LOCUS       sequence-112924-        3491 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 3491)
  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 3491)
  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/LacZ
FEATURES             Location/Qualifiers
     source          1..3491
                     /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(845..861)
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(1070..1089)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     1189..1211
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     primer_bind     complement(1249..1267)
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     promoter        1335..1439
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(1440..1508,1509..2300)
                     /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(1658..1677)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      2471..3059
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     2960..2979
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     3213..3230
                     /label=L4440
                     /note="L4440 vector, forward primer"
     protein_bind    3347..3368
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     promoter        join(3383..3388,3389..3406,3407..3413)
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    3421..3437
                     /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     3426..3448
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     primer_bind     3445..3461
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     3445..3461
                     /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 gacgcgttga cattgtagga
      181 ctatattgct ctaataaagg aggcagctat gctggccgtc gttttacaac gtcgtgactg
      241 ggaaaaccct ggcgttaccc aacttaatcg ccttgcagca catccccctt tcgccagctg
      301 gcgtaatagc gaagaggccc gcaccgatcg cccttcccaa cagttgcgca gcctgaatgg
      361 ctaatttttt cctgtagttt tcccacaacc attttttacc atccgaatga taggatagga
      421 aaaatatcca agtgaacagt attcctataa aattcccgta aaaagcctgc aatccgaatg
      481 agccctgaag tctgaactag ccggtcacct gtacaggcta tcgagatgcc atacaagaga
      541 cggtagtagg aactaggaag acgatggttg attcgtcagg cgaaatcgtc gtcctgcagt
      601 cgcatctatg ggcctggacg gaatagggga aaaagttggc cggataggag ggaaaggccc
      661 aggtgcttac gtgcgaggta ggcctgggct ctcagcactt cgattcgttg gcaccggggt
      721 aggatgcaat agagagcaac gtttagtacc acctcgctta gctagagcaa actggactgc
      781 cttatatgcg cgggtgctgg cttggctgcc gagagacctc tgaagataac atactaagct
      841 tggcactggc cgtcgtttta caacgtcgtg actgggaaaa ccctggcgtt acccaactta
      901 atcgccttgc agcacatccc cctttcgcca gctggcgtaa tagcgaagag gcccgcaccg
      961 atcgcccttc ccaacagttg cgcagcctga atggcgaatg gcgcctgatg cggtattttc
     1021 tccttacgca tctgtgcggt atttcacacc gcatatggtg cactctcagt acaatctgct
     1081 ctgatgccgc atagttaagc cagccccgac acccgccaac acccgctgac gcgccctgac
     1141 gggcttgtct gctcccggca tccgcttaca gacaagctgt gaccgtctcc gggagctgca
     1201 tgtgtcagag gttttcaccg tcatcaccga aacgcgcgag acgaaagggc ctcgtgatac
     1261 gcctattttt ataggttaat gtcatgataa taatggtttc ttagacgtca ggtggcactt
     1321 ttcggggaaa tgtgcgcgga acccctattt gtttattttt ctaaatacat tcaaatatgt
     1381 atccgctcat gagacaataa ccctgataaa tgcttcaata atattgaaaa aggaagagta
     1441 tgagtattca acatttccgt gtcgccctta ttcccttttt tgcggcattt tgccttcctg
     1501 tttttgctca cccagaaacg ctggtgaaag taaaagatgc tgaagatcag ttgggtgcac
     1561 gagtgggtta catcgaactg gatctcaaca gcggtaagat ccttgagagt tttcgccccg
     1621 aagaacgttt tccaatgatg agcactttta aagttctgct atgtggcgcg gtattatccc
     1681 gtattgacgc cgggcaagag caactcggtc gccgcataca ctattctcag aatgacttgg
     1741 ttgagtactc accagtcaca gaaaagcatc ttacggatgg catgacagta agagaattat
     1801 gcagtgctgc cataaccatg agtgataaca ctgcggccaa cttacttctg acaacgatcg
     1861 gaggaccgaa ggagctaacc gcttttttgc acaacatggg ggatcatgta actcgccttg
     1921 atcgttggga accggagctg aatgaagcca taccaaacga cgagcgtgac accacgatgc
     1981 ctgtagcaat ggcaacaacg ttgcgcaaac tattaactgg cgaactactt actctagctt
     2041 cccggcaaca attaatagac tggatggagg cggataaagt tgcaggacca cttctgcgct
     2101 cggcccttcc ggctggctgg tttattgctg ataaatctgg agccggtgag cgtgggtctc
     2161 gcggtatcat tgcagcactg gggccagatg gtaagccctc ccgtatcgta gttatctaca
     2221 cgacggggag tcaggcaact atggatgaac gaaatagaca gatcgctgag ataggtgcct
     2281 cactgattaa gcattggtaa ctgtcagacc aagtttactc atatatactt tagattgatt
     2341 taaaacttca tttttaattt aaaaggatct aggtgaagat cctttttgat aatctcatga
     2401 ccaaaatccc ttaacgtgag ttttcgttcc actgagcgtc agaccccgta gaaaagatca
     2461 aaggatcttc ttgagatcct ttttttctgc gcgtaatctg ctgcttgcaa acaaaaaaac
     2521 caccgctacc agcggtggtt tgtttgccgg atcaagagct accaactctt tttccgaagg
     2581 taactggctt cagcagagcg cagataccaa atactgttct tctagtgtag ccgtagttag
     2641 gccaccactt caagaactct gtagcaccgc ctacatacct cgctctgcta atcctgttac
     2701 cagtggctgc tgccagtggc gataagtcgt gtcttaccgg gttggactca agacgatagt
     2761 taccggataa ggcgcagcgg tcgggctgaa cggggggttc gtgcacacag cccagcttgg
     2821 agcgaacgac ctacaccgaa ctgagatacc tacagcgtga gctatgagaa agcgccacgc
     2881 ttcccgaagg gagaaaggcg gacaggtatc cggtaagcgg cagggtcgga acaggagagc
     2941 gcacgaggga gcttccaggg ggaaacgcct ggtatcttta tagtcctgtc gggtttcgcc
     3001 acctctgact tgagcgtcga tttttgtgat gctcgtcagg ggggcggagc ctatggaaaa
     3061 acgccagcaa cgcggccttt ttacggttcc tggccttttg ctggcctttt gctcacatgt
     3121 tctttcctgc gttatcccct gattctgtgg ataaccgtat taccgccttt gagtgagctg
     3181 ataccgctcg ccgcagccga acgaccgagc gcagcgagtc agtgagcgag gaagcggaag
     3241 agcgcccaat acgcaaaccg cctctccccg cgcgttggcc gattcattaa tgcagctggc
     3301 acgacaggtt tcccgactgg aaagcgggca gtgagcgcaa cgcaattaat gtgagttagc
     3361 tcactcatta ggcaccccag gctttacact ttatgcttcc ggctcgtatg ttgtgtggaa
     3421 ttgtgagcgg ataacaattt cacacaggaa acagctatga ccatgattac gaattcgagc
     3481 tcggtacccg g
//
