LOCUS       sequence-112925-        3175 bp    DNA     circular SYN 14-APR-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 3175)
  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 3175)
  AUTHORS   .
  TITLE     Direct Submission
  JOURNAL   Exported Apr 14, 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-OsU6b
FEATURES             Location/Qualifiers
     source          1..3175
                     /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(529..546)
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     complement(529..545)
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(538..560)
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     primer_bind     complement(754..773)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     873..895
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     primer_bind     complement(933..951)
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     promoter        1019..1123
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(1124..1192,1193..1984)
                     /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(1342..1361)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      2155..2743
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     2644..2663
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     2897..2914
                     /label=L4440
                     /note="L4440 vector, forward primer"
     protein_bind    3031..3052
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     promoter        join(3067..3072,3073..3090,3091..3097)
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    3105..3121
                     /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     3110..3132
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     primer_bind     3129..3145
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     3129..3145
                     /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 gatgcaagaa cgaactaagc
      181 cggacaaaaa aaaaaggagc acatatacaa accggtttta ttcatgaatg gtcacgatgg
      241 atgatggggc tcagacttga gctacgaggc cgcaggcgag agaagcctag tgtgctctct
      301 gcttgtttgg gccgtaacgg aggatacggc ccacgagcgt gtactaccgc gcgggatgcc
      361 gctgggcgct gcgggggccg ttggatgggg atcggtgggt cgcgggagcg ttgaggggag
      421 acaggtttag taccacctcg cctaccgaac aatgaagaac ccaccttata accccgcgcg
      481 ctgccgcttg tgttgagaga cctctgaaga taacatacta agcttggcac tggccgtcgt
      541 tttacaacgt cgtgactggg aaaaccctgg cgttacccaa cttaatcgcc ttgcagcaca
      601 tccccctttc gccagctggc gtaatagcga agaggcccgc accgatcgcc cttcccaaca
      661 gttgcgcagc ctgaatggcg aatggcgcct gatgcggtat tttctcctta cgcatctgtg
      721 cggtatttca caccgcatat ggtgcactct cagtacaatc tgctctgatg ccgcatagtt
      781 aagccagccc cgacacccgc caacacccgc tgacgcgccc tgacgggctt gtctgctccc
      841 ggcatccgct tacagacaag ctgtgaccgt ctccgggagc tgcatgtgtc agaggttttc
      901 accgtcatca ccgaaacgcg cgagacgaaa gggcctcgtg atacgcctat ttttataggt
      961 taatgtcatg ataataatgg tttcttagac gtcaggtggc acttttcggg gaaatgtgcg
     1021 cggaacccct atttgtttat ttttctaaat acattcaaat atgtatccgc tcatgagaca
     1081 ataaccctga taaatgcttc aataatattg aaaaaggaag agtatgagta ttcaacattt
     1141 ccgtgtcgcc cttattccct tttttgcggc attttgcctt cctgtttttg ctcacccaga
     1201 aacgctggtg aaagtaaaag atgctgaaga tcagttgggt gcacgagtgg gttacatcga
     1261 actggatctc aacagcggta agatccttga gagttttcgc cccgaagaac gttttccaat
     1321 gatgagcact tttaaagttc tgctatgtgg cgcggtatta tcccgtattg acgccgggca
     1381 agagcaactc ggtcgccgca tacactattc tcagaatgac ttggttgagt actcaccagt
     1441 cacagaaaag catcttacgg atggcatgac agtaagagaa ttatgcagtg ctgccataac
     1501 catgagtgat aacactgcgg ccaacttact tctgacaacg atcggaggac cgaaggagct
     1561 aaccgctttt ttgcacaaca tgggggatca tgtaactcgc cttgatcgtt gggaaccgga
     1621 gctgaatgaa gccataccaa acgacgagcg tgacaccacg atgcctgtag caatggcaac
     1681 aacgttgcgc aaactattaa ctggcgaact acttactcta gcttcccggc aacaattaat
     1741 agactggatg gaggcggata aagttgcagg accacttctg cgctcggccc ttccggctgg
     1801 ctggtttatt gctgataaat ctggagccgg tgagcgtggg tctcgcggta tcattgcagc
     1861 actggggcca gatggtaagc cctcccgtat cgtagttatc tacacgacgg ggagtcaggc
     1921 aactatggat gaacgaaata gacagatcgc tgagataggt gcctcactga ttaagcattg
     1981 gtaactgtca gaccaagttt actcatatat actttagatt gatttaaaac ttcattttta
     2041 atttaaaagg atctaggtga agatcctttt tgataatctc atgaccaaaa tcccttaacg
     2101 tgagttttcg ttccactgag cgtcagaccc cgtagaaaag atcaaaggat cttcttgaga
     2161 tccttttttt ctgcgcgtaa tctgctgctt gcaaacaaaa aaaccaccgc taccagcggt
     2221 ggtttgtttg ccggatcaag agctaccaac tctttttccg aaggtaactg gcttcagcag
     2281 agcgcagata ccaaatactg ttcttctagt gtagccgtag ttaggccacc acttcaagaa
     2341 ctctgtagca ccgcctacat acctcgctct gctaatcctg ttaccagtgg ctgctgccag
     2401 tggcgataag tcgtgtctta ccgggttgga ctcaagacga tagttaccgg ataaggcgca
     2461 gcggtcgggc tgaacggggg gttcgtgcac acagcccagc ttggagcgaa cgacctacac
     2521 cgaactgaga tacctacagc gtgagctatg agaaagcgcc acgcttcccg aagggagaaa
     2581 ggcggacagg tatccggtaa gcggcagggt cggaacagga gagcgcacga gggagcttcc
     2641 agggggaaac gcctggtatc tttatagtcc tgtcgggttt cgccacctct gacttgagcg
     2701 tcgatttttg tgatgctcgt caggggggcg gagcctatgg aaaaacgcca gcaacgcggc
     2761 ctttttacgg ttcctggcct tttgctggcc ttttgctcac atgttctttc ctgcgttatc
     2821 ccctgattct gtggataacc gtattaccgc ctttgagtga gctgataccg ctcgccgcag
     2881 ccgaacgacc gagcgcagcg agtcagtgag cgaggaagcg gaagagcgcc caatacgcaa
     2941 accgcctctc cccgcgcgtt ggccgattca ttaatgcagc tggcacgaca ggtttcccga
     3001 ctggaaagcg ggcagtgagc gcaacgcaat taatgtgagt tagctcactc attaggcacc
     3061 ccaggcttta cactttatgc ttccggctcg tatgttgtgt ggaattgtga gcggataaca
     3121 atttcacaca ggaaacagct atgaccatga ttacgaattc gagctcggta cccgg
//
