LOCUS       sequence-112926-        3584 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 3584)
  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 3584)
  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-OsU6c
FEATURES             Location/Qualifiers
     source          1..3584
                     /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(938..955)
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     complement(938..954)
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(947..969)
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     primer_bind     complement(1163..1182)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     1282..1304
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     primer_bind     complement(1342..1360)
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     promoter        1428..1532
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(1533..1601,1602..2393)
                     /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(1751..1770)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      2564..3152
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     3053..3072
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     3306..3323
                     /label=L4440
                     /note="L4440 vector, forward primer"
     protein_bind    3440..3461
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     promoter        join(3476..3481,3482..3499,3500..3506)
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    3514..3530
                     /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     3519..3541
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     primer_bind     3538..3554
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     3538..3554
                     /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 gactcattag cggtatgcat
      181 gttggtagaa gtcggagatg taaataattt tcattatata aaaaaggtac ttcgagaaaa
      241 ataaatgcat acgaattaat tctttttatg ttttttaaac caagtatata gaatttattg
      301 atggttaaaa tttcaaaaat atgacgagag aaaggttaaa cgtacggcat atacttctga
      361 acagagaggg aatatggggt ttttgttgct cccaacaatt cttaagcacg taaaggaaaa
      421 aagcacatta tccacattgt acttccagag atatgtacag cattacgtag gtacgttttc
      481 tttttcttcc cggagagatg atacaataat catgtaaacc cagaatttaa aaaatattct
      541 ttactataaa aattttaatt agggaacgta ttatttttta catgacacct tttgagaaag
      601 agggacttgt aatatgggac aaatgaacaa tttctaagaa atgggcatat gactctcagt
      661 acaatggacc aaattccctc cagtcggccc agcaatacaa agggaaagaa atgagggggc
      721 ccacaggcca cggcccactt ttctccgtgg tggggagatc cagctagagg tccggcccac
      781 aagtggccct tgccccgtgg gacggtggga ttgcagagcg cgtgggcgga aacaacagtt
      841 tagtaccacc tcgctcacgc aacgacgcga ccacttgctt ataagctgct gcgctgaggc
      901 tcagagagac ctctgaagat aacatactaa gcttggcact ggccgtcgtt ttacaacgtc
      961 gtgactggga aaaccctggc gttacccaac ttaatcgcct tgcagcacat ccccctttcg
     1021 ccagctggcg taatagcgaa gaggcccgca ccgatcgccc ttcccaacag ttgcgcagcc
     1081 tgaatggcga atggcgcctg atgcggtatt ttctccttac gcatctgtgc ggtatttcac
     1141 accgcatatg gtgcactctc agtacaatct gctctgatgc cgcatagtta agccagcccc
     1201 gacacccgcc aacacccgct gacgcgccct gacgggcttg tctgctcccg gcatccgctt
     1261 acagacaagc tgtgaccgtc tccgggagct gcatgtgtca gaggttttca ccgtcatcac
     1321 cgaaacgcgc gagacgaaag ggcctcgtga tacgcctatt tttataggtt aatgtcatga
     1381 taataatggt ttcttagacg tcaggtggca cttttcgggg aaatgtgcgc ggaaccccta
     1441 tttgtttatt tttctaaata cattcaaata tgtatccgct catgagacaa taaccctgat
     1501 aaatgcttca ataatattga aaaaggaaga gtatgagtat tcaacatttc cgtgtcgccc
     1561 ttattccctt ttttgcggca ttttgccttc ctgtttttgc tcacccagaa acgctggtga
     1621 aagtaaaaga tgctgaagat cagttgggtg cacgagtggg ttacatcgaa ctggatctca
     1681 acagcggtaa gatccttgag agttttcgcc ccgaagaacg ttttccaatg atgagcactt
     1741 ttaaagttct gctatgtggc gcggtattat cccgtattga cgccgggcaa gagcaactcg
     1801 gtcgccgcat acactattct cagaatgact tggttgagta ctcaccagtc acagaaaagc
     1861 atcttacgga tggcatgaca gtaagagaat tatgcagtgc tgccataacc atgagtgata
     1921 acactgcggc caacttactt ctgacaacga tcggaggacc gaaggagcta accgcttttt
     1981 tgcacaacat gggggatcat gtaactcgcc ttgatcgttg ggaaccggag ctgaatgaag
     2041 ccataccaaa cgacgagcgt gacaccacga tgcctgtagc aatggcaaca acgttgcgca
     2101 aactattaac tggcgaacta cttactctag cttcccggca acaattaata gactggatgg
     2161 aggcggataa agttgcagga ccacttctgc gctcggccct tccggctggc tggtttattg
     2221 ctgataaatc tggagccggt gagcgtgggt ctcgcggtat cattgcagca ctggggccag
     2281 atggtaagcc ctcccgtatc gtagttatct acacgacggg gagtcaggca actatggatg
     2341 aacgaaatag acagatcgct gagataggtg cctcactgat taagcattgg taactgtcag
     2401 accaagttta ctcatatata ctttagattg atttaaaact tcatttttaa tttaaaagga
     2461 tctaggtgaa gatccttttt gataatctca tgaccaaaat cccttaacgt gagttttcgt
     2521 tccactgagc gtcagacccc gtagaaaaga tcaaaggatc ttcttgagat cctttttttc
     2581 tgcgcgtaat ctgctgcttg caaacaaaaa aaccaccgct accagcggtg gtttgtttgc
     2641 cggatcaaga gctaccaact ctttttccga aggtaactgg cttcagcaga gcgcagatac
     2701 caaatactgt tcttctagtg tagccgtagt taggccacca cttcaagaac tctgtagcac
     2761 cgcctacata cctcgctctg ctaatcctgt taccagtggc tgctgccagt ggcgataagt
     2821 cgtgtcttac cgggttggac tcaagacgat agttaccgga taaggcgcag cggtcgggct
     2881 gaacgggggg ttcgtgcaca cagcccagct tggagcgaac gacctacacc gaactgagat
     2941 acctacagcg tgagctatga gaaagcgcca cgcttcccga agggagaaag gcggacaggt
     3001 atccggtaag cggcagggtc ggaacaggag agcgcacgag ggagcttcca gggggaaacg
     3061 cctggtatct ttatagtcct gtcgggtttc gccacctctg acttgagcgt cgatttttgt
     3121 gatgctcgtc aggggggcgg agcctatgga aaaacgccag caacgcggcc tttttacggt
     3181 tcctggcctt ttgctggcct tttgctcaca tgttctttcc tgcgttatcc cctgattctg
     3241 tggataaccg tattaccgcc tttgagtgag ctgataccgc tcgccgcagc cgaacgaccg
     3301 agcgcagcga gtcagtgagc gaggaagcgg aagagcgccc aatacgcaaa ccgcctctcc
     3361 ccgcgcgttg gccgattcat taatgcagct ggcacgacag gtttcccgac tggaaagcgg
     3421 gcagtgagcg caacgcaatt aatgtgagtt agctcactca ttaggcaccc caggctttac
     3481 actttatgct tccggctcgt atgttgtgtg gaattgtgag cggataacaa tttcacacag
     3541 gaaacagcta tgaccatgat tacgaattcg agctcggtac ccgg
//
