LOCUS       sequence-82482-e        3193 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Transcription of guide RNA (gRNA) in rice cells.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 3193)
  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 3193)
  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: pU3-gRNA
FEATURES             Location/Qualifiers
     source          1..3193
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     complement(29..51)
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     primer_bind     151..170
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     364..386
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     primer_bind     378..395
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     379..395
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     promoter        427..807
                     /label=OsU3 promoter
                     /note="Oryza sativa (rice) snRNA U3 promoter"
     misc_RNA        837..912
                     /label=gRNA scaffold
                     /note="guide RNA scaffold for the Streptococcus pyogenes 
                     CRISPR/Cas9 system"
     primer_bind     complement(972..988)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(972..988)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(985..1007)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    996..1012
                     /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(1020..1026,1027..1044,1045..1050))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    1065..1086
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(1203..1220)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(1374..1962)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(1454..1473)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(2133..2924,2925..2993))
                     /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     2756..2775
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(2994..3098)
                     /gene="bla"
                     /label=AmpR promoter
     primer_bind     3166..3184
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
ORIGIN
        1 tcgcgcgttt cggtgatgac ggtgaaaacc tctgacacat gcagctcccg gagacggtca
       61 cagcttgtct gtaagcggat gccgggagca gacaagcccg tcagggcgcg tcagcgggtg
      121 ttggcgggtg tcggggctgg cttaactatg cggcatcaga gcagattgta ctgagagtgc
      181 accatatgcg gtgtgaaata ccgcacagat gcgtaaggag aaaataccgc atcaggcgcc
      241 attcgccatt caggctgcgc aactgttggg aagggcgatc ggtgcgggcc tcttcgctat
      301 tacgccagct ggcgaaaggg ggatgtgctg caaggcgatt aagttgggta acgccagggt
      361 tttcccagtc acgacgttgt aaaacgacgg ccagtgccaa gcttgcatgc ctgcaggtcg
      421 acgattaagg aatctttaaa catacgaaca gatcacttaa agttcttctg aagcaactta
      481 aagttatcag gcatgcatgg atcttggagg aatcagatgt gcagtcaggg accatagcac
      541 aagacaggcg tcttctactg gtgctaccag caaatgctgg aagccgggaa cactgggtac
      601 gtcggaaacc acgtgatgtg aagaagtaag ataaactgta ggagaaaagc atttcgtagt
      661 gggccatgaa gcctttcagg acatgtattg cagtatgggc cggcccatta cgcaattgga
      721 cgacaacaaa gactagtatt agtaccacct cggctatcca catagatcaa agctgattta
      781 aaagagttgt gcagatgatc cgtggcagct cgcaggtgaa cacaacacct gcacacgttt
      841 tagagctaga aatagcaagt taaaataagg ctagtccgtt atcaacttga aaaagtggca
      901 ccgagtcggt gctttttttc cacataatct ctagaggatc cccgggtacc gagctcgaat
      961 tcgtaatcat ggtcatagct gtttcctgtg tgaaattgtt atccgctcac aattccacac
     1021 aacatacgag ccggaagcat aaagtgtaaa gcctggggtg cctaatgagt gagctaactc
     1081 acattaattg cgttgcgctc actgcccgct ttccagtcgg gaaacctgtc gtgccagctg
     1141 cattaatgaa tcggccaacg cgcggggaga ggcggtttgc gtattgggcg ctcttccgct
     1201 tcctcgctca ctgactcgct gcgctcggtc gttcggctgc ggcgagcggt atcagctcac
     1261 tcaaaggcgg taatacggtt atccacagaa tcaggggata acgcaggaaa gaacatgtga
     1321 gcaaaaggcc agcaaaaggc caggaaccgt aaaaaggccg cgttgctggc gtttttccat
     1381 aggctccgcc cccctgacga gcatcacaaa aatcgacgct caagtcagag gtggcgaaac
     1441 ccgacaggac tataaagata ccaggcgttt ccccctggaa gctccctcgt gcgctctcct
     1501 gttccgaccc tgccgcttac cggatacctg tccgcctttc tcccttcggg aagcgtggcg
     1561 ctttctcaaa gctcacgctg taggtatctc agttcggtgt aggtcgttcg ctccaagctg
     1621 ggctgtgtgc acgaaccccc cgttcagccc gaccgctgcg ccttatccgg taactatcgt
     1681 cttgagtcca acccggtaag acacgactta tcgccactgg cagcagccac tggtaacagg
     1741 attagcagag cgaggtatgt aggcggtgct acagagttct tgaagtggtg gcctaactac
     1801 ggctacacta gaagaacagt atttggtatc tgcgctctgc tgaagccagt taccttcgga
     1861 aaaagagttg gtagctcttg atccggcaaa caaaccaccg ctggtagcgg tggttttttt
     1921 gtttgcaagc agcagattac gcgcagaaaa aaaggatctc aagaagatcc tttgatcttt
     1981 tctacggggt ctgacgctca gtggaacgaa aactcacgtt aagggatttt ggtcatgaga
     2041 ttatcaaaaa ggatcttcac ctagatcctt ttaaattaaa aatgaagttt taaatcaatc
     2101 taaagtatat atgagtaaac ttggtctgac agttaccaat gcttaatcag tgaggcacct
     2161 atctcagcga tctgtctatt tcgttcatcc atagttgcct gactccccgt cgtgtagata
     2221 actacgatac gggagggctt accatctggc cccagtgctg caatgatacc gcgagaccca
     2281 cgctcaccgg ctccagattt atcagcaata aaccagccag ccggaagggc cgagcgcaga
     2341 agtggtcctg caactttatc cgcctccatc cagtctatta attgttgccg ggaagctaga
     2401 gtaagtagtt cgccagttaa tagtttgcgc aacgttgttg ccattgctac aggcatcgtg
     2461 gtgtcacgct cgtcgtttgg tatggcttca ttcagctccg gttcccaacg atcaaggcga
     2521 gttacatgat cccccatgtt gtgcaaaaaa gcggttagct ccttcggtcc tccgatcgtt
     2581 gtcagaagta agttggccgc agtgttatca ctcatggtta tggcagcact gcataattct
     2641 cttactgtca tgccatccgt aagatgcttt tctgtgactg gtgagtactc aaccaagtca
     2701 ttctgagaat agtgtatgcg gcgaccgagt tgctcttgcc cggcgtcaat acgggataat
     2761 accgcgccac atagcagaac tttaaaagtg ctcatcattg gaaaacgttc ttcggggcga
     2821 aaactctcaa ggatcttacc gctgttgaga tccagttcga tgtaacccac tcgtgcaccc
     2881 aactgatctt cagcatcttt tactttcacc agcgtttctg ggtgagcaaa aacaggaagg
     2941 caaaatgccg caaaaaaggg aataagggcg acacggaaat gttgaatact catactcttc
     3001 ctttttcaat attattgaag catttatcag ggttattgtc tcatgagcgg atacatattt
     3061 gaatgtattt agaaaaataa acaaataggg gttccgcgca catttccccg aaaagtgcca
     3121 cctgacgtct aagaaaccat tattatcatg acattaacct ataaaaatag gcgtatcacg
     3181 aggccctttc gtc
//
