LOCUS       sequence-333608-        3195 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  For sgRNA expression in monocotyledons protoplasts.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 3195)
  AUTHORS   Lin Q, Jin S, Zong Y, Yu H, Zhu Z, Liu G, Kou L, Wang Y, Qiu JL, Li 
            J, Gao C
  TITLE     High-efficiency prime editing with optimized, paired pegRNAs in 
            plants.
  JOURNAL   Nat Biotechnol. 2021 Mar 25. pii: 10.1038/s41587-021-00868-w. doi: 
            10.1038/s41587-021-00868-w.
  PUBMED    33767395
REFERENCE   2  (bases 1 to 3195)
  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: "Nat 
            Biotechnol. 2021 Mar 25. pii: 10.1038/s41587-021-00868-w. doi: 
            10.1038/s41587-021-00868-w."
COMMENT     Sequence Label: pOsU3
FEATURES             Location/Qualifiers
     source          1..3195
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     complement(19..41)
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     primer_bind     141..160
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     354..376
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     primer_bind     368..385
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     369..385
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     promoter        449..828
                     /label=OsU3 promoter
                     /note="Oryza sativa (rice) snRNA U3 promoter"
     misc_RNA        850..925
                     /label=gRNA scaffold
                     /note="guide RNA scaffold for the Streptococcus pyogenes 
                     CRISPR/Cas9 system"
     terminator      926..945
                     /label=SUP4 terminator
                     /note="transcription terminator for the S. cerevisiae SUP4 
                     tRNA gene"
     primer_bind     complement(964..980)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(964..980)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(977..999)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    988..1004
                     /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(1012..1018,1019..1036,1037..1042))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    1057..1078
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(1195..1212)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(1366..1954)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(1446..1465)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(2125..2916,2917..2985))
                     /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     2748..2767
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(2986..3090)
                     /gene="bla"
                     /label=AmpR promoter
     primer_bind     3158..3176
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
ORIGIN
        1 cggtgatgac ggtgaaaacc tctgacacat gcagctcccg gagacggtca cagcttgtct
       61 gtaagcggat gccgggagca gacaagcccg tcagggcgcg tcagcgggtg ttggcgggtg
      121 tcggggctgg cttaactatg cggcatcaga gcagattgta ctgagagtgc accatatgcg
      181 gtgtgaaata ccgcacagat gcgtaaggag aaaataccgc atcaggcgcc attcgccatt
      241 caggctgcgc aactgttggg aagggcgatc ggtgcgggcc tcttcgctat tacgccagct
      301 ggcgaaaggg ggatgtgctg caaggcgatt aagttgggta acgccagggt tttcccagtc
      361 acgacgttgt aaaacgacgg ccagtgaatt cagtaattca tccaggtcac caagttctag
      421 gattttcaga actgcaactt attttatcaa ggaatcttta aacatacgaa cagatcactt
      481 aaagttcttc tgaagcaact taaagttatc aggcatgcat ggatcttgga ggaatcagat
      541 gtgcagtcag ggaccatagc acaagacagg cgtcttctac tggtgctacc agcaaatgct
      601 ggaagccggg aacactgggt acgttggaaa ccacgtgatg tgaagaagta agataaactg
      661 taggagaaaa gcatttcgta gtgggccatg aagcctttca ggacatgtat tgcagtatgg
      721 gccggcccat tacgcaattg gacgacaaca aagactagta ttagtaccac ctcggctatc
      781 cacatagatc aaagctgatt taaaagagtt gtgcagatga tccgtggcgt gagaccctgc
      841 agggtctctg ttttagagct agaaatagca agttaaaata aggctagtcc gttatcaact
      901 tgaaaaagtg gcaccgagtc ggtgcttttt ttgtttttta tgtctaagct tggcgtaatc
      961 atggtcatag ctgtttcctg tgtgaaattg ttatccgctc acaattccac acaacatacg
     1021 agccggaagc ataaagtgta aagcctgggg tgcctaatga gtgagctaac tcacattaat
     1081 tgcgttgcgc tcactgcccg ctttccagtc gggaaacctg tcgtgccagc tgcattaatg
     1141 aatcggccaa cgcgcgggga gaggcggttt gcgtattggg cgctcttccg cttcctcgct
     1201 cactgactcg ctgcgctcgg tcgttcggct gcggcgagcg gtatcagctc actcaaaggc
     1261 ggtaatacgg ttatccacag aatcagggga taacgcagga aagaacatgt gagcaaaagg
     1321 ccagcaaaag gccaggaacc gtaaaaaggc cgcgttgctg gcgtttttcc ataggctccg
     1381 cccccctgac gagcatcaca aaaatcgacg ctcaagtcag aggtggcgaa acccgacagg
     1441 actataaaga taccaggcgt ttccccctgg aagctccctc gtgcgctctc ctgttccgac
     1501 cctgccgctt accggatacc tgtccgcctt tctcccttcg ggaagcgtgg cgctttctca
     1561 tagctcacgc tgtaggtatc tcagttcggt gtaggtcgtt cgctccaagc tgggctgtgt
     1621 gcacgaaccc cccgttcagc ccgaccgctg cgccttatcc ggtaactatc gtcttgagtc
     1681 caacccggta agacacgact tatcgccact ggcagcagcc actggtaaca ggattagcag
     1741 agcgaggtat gtaggcggtg ctacagagtt cttgaagtgg tggcctaact acggctacac
     1801 tagaagaaca gtatttggta tctgcgctct gctgaagcca gttaccttcg gaaaaagagt
     1861 tggtagctct tgatccggca aacaaaccac cgctggtagc ggtggttttt ttgtttgcaa
     1921 gcagcagatt acgcgcagaa aaaaaggatc tcaagaagat cctttgatct tttctacggg
     1981 gtctgacgct cagtggaacg aaaactcacg ttaagggatt ttggtcatga gattatcaaa
     2041 aaggatcttc acctagatcc ttttaaatta aaaatgaagt tttaaatcaa tctaaagtat
     2101 atatgagtaa acttggtctg acagttacca atgcttaatc agtgaggcac ctatctcagc
     2161 gatctgtcta tttcgttcat ccatagttgc ctgactcccc gtcgtgtaga taactacgat
     2221 acgggagggc ttaccatctg gccccagtgc tgcaatgata ccgcgagaac cacgctcacc
     2281 ggctccagat ttatcagcaa taaaccagcc agccggaagg gccgagcgca gaagtggtcc
     2341 tgcaacttta tccgcctcca tccagtctat taattgttgc cgggaagcta gagtaagtag
     2401 ttcgccagtt aatagtttgc gcaacgttgt tgccattgct acaggcatcg tggtgtcacg
     2461 ctcgtcgttt ggtatggctt cattcagctc cggttcccaa cgatcaaggc gagttacatg
     2521 atcccccatg ttgtgcaaaa aagcggttag ctccttcggt cctccgatcg ttgtcagaag
     2581 taagttggcc gcagtgttat cactcatggt tatggcagca ctgcataatt ctcttactgt
     2641 catgccatcc gtaagatgct tttctgtgac tggtgagtac tcaaccaagt cattctgaga
     2701 atagtgtatg cggcgaccga gttgctcttg cccggcgtca atacgggata ataccgcgcc
     2761 acatagcaga actttaaaag tgctcatcat tggaaaacgt tcttcggggc gaaaactctc
     2821 aaggatctta ccgctgttga gatccagttc gatgtaaccc actcgtgcac ccaactgatc
     2881 ttcagcatct tttactttca ccagcgtttc tgggtgagca aaaacaggaa ggcaaaatgc
     2941 cgcaaaaaag ggaataaggg cgacacggaa atgttgaata ctcatactct tcctttttca
     3001 atattattga agcatttatc agggttattg tctcatgagc ggatacatat ttgaatgtat
     3061 ttagaaaaat aaacaaatag gggttccgcg cacatttccc cgaaaagtgc cacctgacgt
     3121 ctaagaaacc attattatca tgacattaac ctataaaaat aggcgtatca cgaggccctt
     3181 tcgtctcgcg cgttt
//
