LOCUS       sequence-444147-        3120 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Transcription of guide RNA (gRNA) in wheat cells.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 3120)
  AUTHORS   Shan Q, Wang Y, Li J, Zhang Y, Chen K, Liang Z, Zhang K, Liu J, Xi 
            JJ, Qiu JL, Gao C
  TITLE     Targeted genome modification of crop plants using a CRISPR-Cas 
            system.
  JOURNAL   Nat Biotechnol. 2013 Aug;31(8):686-8. doi: 10.1038/nbt.2650.
  PUBMED    23929338
REFERENCE   2  (bases 1 to 3120)
  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"; doi: "10.1038/nbt"; 
            journalName: "Nat Biotechnol"; date: "2013-08"; volume: "31"; issue:
            "8"; pages: "686-8"
COMMENT     Sequence Label: pU6-gRNA
FEATURES             Location/Qualifiers
     source          1..3120
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     88..107
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     301..323
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     primer_bind     315..332
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     316..332
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     misc_RNA        726..801
                     /label=gRNA scaffold
                     /note="guide RNA scaffold for the Streptococcus pyogenes 
                     CRISPR/Cas9 system"
     primer_bind     complement(836..852)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(836..852)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(849..871)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    860..876
                     /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(884..890,891..908,909..914))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    929..950
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(1067..1084)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(1238..1826)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(1318..1337)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(1997..2788,2789..2857))
                     /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     2620..2639
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(2858..2962)
                     /gene="bla"
                     /label=AmpR promoter
     primer_bind     3030..3048
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     primer_bind     complement(3086..3108)
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
ORIGIN
        1 cttgtctgta agcggatgcc gggagcagac aagcccgtca gggcgcgtca gcgggtgttg
       61 gcgggtgtcg gggctggctt aactatgcgg catcagagca gattgtactg agagtgcacc
      121 atatgcggtg tgaaataccg cacagatgcg taaggagaaa ataccgcatc aggcgccatt
      181 cgccattcag gctgcgcaac tgttgggaag ggcgatcggt gcgggcctct tcgctattac
      241 gccagctggc gaaaggggga tgtgctgcaa ggcgattaag ttgggtaacg ccagggtttt
      301 cccagtcacg acgttgtaaa acgacggcca gtgccaagct tgaccaagcc cgttattctg
      361 acagttctgg tgctcaacac atttatattt atcaaggagc acattgttac tcactgctag
      421 gagggaatcg aactaggaat attgatcaga ggaactacga gagagctgaa gataactgcc
      481 ctctagctct cactgatctg ggcgcatagt gagatgcagc ccacgtgagt tcagcaacgg
      541 tctagcgctg ggcttttagg cccgcatgat cgggctttgt cgggtggtcg acgtgttcac
      601 gattggggag agcaacgcag cagttcctct tagtttagtc ccacctcgcc tgtccagcag
      661 agttctgacc ggtttataaa ctcgcttgct gcatcagact tgctgtcttc aacacaagaa
      721 gacacgtttt agagctagaa atagcaagtt aaaataaggc tagtccgtta tcaacttgaa
      781 aaagtggcac cgagtcggtg ctttttttgg taccgagctc gaattcgtaa tcatggtcat
      841 agctgtttcc tgtgtgaaat tgttatccgc tcacaattcc acacaacata cgagccggaa
      901 gcataaagtg taaagcctgg ggtgcctaat gagtgagcta actcacatta attgcgttgc
      961 gctcactgcc cgctttccag tcgggaaacc tgtcgtgcca gctgcattaa tgaatcggcc
     1021 aacgcgcggg gagaggcggt ttgcgtattg ggcgctcttc cgcttcctcg ctcactgact
     1081 cgctgcgctc ggtcgttcgg ctgcggcgag cggtatcagc tcactcaaag gcggtaatac
     1141 ggttatccac agaatcaggg gataacgcag gaaagaacat gtgagcaaaa ggccagcaaa
     1201 aggccaggaa ccgtaaaaag gccgcgttgc tggcgttttt ccataggctc cgcccccctg
     1261 acgagcatca caaaaatcga cgctcaagtc agaggtggcg aaacccgaca ggactataaa
     1321 gataccaggc gtttccccct ggaagctccc tcgtgcgctc tcctgttccg accctgccgc
     1381 ttaccggata cctgtccgcc tttctccctt cgggaagcgt ggcgctttct catagctcac
     1441 gctgtaggta tctcagttcg gtgtaggtcg ttcgctccaa gctgggctgt gtgcacgaac
     1501 cccccgttca gcccgaccgc tgcgccttat ccggtaacta tcgtcttgag tccaacccgg
     1561 taagacacga cttatcgcca ctggcagcag ccactggtaa caggattagc agagcgaggt
     1621 atgtaggcgg tgctacagag ttcttgaagt ggtggcctaa ctacggctac actagaagaa
     1681 cagtatttgg tatctgcgct ctgctgaagc cagttacctt cggaaaaaga gttggtagct
     1741 cttgatccgg caaacaaacc accgctggta gcggtggttt ttttgtttgc aagcagcaga
     1801 ttacgcgcag aaaaaaagga tctcaagaag atcctttgat cttttctacg gggtctgacg
     1861 ctcagtggaa cgaaaactca cgttaaggga ttttggtcat gagattatca aaaaggatct
     1921 tcacctagat ccttttaaat taaaaatgaa gttttaaatc aatctaaagt atatatgagt
     1981 aaacttggtc tgacagttac caatgcttaa tcagtgaggc acctatctca gcgatctgtc
     2041 tatttcgttc atccatagtt gcctgactcc ccgtcgtgta gataactacg atacgggagg
     2101 gcttaccatc tggccccagt gctgcaatga taccgcgaga cccacgctca ccggctccag
     2161 atttatcagc aataaaccag ccagccggaa gggccgagcg cagaagtggt cctgcaactt
     2221 tatccgcctc catccagtct attaattgtt gccgggaagc tagagtaagt agttcgccag
     2281 ttaatagttt gcgcaacgtt gttgccattg ctacaggcat cgtggtgtca cgctcgtcgt
     2341 ttggtatggc ttcattcagc tccggttccc aacgatcaag gcgagttaca tgatccccca
     2401 tgttgtgcaa aaaagcggtt agctccttcg gtcctccgat cgttgtcaga agtaagttgg
     2461 ccgcagtgtt atcactcatg gttatggcag cactgcataa ttctcttact gtcatgccat
     2521 ccgtaagatg cttttctgtg actggtgagt actcaaccaa gtcattctga gaatagtgta
     2581 tgcggcgacc gagttgctct tgcccggcgt caatacggga taataccgcg ccacatagca
     2641 gaactttaaa agtgctcatc attggaaaac gttcttcggg gcgaaaactc tcaaggatct
     2701 taccgctgtt gagatccagt tcgatgtaac ccactcgtgc acccaactga tcttcagcat
     2761 cttttacttt caccagcgtt tctgggtgag caaaaacagg aaggcaaaat gccgcaaaaa
     2821 agggaataag ggcgacacgg aaatgttgaa tactcatact cttccttttt caatattatt
     2881 gaagcattta tcagggttat tgtctcatga gcggatacat atttgaatgt atttagaaaa
     2941 ataaacaaat aggggttccg cgcacatttc cccgaaaagt gccacctgac gtctaagaaa
     3001 ccattattat catgacatta acctataaaa ataggcgtat cacgaggccc tttcgtctcg
     3061 cgcgtttcgg tgatgacggt gaaaacctct gacacatgca gctcccggag acggtcacag
//
