LOCUS       sequence-254134-        4440 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Cas9 R promoter targeting sgRNA1 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: PV521
FEATURES             Location/Qualifiers
     source          1..4440
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     10..27
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     11..27
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     34..53
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        34..52
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     primer_bind     77..93
                     /label=pBluescriptKS
                     /note="For pBluescript vector"
     primer_bind     78..94
                     /label=KS primer
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     misc_RNA        1123..1198
                     /label=gRNA scaffold
                     /note="guide RNA scaffold for the Streptococcus pyogenes 
                     CRISPR/Cas9 system"
     promoter        complement(1663..1681)
                     /label=T3 promoter
                     /note="promoter for bacteriophage T3 RNA polymerase"
     primer_bind     complement(1702..1718)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(1702..1718)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(1715..1737)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    1726..1742
                     /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(1750..1756,1757..1774,1775..1780))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    1795..1816
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(1933..1950)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(2104..2692)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(2184..2203)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(2863..3654,3655..3723))
                     /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     3486..3505
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(3724..3828)
                     /gene="bla"
                     /label=AmpR promoter
     rep_origin      complement(3854..4309)
                     /direction=LEFT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(3991..4012)
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     4203..4222
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     join(4436..4440,1..18)
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
ORIGIN
        1 tcacgacgtt gtaaaacgac ggccagtgaa ttgtaatacg actcactata gggcgaattg
       61 ggtaccgggc cccccctcga ggtcgacggt atcgataagc tttgagagta caatgatgaa
      121 cctagattaa tcaatgccaa agtctgaaaa atgcaccctc agtctatgat ccagaaaatc
      181 aagattgctt gaggccctgt tcggttgttc cggattagag ccccggatta attcctagcc
      241 ggattacttc tctaatttat atagattttg atgagctgga atgaatcctg gcttattccg
      301 gtacaaccga acaggccctg aaggatacca gtaatcgctg agctaaattg gcatgctgtc
      361 agagtgtcag tattgcagca aggtagtgag ataaccggca tcatggtgcc agtttgatgg
      421 caccattagg gttagagatg gtggccatgg gcgcatgtcc tggccaactt tgtatgatat
      481 atggcagggt gaataggaaa gtaaaattgt attgtaaaaa gggatttctt ctgtttgtta
      541 gcgcatgtac aaggaatgca agttttgagc gagggggcat caaagatctg gctgtgtttc
      601 cagctgtttt tgttagcccc atcgaatcct tgacataatg atcccgctta aataagcaac
      661 ctcgcttgta tagttccttg tgctctaaca cacgatgatg ataagtcgta aaatagtggt
      721 gtccaaagaa tttccaggcc cagttgtaaa agctaaaatg ctattcgaat ttctactagc
      781 agtaagtcgt gtttagaaat tattttttta tatacctttt ttccttctat gtacagtagg
      841 acacagtgtc agcgccgcgt tgacggagaa tatttgcaaa aaagtaaaag agaaagtcat
      901 agcggcgtat gtgccaaaaa cttcgtcaca gagagggcca taagaaacat ggcccacggc
      961 ccaatacgaa gcaccgcgac gaagcccaaa cagcagtccg taggtggagc aaagcgctgg
     1021 gtaatacgca aacgttttgt cccaccttga ctaatcacaa gagtggagcg taccttataa
     1081 accgagccgc aagcaccgaa ttgagctcca ccaaagacaa aggttttaga gctagaaata
     1141 gcaagttaaa ataaggctag tccgttatca acttgaaaaa gtggcaccga gtcggtgctt
     1201 tttttttgcg gccgcgaatt cctgcagggc cctcttgtcg gaccagttgc ccaccacgtt
     1261 ggtgagctcg gtgaggccct tcattgagag gaaggaggtc atgaggtgcc taccgatgtg
     1321 ggacttgggg ccgttcttga tggcgaagat ggagtagggg gcgttcttct tgagggcctt
     1381 gttgtaggac ctcacgaggt tgtccttgag gagctggtac tcctgcttgt tggaggagga
     1441 gttgccggtc ctgttcaccc tcttgagcac gggctctgag ttcctgagga actcgtcgag
     1501 gtacacgagg gggtcgatcc tgccgcgagc ggagaagaag tagatgtgcc tggacacgga
     1561 ggtcttggtc tcggtcacga ggcactggat gatcacgccg aggtacttgt tctgactagt
     1621 tctagagcgg ccgccaccgc ggtggagctc cagcttttgt tccctttagt gagggttaat
     1681 ttcgagcttg gcgtaatcat ggtcatagct gtttcctgtg tgaaattgtt atccgctcac
     1741 aattccacac aacatacgag ccggaagcat aaagtgtaaa gcctggggtg cctaatgagt
     1801 gagctaactc acattaattg cgttgcgctc actgcccgct ttccagtcgg gaaacctgtc
     1861 gtgccagctg cattaatgaa tcggccaacg cgcggggaga ggcggtttgc gtattgggcg
     1921 ctcttccgct tcctcgctca ctgactcgct gcgctcggtc gttcggctgc ggcgagcggt
     1981 atcagctcac tcaaaggcgg taatacggtt atccacagaa tcaggggata acgcaggaaa
     2041 gaacatgtga gcaaaaggcc agcaaaaggc caggaaccgt aaaaaggccg cgttgctggc
     2101 gtttttccat aggctccgcc cccctgacga gcatcacaaa aatcgacgct caagtcagag
     2161 gtggcgaaac ccgacaggac tataaagata ccaggcgttt ccccctggaa gctccctcgt
     2221 gcgctctcct gttccgaccc tgccgcttac cggatacctg tccgcctttc tcccttcggg
     2281 aagcgtggcg ctttctcata gctcacgctg taggtatctc agttcggtgt aggtcgttcg
     2341 ctccaagctg ggctgtgtgc acgaaccccc cgttcagccc gaccgctgcg ccttatccgg
     2401 taactatcgt cttgagtcca acccggtaag acacgactta tcgccactgg cagcagccac
     2461 tggtaacagg attagcagag cgaggtatgt aggcggtgct acagagttct tgaagtggtg
     2521 gcctaactac ggctacacta gaaggacagt atttggtatc tgcgctctgc tgaagccagt
     2581 taccttcgga aaaagagttg gtagctcttg atccggcaaa caaaccaccg ctggtagcgg
     2641 tggttttttt gtttgcaagc agcagattac gcgcagaaaa aaaggatctc aagaagatcc
     2701 tttgatcttt tctacggggt ctgacgctca gtggaacgaa aactcacgtt aagggatttt
     2761 ggtcatgaga ttatcaaaaa ggatcttcac ctagatcctt ttaaattaaa aatgaagttt
     2821 taaatcaatc taaagtatat atgagtaaac ttggtctgac agttaccaat gcttaatcag
     2881 tgaggcacct atctcagcga tctgtctatt tcgttcatcc atagttgcct gactccccgt
     2941 cgtgtagata actacgatac gggagggctt accatctggc cccagtgctg caatgatacc
     3001 gcgagaccca cgctcaccgg ctccagattt atcagcaata aaccagccag ccggaagggc
     3061 cgagcgcaga agtggtcctg caactttatc cgcctccatc cagtctatta attgttgccg
     3121 ggaagctaga gtaagtagtt cgccagttaa tagtttgcgc aacgttgttg ccattgctac
     3181 aggcatcgtg gtgtcacgct cgtcgtttgg tatggcttca ttcagctccg gttcccaacg
     3241 atcaaggcga gttacatgat cccccatgtt gtgcaaaaaa gcggttagct ccttcggtcc
     3301 tccgatcgtt gtcagaagta agttggccgc agtgttatca ctcatggtta tggcagcact
     3361 gcataattct cttactgtca tgccatccgt aagatgcttt tctgtgactg gtgagtactc
     3421 aaccaagtca ttctgagaat agtgtatgcg gcgaccgagt tgctcttgcc cggcgtcaat
     3481 acgggataat accgcgccac atagcagaac tttaaaagtg ctcatcattg gaaaacgttc
     3541 ttcggggcga aaactctcaa ggatcttacc gctgttgaga tccagttcga tgtaacccac
     3601 tcgtgcaccc aactgatctt cagcatcttt tactttcacc agcgtttctg ggtgagcaaa
     3661 aacaggaagg caaaatgccg caaaaaaggg aataagggcg acacggaaat gttgaatact
     3721 catactcttc ctttttcaat attattgaag catttatcag ggttattgtc tcatgagcgg
     3781 atacatattt gaatgtattt agaaaaataa acaaataggg gttccgcgca catttccccg
     3841 aaaagtgcca cctaaattgt aagcgttaat attttgttaa aattcgcgtt aaatttttgt
     3901 taaatcagct cattttttaa ccaataggcc gaaatcggca aaatccctta taaatcaaaa
     3961 gaatagaccg agatagggtt gagtgttgtt ccagtttgga acaagagtcc actattaaag
     4021 aacgtggact ccaacgtcaa agggcgaaaa accgtctatc agggcgatgg cccactacgt
     4081 gaaccatcac cctaatcaag ttttttgggg tcgaggtgcc gtaaagcact aaatcggaac
     4141 cctaaaggga gcccccgatt tagagcttga cggggaaagc cggcgaacgt ggcgagaaag
     4201 gaagggaaga aagcgaaagg agcgggcgct agggcgctgg caagtgtagc ggtcacgctg
     4261 cgcgtaacca ccacacccgc cgcgcttaat gcgccgctac agggcgcgtc ccattcgcca
     4321 ttcaggctgc gcaactgttg ggaagggcga tcggtgcggg cctcttcgct attacgccag
     4381 ctggcgaaag ggggatgtgc tgcaaggcga ttaagttggg taacgccagg gttttcccag
//
