LOCUS       sequence-254137-        4440 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Cas9 R promoter targeting sgRNA3 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: PV528
FEATURES             Location/Qualifiers
     source          1..4440
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     3..25
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     primer_bind     17..34
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     18..34
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     41..60
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        41..59
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     primer_bind     84..100
                     /label=pBluescriptKS
                     /note="For pBluescript vector"
     primer_bind     85..101
                     /label=KS primer
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     misc_RNA        1130..1205
                     /label=gRNA scaffold
                     /note="guide RNA scaffold for the Streptococcus pyogenes 
                     CRISPR/Cas9 system"
     promoter        complement(1670..1688)
                     /label=T3 promoter
                     /note="promoter for bacteriophage T3 RNA polymerase"
     primer_bind     complement(1709..1725)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(1709..1725)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(1722..1744)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    1733..1749
                     /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(1757..1763,1764..1781,1782..1787))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    1802..1823
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(1940..1957)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(2111..2699)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(2191..2210)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(2870..3661,3662..3730))
                     /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     3493..3512
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(3731..3835)
                     /gene="bla"
                     /label=AmpR promoter
     rep_origin      complement(3861..4316)
                     /direction=LEFT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(3998..4019)
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     4210..4229
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
ORIGIN
        1 ttcccagtca cgacgttgta aaacgacggc cagtgaattg taatacgact cactataggg
       61 cgaattgggt accgggcccc ccctcgaggt cgacggtatc gataagcttt gagagtacaa
      121 tgatgaacct agattaatca atgccaaagt ctgaaaaatg caccctcagt ctatgatcca
      181 gaaaatcaag attgcttgag gccctgttcg gttgttccgg attagagccc cggattaatt
      241 cctagccgga ttacttctct aatttatata gattttgatg agctggaatg aatcctggct
      301 tattccggta caaccgaaca ggccctgaag gataccagta atcgctgagc taaattggca
      361 tgctgtcaga gtgtcagtat tgcagcaagg tagtgagata accggcatca tggtgccagt
      421 ttgatggcac cattagggtt agagatggtg gccatgggcg catgtcctgg ccaactttgt
      481 atgatatatg gcagggtgaa taggaaagta aaattgtatt gtaaaaaggg atttcttctg
      541 tttgttagcg catgtacaag gaatgcaagt tttgagcgag ggggcatcaa agatctggct
      601 gtgtttccag ctgtttttgt tagccccatc gaatccttga cataatgatc ccgcttaaat
      661 aagcaacctc gcttgtatag ttccttgtgc tctaacacac gatgatgata agtcgtaaaa
      721 tagtggtgtc caaagaattt ccaggcccag ttgtaaaagc taaaatgcta ttcgaatttc
      781 tactagcagt aagtcgtgtt tagaaattat ttttttatat accttttttc cttctatgta
      841 cagtaggaca cagtgtcagc gccgcgttga cggagaatat ttgcaaaaaa gtaaaagaga
      901 aagtcatagc ggcgtatgtg ccaaaaactt cgtcacagag agggccataa gaaacatggc
      961 ccacggccca atacgaagca ccgcgacgaa gcccaaacag cagtccgtag gtggagcaaa
     1021 gcgctgggta atacgcaaac gttttgtccc accttgacta atcacaagag tggagcgtac
     1081 cttataaacc gagccgcaag caccgaattg tccaagtgac cgagcaagag ttttagagct
     1141 agaaatagca agttaaaata aggctagtcc gttatcaact tgaaaaagtg gcaccgagtc
     1201 ggtgcttttt ttttgcggcc gcgaattcct gcagggccct cttgtcggac cagttgccca
     1261 ccacgttggt gagctcggtg aggcccttca ttgagaggaa ggaggtcatg aggtgcctac
     1321 cgatgtggga cttggggccg ttcttgatgg cgaagatgga gtagggggcg ttcttcttga
     1381 gggccttgtt gtaggacctc acgaggttgt ccttgaggag ctggtactcc tgcttgttgg
     1441 aggaggagtt gccggtcctg ttcaccctct tgagcacggg ctctgagttc ctgaggaact
     1501 cgtcgaggta cacgaggggg tcgatcctgc cgcgagcgga gaagaagtag atgtgcctgg
     1561 acacggaggt cttggtctcg gtcacgaggc actggatgat cacgccgagg tacttgttct
     1621 gactagttct agagcggccg ccaccgcggt ggagctccag cttttgttcc ctttagtgag
     1681 ggttaatttc gagcttggcg taatcatggt catagctgtt tcctgtgtga aattgttatc
     1741 cgctcacaat tccacacaac atacgagccg gaagcataaa gtgtaaagcc tggggtgcct
     1801 aatgagtgag ctaactcaca ttaattgcgt tgcgctcact gcccgctttc cagtcgggaa
     1861 acctgtcgtg ccagctgcat taatgaatcg gccaacgcgc ggggagaggc ggtttgcgta
     1921 ttgggcgctc ttccgcttcc tcgctcactg actcgctgcg ctcggtcgtt cggctgcggc
     1981 gagcggtatc agctcactca aaggcggtaa tacggttatc cacagaatca ggggataacg
     2041 caggaaagaa catgtgagca aaaggccagc aaaaggccag gaaccgtaaa aaggccgcgt
     2101 tgctggcgtt tttccatagg ctccgccccc ctgacgagca tcacaaaaat cgacgctcaa
     2161 gtcagaggtg gcgaaacccg acaggactat aaagatacca ggcgtttccc cctggaagct
     2221 ccctcgtgcg ctctcctgtt ccgaccctgc cgcttaccgg atacctgtcc gcctttctcc
     2281 cttcgggaag cgtggcgctt tctcatagct cacgctgtag gtatctcagt tcggtgtagg
     2341 tcgttcgctc caagctgggc tgtgtgcacg aaccccccgt tcagcccgac cgctgcgcct
     2401 tatccggtaa ctatcgtctt gagtccaacc cggtaagaca cgacttatcg ccactggcag
     2461 cagccactgg taacaggatt agcagagcga ggtatgtagg cggtgctaca gagttcttga
     2521 agtggtggcc taactacggc tacactagaa ggacagtatt tggtatctgc gctctgctga
     2581 agccagttac cttcggaaaa agagttggta gctcttgatc cggcaaacaa accaccgctg
     2641 gtagcggtgg tttttttgtt tgcaagcagc agattacgcg cagaaaaaaa ggatctcaag
     2701 aagatccttt gatcttttct acggggtctg acgctcagtg gaacgaaaac tcacgttaag
     2761 ggattttggt catgagatta tcaaaaagga tcttcaccta gatcctttta aattaaaaat
     2821 gaagttttaa atcaatctaa agtatatatg agtaaacttg gtctgacagt taccaatgct
     2881 taatcagtga ggcacctatc tcagcgatct gtctatttcg ttcatccata gttgcctgac
     2941 tccccgtcgt gtagataact acgatacggg agggcttacc atctggcccc agtgctgcaa
     3001 tgataccgcg agacccacgc tcaccggctc cagatttatc agcaataaac cagccagccg
     3061 gaagggccga gcgcagaagt ggtcctgcaa ctttatccgc ctccatccag tctattaatt
     3121 gttgccggga agctagagta agtagttcgc cagttaatag tttgcgcaac gttgttgcca
     3181 ttgctacagg catcgtggtg tcacgctcgt cgtttggtat ggcttcattc agctccggtt
     3241 cccaacgatc aaggcgagtt acatgatccc ccatgttgtg caaaaaagcg gttagctcct
     3301 tcggtcctcc gatcgttgtc agaagtaagt tggccgcagt gttatcactc atggttatgg
     3361 cagcactgca taattctctt actgtcatgc catccgtaag atgcttttct gtgactggtg
     3421 agtactcaac caagtcattc tgagaatagt gtatgcggcg accgagttgc tcttgcccgg
     3481 cgtcaatacg ggataatacc gcgccacata gcagaacttt aaaagtgctc atcattggaa
     3541 aacgttcttc ggggcgaaaa ctctcaagga tcttaccgct gttgagatcc agttcgatgt
     3601 aacccactcg tgcacccaac tgatcttcag catcttttac tttcaccagc gtttctgggt
     3661 gagcaaaaac aggaaggcaa aatgccgcaa aaaagggaat aagggcgaca cggaaatgtt
     3721 gaatactcat actcttcctt tttcaatatt attgaagcat ttatcagggt tattgtctca
     3781 tgagcggata catatttgaa tgtatttaga aaaataaaca aataggggtt ccgcgcacat
     3841 ttccccgaaa agtgccacct aaattgtaag cgttaatatt ttgttaaaat tcgcgttaaa
     3901 tttttgttaa atcagctcat tttttaacca ataggccgaa atcggcaaaa tcccttataa
     3961 atcaaaagaa tagaccgaga tagggttgag tgttgttcca gtttggaaca agagtccact
     4021 attaaagaac gtggactcca acgtcaaagg gcgaaaaacc gtctatcagg gcgatggccc
     4081 actacgtgaa ccatcaccct aatcaagttt tttggggtcg aggtgccgta aagcactaaa
     4141 tcggaaccct aaagggagcc cccgatttag agcttgacgg ggaaagccgg cgaacgtggc
     4201 gagaaaggaa gggaagaaag cgaaaggagc gggcgctagg gcgctggcaa gtgtagcggt
     4261 cacgctgcgc gtaaccacca cacccgccgc gcttaatgcg ccgctacagg gcgcgtccca
     4321 ttcgccattc aggctgcgca actgttggga agggcgatcg gtgcgggcct cttcgctatt
     4381 acgccagctg gcgaaagggg gatgtgctgc aaggcgatta agttgggtaa cgccagggtt
//
