacacta gaagaacagt atttggtatc tgcgctctgc tgaagccagt taccttcgga
     2581 aaaagagttg gtagctcttg atccggcaaa caaaccaccg ctggtagcgg tggttttttt
     2641 gtttgcaagc agcagattac gcgcagaaaa aaaggatctc aagaagatcc tttgatcttt
     2701 tctacggggt ctgacgctca gtggaacgaa aactcacgtt aagggatttt ggtcatgaga
     2761 ttatcaaaaa ggatcttcac ctagatcctt ttaaattaaa aatgaagttt taaatcaatc
     2821 taaagtatat atgagtaaac ttggtctgac agttaccaat gcttaatcag tgaggcacct
     2881 atctcagcga tctgtctatt tcgttcatcc atagttgcct gactccccgt cgtgtagata
     2941 actacgatac gggagggctt accatctggc cccagtgctg caatgatacc gcgagaccca
     3001 cgctcaccgg ctccagattt atcagcaata aaccagccag ccggaagggc cgagcgcaga
     3061 agtggtcctg caactttatc cgcctccatc cagtctatta attgttgccg ggaagctaga
     3121 gtaagtagtt cgccagttaa tagtttgcgc aacgttgttg ccattgctac aggcatcgtg
     3181 gtgtcacgct cgtcgtttgg tatggcttca ttcagctccg gttcccaacg atcaaggcga
     3241 gttacatgat cccccatgtt gtgcaaaaaa gcggttagct ccttcggtcc tccgatcgtt
     3301 gtcagaagta agttggccgc agtgttatca ctcatggtta tggcagcact gcataattct
     3361 cttactgtca tgccatccgt aagatgcttt tctgtgactg gtgagtactc aaccaagtca
     3421 ttctgagaat agtgtatgcg gcgaccgagt tgctcttgcc cggcgtcaat acgggataat
     3481 accgcgccac atagcagaac tttaaaagtg ctcatcattg gaaaacgttc ttcggggcga
     3541 aaactctcaa ggatcttacc gctgttgaga tccagttcga tgtaacccac tcgtgcaccc
     3601 aactgatctt cagcatcttt tactttcacc agcgtttctg ggtgagcaaa aacaggaagg
     3661 caaaatgccg caaaaaaggg aataagggcg acacggaaat gttgaatact catactcttc
     3721 ctttttcaat attattgaag catttatcag ggttattgtc tcatgagcgg atacatattt
     3781 gaatgtattt agaaaaataa acaaataggg gttccgcgca catttccccg aaaagtgcca
     3841 cctaaattgt aagcgttaat attttgttaa aattcgcgtt aaatttttgt taaatcagct
     3901 cattttttaa ccaataggcc gaaatcggca aaatccctta taaatcaaaa gaatagaccg
     3961 agatagggtt gagtgttgtt ccagtttgga acaagagtcc actattaaag aacgtggact
     4021 ccaacgtcaa agggcgaaaa accgtctatc agggcgatgg cccactacgt gaaccatcac
     4081 cctaatcaag ttttttgggg tcgaggtgcc gtaaagcact aaatcggaac cctaaaggga
     4141 gcccccgatt tagagcttga cggggaaagc cggcgaacgt ggcgagaaag gaagggaaga
     4201 aagcgaaagg agcgggcgct agggcgctgg caagtgtagc ggtcacgctg cgcgtaacca
     4261 ccacacccgc cgcgcttaat gcgccgctac agggcgcgtc ccattcgcca ttcaggctgc
     4321 gcaactgttg ggaagggcga tcggtgcggg cctcttcgct attacgccag ctggcgaaag
     4381 ggggatgtgc tgcaaggcga ttaagttggg taacgccagg gttttcccag tcacgacgtt
//
