LOCUS       sequence-112923-        3289 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 3289)
  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 3289)
  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-OsU6a
FEATURES             Location/Qualifiers
     source          1..3289
                     /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"
     primer_bind     complement(643..660)
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     complement(643..659)
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(652..674)
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     primer_bind     complement(868..887)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     987..1009
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     primer_bind     complement(1047..1065)
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     promoter        1133..1237
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(1238..1306,1307..2098)
                     /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(1456..1475)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      2269..2857
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     2758..2777
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     3011..3028
                     /label=L4440
                     /note="L4440 vector, forward primer"
     protein_bind    3145..3166
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     promoter        join(3181..3186,3187..3204,3205..3211)
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    3219..3235
                     /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     3224..3246
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     primer_bind     3243..3259
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     3243..3259
                     /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 gattttttcc tgtagttttc
      181 ccacaaccat tttttaccat ccgaatgata ggataggaaa aatatccaag tgaacagtat
      241 tcctataaaa ttcccgtaaa aagcctgcaa tccgaatgag ccctgaagtc tgaactagcc
      301 ggtcacctgt acaggctatc gagatgccat acaagagacg gtagtaggaa ctaggaagac
      361 gatggttgat tcgtcaggcg aaatcgtcgt cctgcagtcg catctatggg cctggacgga
      421 ataggggaaa aagttggccg gataggaggg aaaggcccag gtgcttacgt gcgaggtagg
      481 cctgggctct cagcacttcg attcgttggc accggggtag gatgcaatag agagcaacgt
      541 ttagtaccac ctcgcttagc tagagcaaac tggactgcct tatatgcgcg ggtgctggct
      601 tggctgccga gagacctctg aagataacat actaagcttg gcactggccg tcgttttaca
      661 acgtcgtgac tgggaaaacc ctggcgttac ccaacttaat cgccttgcag cacatccccc
      721 tttcgccagc tggcgtaata gcgaagaggc ccgcaccgat cgcccttccc aacagttgcg
      781 cagcctgaat ggcgaatggc gcctgatgcg gtattttctc cttacgcatc tgtgcggtat
      841 ttcacaccgc atatggtgca ctctcagtac aatctgctct gatgccgcat agttaagcca
      901 gccccgacac ccgccaacac ccgctgacgc gccctgacgg gcttgtctgc tcccggcatc
      961 cgcttacaga caagctgtga ccgtctccgg gagctgcatg tgtcagaggt tttcaccgtc
     1021 atcaccgaaa cgcgcgagac gaaagggcct cgtgatacgc ctatttttat aggttaatgt
     1081 catgataata atggtttctt agacgtcagg tggcactttt cggggaaatg tgcgcggaac
     1141 ccctatttgt ttatttttct aaatacattc aaatatgtat ccgctcatga gacaataacc
     1201 ctgataaatg cttcaataat attgaaaaag gaagagtatg agtattcaac atttccgtgt
     1261 cgcccttatt cccttttttg cggcattttg ccttcctgtt tttgctcacc cagaaacgct
     1321 ggtgaaagta aaagatgctg aagatcagtt gggtgcacga gtgggttaca tcgaactgga
     1381 tctcaacagc ggtaagatcc ttgagagttt tcgccccgaa gaacgttttc caatgatgag
     1441 cacttttaaa gttctgctat gtggcgcggt attatcccgt attgacgccg ggcaagagca
     1501 actcggtcgc cgcatacact attctcagaa tgacttggtt gagtactcac cagtcacaga
     1561 aaagcatctt acggatggca tgacagtaag agaattatgc agtgctgcca taaccatgag
     1621 tgataacact gcggccaact tacttctgac aacgatcgga ggaccgaagg agctaaccgc
     1681 ttttttgcac aacatggggg atcatgtaac tcgccttgat cgttgggaac cggagctgaa
     1741 tgaagccata ccaaacgacg agcgtgacac cacgatgcct gtagcaatgg caacaacgtt
     1801 gcgcaaacta ttaactggcg aactacttac tctagcttcc cggcaacaat taatagactg
     1861 gatggaggcg gataaagttg caggaccact tctgcgctcg gcccttccgg ctggctggtt
     1921 tattgctgat aaatctggag ccggtgagcg tgggtctcgc ggtatcattg cagcactggg
     1981 gccagatggt aagccctccc gtatcgtagt tatctacacg acggggagtc aggcaactat
     2041 ggatgaacga aatagacaga tcgctgagat aggtgcctca ctgattaagc attggtaact
     2101 gtcagaccaa gtttactcat atatacttta gattgattta aaacttcatt tttaatttaa
     2161 aaggatctag gtgaagatcc tttttgataa tctcatgacc aaaatccctt aacgtgagtt
     2221 ttcgttccac tgagcgtcag accccgtaga aaagatcaaa ggatcttctt gagatccttt
     2281 ttttctgcgc gtaatctgct gcttgcaaac aaaaaaacca ccgctaccag cggtggtttg
     2341 tttgccggat caagagctac caactctttt tccgaaggta actggcttca gcagagcgca
     2401 gataccaaat actgttcttc tagtgtagcc gtagttaggc caccacttca agaactctgt
     2461 agcaccgcct acatacctcg ctctgctaat cctgttacca gtggctgctg ccagtggcga
     2521 taagtcgtgt cttaccgggt tggactcaag acgatagtta ccggataagg cgcagcggtc
     2581 gggctgaacg gggggttcgt gcacacagcc cagcttggag cgaacgacct acaccgaact
     2641 gagataccta cagcgtgagc tatgagaaag cgccacgctt cccgaaggga gaaaggcgga
     2701 caggtatccg gtaagcggca gggtcggaac aggagagcgc acgagggagc ttccaggggg
     2761 aaacgcctgg tatctttata gtcctgtcgg gtttcgccac ctctgacttg agcgtcgatt
     2821 tttgtgatgc tcgtcagggg ggcggagcct atggaaaaac gccagcaacg cggccttttt
     2881 acggttcctg gccttttgct ggccttttgc tcacatgttc tttcctgcgt tatcccctga
     2941 ttctgtggat aaccgtatta ccgcctttga gtgagctgat accgctcgcc gcagccgaac
     3001 gaccgagcgc agcgagtcag tgagcgagga agcggaagag cgcccaatac gcaaaccgcc
     3061 tctccccgcg cgttggccga ttcattaatg cagctggcac gacaggtttc ccgactggaa
     3121 agcgggcagt gagcgcaacg caattaatgt gagttagctc actcattagg caccccaggc
     3181 tttacacttt atgcttccgg ctcgtatgtt gtgtggaatt gtgagcggat aacaatttca
     3241 cacaggaaac agctatgacc atgattacga attcgagctc ggtacccgg
//
