LOCUS       sequence-258131-        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     4..23
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        4..22
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     primer_bind     47..63
                     /label=pBluescriptKS
                     /note="For pBluescript vector"
     primer_bind     48..64
                     /label=KS primer
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     misc_RNA        1093..1168
                     /label=gRNA scaffold
                     /note="guide RNA scaffold for the Streptococcus pyogenes 
                     CRISPR/Cas9 system"
     promoter        complement(1633..1651)
                     /label=T3 promoter
                     /note="promoter for bacteriophage T3 RNA polymerase"
     primer_bind     complement(1672..1688)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(1672..1688)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(1685..1707)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    1696..1712
                     /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(1720..1726,1727..1744,1745..1750))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    1765..1786
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(1903..1920)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(2074..2662)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(2154..2173)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(2833..3624,3625..3693))
                     /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     3456..3475
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(3694..3798)
                     /gene="bla"
                     /label=AmpR promoter
     rep_origin      complement(3824..4279)
                     /direction=LEFT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(3961..3982)
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     4173..4192
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     4406..4428
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     primer_bind     4420..4437
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     4421..4437
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
ORIGIN
        1 ttgtaatacg actcactata gggcgaattg ggtaccgggc cccccctcga ggtcgacggt
       61 atcgataagc tttgagagta caatgatgaa cctagattaa tcaatgccaa agtctgaaaa
      121 atgcaccctc agtctatgat ccagaaaatc aagattgctt gaggccctgt tcggttgttc
      181 cggattagag ccccggatta attcctagcc ggattacttc tctaatttat atagattttg
      241 atgagctgga atgaatcctg gcttattccg gtacaaccga acaggccctg aaggatacca
      301 gtaatcgctg agctaaattg gcatgctgtc agagtgtcag tattgcagca aggtagtgag
      361 ataaccggca tcatggtgcc agtttgatgg caccattagg gttagagatg gtggccatgg
      421 gcgcatgtcc tggccaactt tgtatgatat atggcagggt gaataggaaa gtaaaattgt
      481 attgtaaaaa gggatttctt ctgtttgtta gcgcatgtac aaggaatgca agttttgagc
      541 gagggggcat caaagatctg gctgtgtttc cagctgtttt tgttagcccc atcgaatcct
      601 tgacataatg atcccgctta aataagcaac ctcgcttgta tagttccttg tgctctaaca
      661 cacgatgatg ataagtcgta aaatagtggt gtccaaagaa tttccaggcc cagttgtaaa
      721 agctaaaatg ctattcgaat ttctactagc agtaagtcgt gtttagaaat tattttttta
      781 tatacctttt ttccttctat gtacagtagg acacagtgtc agcgccgcgt tgacggagaa
      841 tatttgcaaa aaagtaaaag agaaagtcat agcggcgtat gtgccaaaaa cttcgtcaca
      901 gagagggcca taagaaacat ggcccacggc ccaatacgaa gcaccgcgac gaagcccaaa
      961 cagcagtccg taggtggagc aaagcgctgg gtaatacgca aacgttttgt cccaccttga
     1021 ctaatcacaa gagtggagcg taccttataa accgagccgc aagcaccgaa ttgagctcca
     1081 ccaaagacaa aggttttaga gctagaaata gcaagttaaa ataaggctag tccgttatca
     1141 acttgaaaaa gtggcaccga gtcggtgctt tttttttgcg gccgcgaatt cctgcagggc
     1201 cctcttgtcg gaccagttgc ccaccacgtt ggtgagctcg gtgaggccct tcattgagag
     1261 gaaggaggtc atgaggtgcc taccgatgtg ggacttgggg ccgttcttga tggcgaagat
     1321 ggagtagggg gcgttcttct tgagggcctt gttgtaggac ctcacgaggt tgtccttgag
     1381 gagctggtac tcctgcttgt tggaggagga gttgccggtc ctgttcaccc tcttgagcac
     1441 gggctctgag ttcctgagga actcgtcgag gtacacgagg gggtcgatcc tgccgcgagc
     1501 ggagaagaag tagatgtgcc tggacacgga ggtcttggtc tcggtcacga ggcactggat
     1561 gatcacgccg aggtacttgt tctgactagt tctagagcgg ccgccaccgc ggtggagctc
     1621 cagcttttgt tccctttagt gagggttaat ttcgagcttg gcgtaatcat ggtcatagct
     1681 gtttcctgtg tgaaattgtt atccgctcac aattccacac aacatacgag ccggaagcat
     1741 aaagtgtaaa gcctggggtg cctaatgagt gagctaactc acattaattg cgttgcgctc
     1801 actgcccgct ttccagtcgg gaaacctgtc gtgccagctg cattaatgaa tcggccaacg
     1861 cgcggggaga ggcggtttgc gtattgggcg ctcttccgct tcctcgctca ctgactcgct
     1921 gcgctcggtc gttcggctgc ggcgagcggt atcagctcac tcaaaggcgg taatacggtt
     1981 atccacagaa tcaggggata acgcaggaaa gaacatgtga gcaaaaggcc agcaaaaggc
     2041 caggaaccgt aaaaaggccg cgttgctggc gtttttccat aggctccgcc cccctgacga
     2101 gcatcacaaa aatcgacgct caagtcagag gtggcgaaac ccgacaggac tataaagata
     2161 ccaggcgttt ccccctggaa gctccctcgt gcgctctcct gttccgaccc tgccgcttac
     2221 cggatacctg tccgcctttc tcccttcggg aagcgtggcg ctttctcata gctcacgctg
     2281 taggtatctc agttcggtgt aggtcgttcg ctccaagctg ggctgtgtgc acgaaccccc
     2341 cgttcagccc gaccgctgcg ccttatccgg taactatcgt cttgagtcca acccggtaag
     2401 acacgactta tcgccactgg cagcagccac tggtaacagg attagcagag cgaggtatgt
     2461 aggcggtgct acagagttct tgaagtggtg gcctaactac ggctacacta gaagaacagt
     2521 atttggtatc tgcgctctgc tgaagccagt taccttcgga aaaagagttg gtagctcttg
     2581 atccggcaaa caaaccaccg ctggtagcgg tggttttttt gtttgcaagc agcagattac
     2641 gcgcagaaaa aaaggatctc aagaagatcc tttgatcttt tctacggggt ctgacgctca
     2701 gtggaacgaa aactcacgtt aagggatttt ggtcatgaga ttatcaaaaa ggatcttcac
     2761 ctagatcctt ttaaattaaa aatgaagttt taaatcaatc taaagtatat atgagtaaac
     2821 ttggtctgac agttaccaat gcttaatcag tgaggcacct atctcagcga tctgtctatt
     2881 tcgttcatcc atagttgcct gactccccgt cgtgtagata actacgatac gggagggctt
     2941 accatctggc cccagtgctg caatgatacc gcgagaccca cgctcaccgg ctccagattt
     3001 atcagcaata aaccagccag ccggaagggc cgagcgcaga agtggtcctg caactttatc
     3061 cgcctccatc cagtctatta attgttgccg ggaagctaga gtaagtagtt cgccagttaa
     3121 tagtttgcgc aacgttgttg ccattgctac aggcatcgtg gtgtcacgct cgtcgtttgg
     3181 tatggcttca ttcagctccg gttcccaacg atcaaggcga gttacatgat cccccatgtt
     3241 gtgcaaaaaa gcggttagct ccttcggtcc tccgatcgtt gtcagaagta agttggccgc
     3301 agtgttatca ctcatggtta tggcagcact gcataattct cttactgtca tgccatccgt
     3361 aagatgcttt tctgtgactg gtgagtactc aaccaagtca ttctgagaat agtgtatgcg
     3421 gcgaccgagt tgctcttgcc cggcgtcaat acgggataat accgcgccac atagcagaac
     3481 tttaaaagtg ctcatcattg gaaaacgttc ttcggggcga aaactctcaa ggatcttacc
     3541 gctgttgaga tccagttcga tgtaacccac tcgtgcaccc aactgatctt cagcatcttt
     3601 tactttcacc agcgtttctg ggtgagcaaa aacaggaagg caaaatgccg caaaaaaggg
     3661 aataagggcg acacggaaat gttgaatact catactcttc ctttttcaat attattgaag
     3721 catttatcag ggttattgtc tcatgagcgg atacatattt gaatgtattt agaaaaataa
     3781 acaaataggg gttccgcgca catttccccg aaaagtgcca cctaaattgt aagcgttaat
     3841 attttgttaa aattcgcgtt aaatttttgt taaatcagct cattttttaa ccaataggcc
     3901 gaaatcggca aaatccctta taaatcaaaa gaatagaccg agatagggtt gagtgttgtt
     3961 ccagtttgga acaagagtcc actattaaag aacgtggact ccaacgtcaa agggcgaaaa
     4021 accgtctatc agggcgatgg cccactacgt gaaccatcac cctaatcaag ttttttgggg
     4081 tcgaggtgcc gtaaagcact aaatcggaac cctaaaggga gcccccgatt tagagcttga
     4141 cggggaaagc cggcgaacgt ggcgagaaag gaagggaaga aagcgaaagg agcgggcgct
     4201 agggcgctgg caagtgtagc ggtcacgctg cgcgtaacca ccacacccgc cgcgcttaat
     4261 gcgccgctac agggcgcgtc ccattcgcca ttcaggctgc gcaactgttg ggaagggcga
     4321 tcggtgcggg cctcttcgct attacgccag ctggcgaaag ggggatgtgc tgcaaggcga
     4381 ttaagttggg taacgccagg gttttcccag tcacgacgtt gtaaaacgac ggccagtgaa
//
