LOCUS       sequence-254136-        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     17..36
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        17..35
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     primer_bind     60..76
                     /label=pBluescriptKS
                     /note="For pBluescript vector"
     primer_bind     61..77
                     /label=KS primer
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     misc_RNA        1106..1181
                     /label=gRNA scaffold
                     /note="guide RNA scaffold for the Streptococcus pyogenes 
                     CRISPR/Cas9 system"
     promoter        complement(1646..1664)
                     /label=T3 promoter
                     /note="promoter for bacteriophage T3 RNA polymerase"
     primer_bind     complement(1685..1701)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(1685..1701)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(1698..1720)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    1709..1725
                     /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(1733..1739,1740..1757,1758..1763))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    1778..1799
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(1916..1933)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(2087..2675)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(2167..2186)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(2846..3637,3638..3706))
                     /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     3469..3488
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(3707..3811)
                     /gene="bla"
                     /label=AmpR promoter
     rep_origin      complement(3837..4292)
                     /direction=LEFT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(3974..3995)
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     4186..4205
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     join(4419..4440,1)
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     primer_bind     join(4433..4440,1..10)
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     join(4434..4440,1..10)
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
ORIGIN
        1 gacggccagt gaattgtaat acgactcact atagggcgaa ttgggtaccg ggccccccct
       61 cgaggtcgac ggtatcgata agctttgaga gtacaatgat gaacctagat taatcaatgc
      121 caaagtctga aaaatgcacc ctcagtctat gatccagaaa atcaagattg cttgaggccc
      181 tgttcggttg ttccggatta gagccccgga ttaattccta gccggattac ttctctaatt
      241 tatatagatt ttgatgagct ggaatgaatc ctggcttatt ccggtacaac cgaacaggcc
      301 ctgaaggata ccagtaatcg ctgagctaaa ttggcatgct gtcagagtgt cagtattgca
      361 gcaaggtagt gagataaccg gcatcatggt gccagtttga tggcaccatt agggttagag
      421 atggtggcca tgggcgcatg tcctggccaa ctttgtatga tatatggcag ggtgaatagg
      481 aaagtaaaat tgtattgtaa aaagggattt cttctgtttg ttagcgcatg tacaaggaat
      541 gcaagttttg agcgaggggg catcaaagat ctggctgtgt ttccagctgt ttttgttagc
      601 cccatcgaat ccttgacata atgatcccgc ttaaataagc aacctcgctt gtatagttcc
      661 ttgtgctcta acacacgatg atgataagtc gtaaaatagt ggtgtccaaa gaatttccag
      721 gcccagttgt aaaagctaaa atgctattcg aatttctact agcagtaagt cgtgtttaga
      781 aattattttt ttatatacct tttttccttc tatgtacagt aggacacagt gtcagcgccg
      841 cgttgacgga gaatatttgc aaaaaagtaa aagagaaagt catagcggcg tatgtgccaa
      901 aaacttcgtc acagagaggg ccataagaaa catggcccac ggcccaatac gaagcaccgc
      961 gacgaagccc aaacagcagt ccgtaggtgg agcaaagcgc tgggtaatac gcaaacgttt
     1021 tgtcccacct tgactaatca caagagtgga gcgtacctta taaaccgagc cgcaagcacc
     1081 gaattgagtg ctataatatg agtgggtttt agagctagaa atagcaagtt aaaataaggc
     1141 tagtccgtta tcaacttgaa aaagtggcac cgagtcggtg cttttttttt gcggccgcga
     1201 attcctgcag ggccctcttg tcggaccagt tgcccaccac gttggtgagc tcggtgaggc
     1261 ccttcattga gaggaaggag gtcatgaggt gcctaccgat gtgggacttg gggccgttct
     1321 tgatggcgaa gatggagtag ggggcgttct tcttgagggc cttgttgtag gacctcacga
     1381 ggttgtcctt gaggagctgg tactcctgct tgttggagga ggagttgccg gtcctgttca
     1441 ccctcttgag cacgggctct gagttcctga ggaactcgtc gaggtacacg agggggtcga
     1501 tcctgccgcg agcggagaag aagtagatgt gcctggacac ggaggtcttg gtctcggtca
     1561 cgaggcactg gatgatcacg ccgaggtact tgttctgact agttctagag cggccgccac
     1621 cgcggtggag ctccagcttt tgttcccttt agtgagggtt aatttcgagc ttggcgtaat
     1681 catggtcata gctgtttcct gtgtgaaatt gttatccgct cacaattcca cacaacatac
     1741 gagccggaag cataaagtgt aaagcctggg gtgcctaatg agtgagctaa ctcacattaa
     1801 ttgcgttgcg ctcactgccc gctttccagt cgggaaacct gtcgtgccag ctgcattaat
     1861 gaatcggcca acgcgcgggg agaggcggtt tgcgtattgg gcgctcttcc gcttcctcgc
     1921 tcactgactc gctgcgctcg gtcgttcggc tgcggcgagc ggtatcagct cactcaaagg
     1981 cggtaatacg gttatccaca gaatcagggg ataacgcagg aaagaacatg tgagcaaaag
     2041 gccagcaaaa ggccaggaac cgtaaaaagg ccgcgttgct ggcgtttttc cataggctcc
     2101 gcccccctga cgagcatcac aaaaatcgac gctcaagtca gaggtggcga aacccgacag
     2161 gactataaag ataccaggcg tttccccctg gaagctccct cgtgcgctct cctgttccga
     2221 ccctgccgct taccggatac ctgtccgcct ttctcccttc gggaagcgtg gcgctttctc
     2281 atagctcacg ctgtaggtat ctcagttcgg tgtaggtcgt tcgctccaag ctgggctgtg
     2341 tgcacgaacc ccccgttcag cccgaccgct gcgccttatc cggtaactat cgtcttgagt
     2401 ccaacccggt aagacacgac ttatcgccac tggcagcagc cactggtaac aggattagca
     2461 gagcgaggta tgtaggcggt gctacagagt tcttgaagtg gtggcctaac tacggctaca
     2521 ctagaaggac agtatttggt atctgcgctc tgctgaagcc agttaccttc ggaaaaagag
     2581 ttggtagctc ttgatccggc aaacaaacca ccgctggtag cggtggtttt tttgtttgca
     2641 agcagcagat tacgcgcaga aaaaaaggat ctcaagaaga tcctttgatc ttttctacgg
     2701 ggtctgacgc tcagtggaac gaaaactcac gttaagggat tttggtcatg agattatcaa
     2761 aaaggatctt cacctagatc cttttaaatt aaaaatgaag ttttaaatca atctaaagta
     2821 tatatgagta aacttggtct gacagttacc aatgcttaat cagtgaggca cctatctcag
     2881 cgatctgtct atttcgttca tccatagttg cctgactccc cgtcgtgtag ataactacga
     2941 tacgggaggg cttaccatct ggccccagtg ctgcaatgat accgcgagac ccacgctcac
     3001 cggctccaga tttatcagca ataaaccagc cagccggaag ggccgagcgc agaagtggtc
     3061 ctgcaacttt atccgcctcc atccagtcta ttaattgttg ccgggaagct agagtaagta
     3121 gttcgccagt taatagtttg cgcaacgttg ttgccattgc tacaggcatc gtggtgtcac
     3181 gctcgtcgtt tggtatggct tcattcagct ccggttccca acgatcaagg cgagttacat
     3241 gatcccccat gttgtgcaaa aaagcggtta gctccttcgg tcctccgatc gttgtcagaa
     3301 gtaagttggc cgcagtgtta tcactcatgg ttatggcagc actgcataat tctcttactg
     3361 tcatgccatc cgtaagatgc ttttctgtga ctggtgagta ctcaaccaag tcattctgag
     3421 aatagtgtat gcggcgaccg agttgctctt gcccggcgtc aatacgggat aataccgcgc
     3481 cacatagcag aactttaaaa gtgctcatca ttggaaaacg ttcttcgggg cgaaaactct
     3541 caaggatctt accgctgttg agatccagtt cgatgtaacc cactcgtgca cccaactgat
     3601 cttcagcatc ttttactttc accagcgttt ctgggtgagc aaaaacagga aggcaaaatg
     3661 ccgcaaaaaa gggaataagg gcgacacgga aatgttgaat actcatactc ttcctttttc
     3721 aatattattg aagcatttat cagggttatt gtctcatgag cggatacata tttgaatgta
     3781 tttagaaaaa taaacaaata ggggttccgc gcacatttcc ccgaaaagtg ccacctaaat
     3841 tgtaagcgtt aatattttgt taaaattcgc gttaaatttt tgttaaatca gctcattttt
     3901 taaccaatag gccgaaatcg gcaaaatccc ttataaatca aaagaataga ccgagatagg
     3961 gttgagtgtt gttccagttt ggaacaagag tccactatta aagaacgtgg actccaacgt
     4021 caaagggcga aaaaccgtct atcagggcga tggcccacta cgtgaaccat caccctaatc
     4081 aagttttttg gggtcgaggt gccgtaaagc actaaatcgg aaccctaaag ggagcccccg
     4141 atttagagct tgacggggaa agccggcgaa cgtggcgaga aaggaaggga agaaagcgaa
     4201 aggagcgggc gctagggcgc tggcaagtgt agcggtcacg ctgcgcgtaa ccaccacacc
     4261 cgccgcgctt aatgcgccgc tacagggcgc gtcccattcg ccattcaggc tgcgcaactg
     4321 ttgggaaggg cgatcggtgc gggcctcttc gctattacgc cagctggcga aagggggatg
     4381 tgctgcaagg cgattaagtt gggtaacgcc agggttttcc cagtcacgac gttgtaaaac
//
