LOCUS       sequence-113663-        3224 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  cloning of sgRNAs for expression in plants.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 3224)
  AUTHORS   Ma X, Zhang Q, Zhu Q, Liu W, Chen Y, Qiu R, Wang B, Yang Z, Li H, 
            Lin Y, Xie Y, Shen R, Chen S, Wang Z, Chen Y, Guo J, Chen L, Zhao X,
            Dong Z, Liu YG
  TITLE     A robust CRISPR/Cas9 system for convenient, high-efficiency 
            multiplex genome editing in monocot and dicot plants.
  JOURNAL   Mol Plant. 2015 Apr 24. pii: S1674-2052(15)00204-X. doi: 
            10.1016/j.molp.2015.04.007.
  PUBMED    25917172
REFERENCE   2  (bases 1 to 3224)
  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: "Mol Plant. 
            2015 Apr 24. pii: S1674-2052(15)00204-X. doi: 
            10.1016/j.molp.2015.04.007."
COMMENT     Sequence Label: pYLsgRNA-OsU3m
FEATURES             Location/Qualifiers
     source          1..3224
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     misc_RNA        20..95
                     /label=gRNA scaffold
                     /note="guide RNA scaffold for the Streptococcus pyogenes 
                     CRISPR/Cas9 system"
     promoter        162..544
                     /label=OsU3 promoter
                     /note="Oryza sativa (rice) snRNA U3 promoter"
     primer_bind     complement(578..595)
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     complement(578..594)
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(587..609)
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     primer_bind     complement(803..822)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     922..944
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     primer_bind     complement(982..1000)
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     promoter        1068..1172
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(1173..1241,1242..2033)
                     /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     complement(1391..1410)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      2204..2792
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     2693..2712
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     2946..2963
                     /label=L4440
                     /note="L4440 vector, forward primer"
     protein_bind    3080..3101
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     promoter        join(3116..3121,3122..3139,3140..3146)
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    3154..3170
                     /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)."
     primer_bind     3159..3181
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     primer_bind     3178..3194
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     3178..3194
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
ORIGIN
        1 ggatccagcg tgggtctcgg ttttagagct agaaatagca agttaaaata aggctagtcc
       61 gttatcaact tgaaaaagtg gcaccgagtc ggtgcttttt ttcaagagct tggagtggat
      121 ggaattttcc tccgttttac ctgtggaatc ggcagcaaag gaaggaatct ttaaacatac
      181 gaacagatca cttaaagttc ttctgaagca acttaaagtt atcaggcatg catggatctt
      241 ggaggaatca gatgtgcagt cagggaccat agcacaagac aggcgtcttc tactggtgct
      301 accagcaaat gctggaagcc gggaacactg ggtacgttgg aaaccacgtg tgatgtgaag
      361 gagtaagata aactgtagga gaaaagcatt tcgtagtggg ccatgaagcc tttcaggaca
      421 tgtattgcag tatgggccgg cccattacgc aattggacga caacaaaggc tagtattagt
      481 accacctcgg ctatccacat agatcaaagc tggtttaaaa gagttgtgca gatgatccgt
      541 ggcaagagac ctctgaagat aacatactaa gcttggcact ggccgtcgtt ttacaacgtc
      601 gtgactggga aaaccctggc gttacccaac ttaatcgcct tgcagcacat ccccctttcg
      661 ccagctggcg taatagcgaa gaggcccgca ccgatcgccc ttcccaacag ttgcgcagcc
      721 tgaatggcga atggcgcctg atgcggtatt ttctccttac gcatctgtgc ggtatttcac
      781 accgcatatg gtgcactctc agtacaatct gctctgatgc cgcatagtta agccagcccc
      841 gacacccgcc aacacccgct gacgcgccct gacgggcttg tctgctcccg gcatccgctt
      901 acagacaagc tgtgaccgtc tccgggagct gcatgtgtca gaggttttca ccgtcatcac
      961 cgaaacgcgc gagacgaaag ggcctcgtga tacgcctatt tttataggtt aatgtcatga
     1021 taataatggt ttcttagacg tcaggtggca cttttcgggg aaatgtgcgc ggaaccccta
     1081 tttgtttatt tttctaaata cattcaaata tgtatccgct catgagacaa taaccctgat
     1141 aaatgcttca ataatattga aaaaggaaga gtatgagtat tcaacatttc cgtgtcgccc
     1201 ttattccctt ttttgcggca ttttgccttc ctgtttttgc tcacccagaa acgctggtga
     1261 aagtaaaaga tgctgaagat cagttgggtg cacgagtggg ttacatcgaa ctggatctca
     1321 acagcggtaa gatccttgag agttttcgcc ccgaagaacg ttttccaatg atgagcactt
     1381 ttaaagttct gctatgtggc gcggtattat cccgtattga cgccgggcaa gagcaactcg
     1441 gtcgccgcat acactattct cagaatgact tggttgagta ctcaccagtc acagaaaagc
     1501 atcttacgga tggcatgaca gtaagagaat tatgcagtgc tgccataacc atgagtgata
     1561 acactgcggc caacttactt ctgacaacga tcggaggacc gaaggagcta accgcttttt
     1621 tgcacaacat gggggatcat gtaactcgcc ttgatcgttg ggaaccggag ctgaatgaag
     1681 ccataccaaa cgacgagcgt gacaccacga tgcctgtagc aatggcaaca acgttgcgca
     1741 aactattaac tggcgaacta cttactctag cttcccggca acaattaata gactggatgg
     1801 aggcggataa agttgcagga ccacttctgc gctcggccct tccggctggc tggtttattg
     1861 ctgataaatc tggagccggt gagcgtgggt ctcgcggtat cattgcagca ctggggccag
     1921 atggtaagcc ctcccgtatc gtagttatct acacgacggg gagtcaggca actatggatg
     1981 aacgaaatag acagatcgct gagataggtg cctcactgat taagcattgg taactgtcag
     2041 accaagttta ctcatatata ctttagattg atttaaaact tcatttttaa tttaaaagga
     2101 tctaggtgaa gatccttttt gataatctca tgaccaaaat cccttaacgt gagttttcgt
     2161 tccactgagc gtcagacccc gtagaaaaga tcaaaggatc ttcttgagat cctttttttc
     2221 tgcgcgtaat ctgctgcttg caaacaaaaa aaccaccgct accagcggtg gtttgtttgc
     2281 cggatcaaga gctaccaact ctttttccga aggtaactgg cttcagcaga gcgcagatac
     2341 caaatactgt tcttctagtg tagccgtagt taggccacca cttcaagaac tctgtagcac
     2401 cgcctacata cctcgctctg ctaatcctgt taccagtggc tgctgccagt ggcgataagt
     2461 cgtgtcttac cgggttggac tcaagacgat agttaccgga taaggcgcag cggtcgggct
     2521 gaacgggggg ttcgtgcaca cagcccagct tggagcgaac gacctacacc gaactgagat
     2581 acctacagcg tgagctatga gaaagcgcca cgcttcccga agggagaaag gcggacaggt
     2641 atccggtaag cggcagggtc ggaacaggag agcgcacgag ggagcttcca gggggaaacg
     2701 cctggtatct ttatagtcct gtcgggtttc gccacctctg acttgagcgt cgatttttgt
     2761 gatgctcgtc aggggggcgg agcctatgga aaaacgccag caacgcggcc tttttacggt
     2821 tcctggcctt ttgctggcct tttgctcaca tgttctttcc tgcgttatcc cctgattctg
     2881 tggataaccg tattaccgcc tttgagtgag ctgataccgc tcgccgcagc cgaacgaccg
     2941 agcgcagcga gtcagtgagc gaggaagcgg aagagcgccc aatacgcaaa ccgcctctcc
     3001 ccgcgcgttg gccgattcat taatgcagct ggcacgacag gtttcccgac tggaaagcgg
     3061 gcagtgagcg caacgcaatt aatgtgagtt agctcactca ttaggcaccc caggctttac
     3121 actttatgct tccggctcgt atgttgtgtg gaattgtgag cggataacaa tttcacacag
     3181 gaaacagcta tgaccatgat tacgaattcg agctcggtac ccgg
//
