LOCUS       sequence-490425-        4679 bp    DNA     circular SYN 09-MAR-2026
DEFINITION  Express gRNA in plant cells (protoplast or callus) for Cas9-mediated
            editing.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 4679)
  TITLE     Gao September 2025 unpublished plasmids
REFERENCE   2  (bases 1 to 4679)
  AUTHORS   .
  TITLE     Direct Submission
  JOURNAL   Exported Mar 9, 2026 from SnapGene Server 8.0.1
            https://www.snapgene.com
COMMENT     SGRef: number: 1; type: "Journal Article"
COMMENT     Sequence Label: p35C-gRNA-ccdB
FEATURES             Location/Qualifiers
     source          1..4679
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     194..213
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     407..429
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     primer_bind     421..438
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     422..438
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     CDS             1812..2117
                     /codon_start=1
                     /gene="ccdB"
                     /product="CcdB, a bacterial toxin that poisons DNA gyrase"
                     /label=ccdB
                     /note="Plasmids containing the ccdB gene cannot be 
                     propagated in standard E. coli strains."
                     /translation="MQFKVYTYKRESRYRLFVDVQSDIIDTPGRRMVIPLASARLLSDK
                     VSRELYPVVHIGDESWRMMTTDMASVPVSVIGEEVADLSHRENDIKNAINLMFWGI"
     misc_RNA        2125..2200
                     /label=gRNA scaffold
                     /note="guide RNA scaffold for the Streptococcus pyogenes 
                     CRISPR/Cas9 system"
     ncRNA           2201..2268
                     /product="hepatitis delta virus self-cleaving ribozyme 
                     (Ferre-D'Amare and Scott, 2010)"
                     /label=HDV ribozyme
                     /ncRNA_class="ribozyme"
     terminator      2299..2547
                     /label=HSP terminator
                     /note="efficient transcription terminator from the 
                     Arabidopsis thaliana heat shock protein 18.2 gene (Nagaya 
                     et al., 2010)"
     primer_bind     complement(2566..2582)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(2566..2582)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(2579..2601)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    2590..2606
                     /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(2614..2620,2621..2638,2639..2644))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    2659..2680
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(2797..2814)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(2968..3556)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(3048..3067)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(3727..4518,4519..4587))
                     /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     4350..4369
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(join(4588..4679,1..13))
                     /gene="bla"
                     /label=AmpR promoter
ORIGIN
        1 taggggttcc gcgcacattt ccccgaaaag tgccacctga cgtctaagaa accattatta
       61 tcatgacatt aacctataaa aataggcgta tcacggacgg tcacagcttg tctgtaagcg
      121 gatgccggga gcagacaagc ccgtcagggc gcgtcagcgg gtgttggcgg gtgtcggggc
      181 tggcttaact atgcggcatc agagcagatt gtactgagag tgcaccatat gcggtgtgaa
      241 ataccgcaca gatgcgtaag gagaaaatac cgcatcaggc gccattcgcc attcaggctg
      301 cgcaactgtt gggaagggcg atcggtgcgg gcctcttcgc tattacgcca gctggcgaaa
      361 gggggatgtg ctgcaaggcg attaagttgg gtaacgccag ggttttccca gtcacgacgt
      421 tgtaaaacga cggccagtga attcatggag tcaaagattc aaatagagga cctaacagaa
      481 ctcgccgtaa agactggcga acagttcata cagagtctct tacgactcaa tgacaagaag
      541 aaaatcttcg tcaacatggt ggagcacgac acacttgtct actccaaaaa tatcaaagat
      601 acagtctcag aagaccaaag ggcaattgag acttttcaac aaagggtaat atccggaaac
      661 ctcctcggat tccattgccc agctatctgt cactttattg tgaagatagt ggaaaaggaa
      721 ggtggctcct acaaatgcca tcattgcgat aaaggaaagg ccatcgttga agatgcctct
      781 gccgacagtg gtcccaaaga tggaccccca cccacgagga gcatcgtgga aaaagaagac
      841 gttccaacca cgtcttcaaa gcaagtggat tgatgtgatt ggcagacata ctgtcccaca
      901 aatgaagatg gaatctgtaa aagaaaacgc gtgaaataat gcgtctgaca aaggttaggt
      961 cggctgcctt taatcaatac caaagtggtc cctaccacga tggaaaaact gtgcagtcgg
     1021 tttggctttt tctgacgaac aaataagatt cgtggccgac aggtgggggt ccaccatgtg
     1081 aaggcatctt cagactccaa taatggagca atgacgtaag ggcttacgaa ataagtaagg
     1141 gtagtttggg aaatgtccac tcacccgtca gtctataaat acttagcccc tccctcattg
     1201 ttaagggagc aaaatctcag agagatagtc ctagagagag aaagagagca agtagcctag
     1261 aagtagtcaa ggcggcgaag tattcaggca cgtggccagg aagaagaaaa gccaagacga
     1321 cgaaaacagg taagagctaa gcatctagat aagttgaaaa caatcttcaa aagtcccaca
     1381 tcgcttagat aagaaaacga agctgagttt atatacagct agagtcgaag tagtgattga
     1441 acaaagcacc agtggtctag tggtagaata gtaccctgcc acggtacaga cccgggttcg
     1501 attcccggct ggtgtgagac cgctgatttt tgcggtataa gaatatatac tgatatgtat
     1561 acccgaagta tgtcaaaagg aggtatgcta tgaagcagcg tattacagtg acagttgaca
     1621 gcgacagcta tcagttgctc aaggcatata tgatgtcaat atctccggtc tggtaagcac
     1681 aaccatgcag aatgaagccc gtcgtctgcg tgccgaacgc tggaaagcgg aaaatcagga
     1741 agggatggct gaggtcgccc ggtttattga aatgaacggc tcttttgctg acgagaacag
     1801 gggctggtga aatgcagttt aaggtttaca cctataaaag agagagccgt tatcgtctgt
     1861 ttgtggatgt tcagagtgat attattgaca cgcccgggcg acggatggtg atccccctgg
     1921 ccagtgcacg tctgctgtca gataaagtct cccgtgaact ttacccggtg gtgcatatcg
     1981 gggatgaaag ctggcgtatg atgacaaccg atatggccag tgtgccggtt tccgttatcg
     2041 gggaagaagt ggctgatctc agccaccgcg aaaatgacat caaaaacgcc attaacctga
     2101 tgttctgggg aatataaggt ctctgtttta gagctagaaa tagcaagtta aaataaggct
     2161 agtccgttat caacttgaaa aagtggcacc gagtcggtgc ggccggcatg gtcccagcct
     2221 cctcgctggc gccggctggg caacatgctt cggcatggcg aatgggactt ttttttgata
     2281 tctccggggc taattgaata tgaagatgaa gatgaaatat ttggtgtgtc aaataaaaag
     2341 ctggtgtgct taagtttgtg tttttttctt ggcttgttgt gttatgaatt tgtggctttt
     2401 tctaatatta aatgaatgta agatctcatt ataatgaata aacaaatgtt tctataatcc
     2461 attgtgaatg ttttgttgga tctcttctgc agcatataac tactgtatgt gctatggtat
     2521 ggactatgga atatgattaa agataagaag cttggcgtaa tcatggtcat agctgtttcc
     2581 tgtgtgaaat tgttatccgc tcacaattcc acacaacata cgagccggaa gcataaagtg
     2641 taaagcctgg ggtgcctaat gagtgagcta actcacatta attgcgttgc gctcactgcc
     2701 cgctttccag tcgggaaacc tgtcgtgcca gctgcattaa tgaatcggcc aacgcgcggg
     2761 gagaggcggt ttgcgtattg ggcgctcttc cgcttcctcg ctcactgact cgctgcgctc
     2821 ggtcgttcgg ctgcggcgag cggtatcagc tcactcaaag gcggtaatac ggttatccac
     2881 agaatcaggg gataacgcag gaaagaacat gtgagcaaaa ggccagcaaa aggccaggaa
     2941 ccgtaaaaag gccgcgttgc tggcgttttt ccataggctc cgcccccctg acgagcatca
     3001 caaaaatcga cgctcaagtc agaggtggcg aaacccgaca ggactataaa gataccaggc
     3061 gtttccccct ggaagctccc tcgtgcgctc tcctgttccg accctgccgc ttaccggata
     3121 cctgtccgcc tttctccctt cgggaagcgt ggcgctttct catagctcac gctgtaggta
     3181 tctcagttcg gtgtaggtcg ttcgctccaa gctgggctgt gtgcacgaac cccccgttca
     3241 gcccgaccgc tgcgccttat ccggtaacta tcgtcttgag tccaacccgg taagacacga
     3301 cttatcgcca ctggcagcag ccactggtaa caggattagc agagcgaggt atgtaggcgg
     3361 tgctacagag ttcttgaagt ggtggcctaa ctacggctac actagaagaa cagtatttgg
     3421 tatctgcgct ctgctgaagc cagttacctt cggaaaaaga gttggtagct cttgatccgg
     3481 caaacaaacc accgctggta gcggtggttt ttttgtttgc aagcagcaga ttacgcgcag
     3541 aaaaaaagga tctcaagaag atcctttgat cttttctacg gggtctgacg ctcagtggaa
     3601 cgaaaactca cgttaaggga ttttggtcat gagattatca aaaaggatct tcacctagat
     3661 ccttttaaat taaaaatgaa gttttaaatc aatctaaagt atatatgagt aaacttggtc
     3721 tgacagttac caatgcttaa tcagtgaggc acctatctca gcgatctgtc tatttcgttc
     3781 atccatagtt gcctgactcc ccgtcgtgta gataactacg atacgggagg gcttaccatc
     3841 tggccccagt gctgcaatga taccgcgaga accacgctca ccggctccag atttatcagc
     3901 aataaaccag ccagccggaa gggccgagcg cagaagtggt cctgcaactt tatccgcctc
     3961 catccagtct attaattgtt gccgggaagc tagagtaagt agttcgccag ttaatagttt
     4021 gcgcaacgtt gttgccattg ctacaggcat cgtggtgtca cgctcgtcgt ttggtatggc
     4081 ttcattcagc tccggttccc aacgatcaag gcgagttaca tgatccccca tgttgtgcaa
     4141 aaaagcggtt agctccttcg gtcctccgat cgttgtcaga agtaagttgg ccgcagtgtt
     4201 atcactcatg gttatggcag cactgcataa ttctcttact gtcatgccat ccgtaagatg
     4261 cttttctgtg actggtgagt actcaaccaa gtcattctga gaatagtgta tgcggcgacc
     4321 gagttgctct tgcccggcgt caatacggga taataccgcg ccacatagca gaactttaaa
     4381 agtgctcatc attggaaaac gttcttcggg gcgaaaactc tcaaggatct taccgctgtt
     4441 gagatccagt tcgatgtaac ccactcgtgc acccaactga tcttcagcat cttttacttt
     4501 caccagcgtt tctgggtgag caaaaacagg aaggcaaaat gccgcaaaaa agggaataag
     4561 ggcgacacgg aaatgttgaa tactcatact cttccttttt caatattatt gaagcattta
     4621 tcagggttat tgtctcatga gcggatacat atttgaatgt atttagaaaa ataaacaaa
//
