LOCUS       sequence-112932-        3162 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 3162)
  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 3162)
  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-AtU6-29
FEATURES             Location/Qualifiers
     source          1..3162
                     /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(516..533)
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     complement(516..532)
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(525..547)
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     primer_bind     complement(741..760)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     860..882
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     primer_bind     complement(920..938)
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     promoter        1006..1110
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(1111..1179,1180..1971)
                     /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(1329..1348)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      2142..2730
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     2631..2650
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     2884..2901
                     /label=L4440
                     /note="L4440 vector, forward primer"
     protein_bind    3018..3039
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     promoter        join(3054..3059,3060..3077,3078..3084)
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    3092..3108
                     /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     3097..3119
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     primer_bind     3116..3132
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     3116..3132
                     /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 gaaaatatca gagatctctt
      181 acagttagtt tcgttcttaa tccaaactac tgcagcctga cagacaaatg aggatgcaaa
      241 caattttaaa gtttatctaa cgctagctgt tttgtttctt ctctctggtg caccaacgac
      301 ggcgttttct caatcataaa gaggcttgtt ttacttaagg ccaataatgt tgatggatcg
      361 aaagaagagg gcttttaata aacgagcccg tttaagctgt aaacgatgtc aaaaacatcc
      421 cacatcgttc agttgaaaat agtagctctg tttatatatt ggtagagtcg actaagagat
      481 tgagagacct ctgaagataa catactaagc ttggcactgg ccgtcgtttt acaacgtcgt
      541 gactgggaaa accctggcgt tacccaactt aatcgccttg cagcacatcc ccctttcgcc
      601 agctggcgta atagcgaaga ggcccgcacc gatcgccctt cccaacagtt gcgcagcctg
      661 aatggcgaat ggcgcctgat gcggtatttt ctccttacgc atctgtgcgg tatttcacac
      721 cgcatatggt gcactctcag tacaatctgc tctgatgccg catagttaag ccagccccga
      781 cacccgccaa cacccgctga cgcgccctga cgggcttgtc tgctcccggc atccgcttac
      841 agacaagctg tgaccgtctc cgggagctgc atgtgtcaga ggttttcacc gtcatcaccg
      901 aaacgcgcga gacgaaaggg cctcgtgata cgcctatttt tataggttaa tgtcatgata
      961 ataatggttt cttagacgtc aggtggcact tttcggggaa atgtgcgcgg aacccctatt
     1021 tgtttatttt tctaaataca ttcaaatatg tatccgctca tgagacaata accctgataa
     1081 atgcttcaat aatattgaaa aaggaagagt atgagtattc aacatttccg tgtcgccctt
     1141 attccctttt ttgcggcatt ttgccttcct gtttttgctc acccagaaac gctggtgaaa
     1201 gtaaaagatg ctgaagatca gttgggtgca cgagtgggtt acatcgaact ggatctcaac
     1261 agcggtaaga tccttgagag ttttcgcccc gaagaacgtt ttccaatgat gagcactttt
     1321 aaagttctgc tatgtggcgc ggtattatcc cgtattgacg ccgggcaaga gcaactcggt
     1381 cgccgcatac actattctca gaatgacttg gttgagtact caccagtcac agaaaagcat
     1441 cttacggatg gcatgacagt aagagaatta tgcagtgctg ccataaccat gagtgataac
     1501 actgcggcca acttacttct gacaacgatc ggaggaccga aggagctaac cgcttttttg
     1561 cacaacatgg gggatcatgt aactcgcctt gatcgttggg aaccggagct gaatgaagcc
     1621 ataccaaacg acgagcgtga caccacgatg cctgtagcaa tggcaacaac gttgcgcaaa
     1681 ctattaactg gcgaactact tactctagct tcccggcaac aattaataga ctggatggag
     1741 gcggataaag ttgcaggacc acttctgcgc tcggcccttc cggctggctg gtttattgct
     1801 gataaatctg gagccggtga gcgtgggtct cgcggtatca ttgcagcact ggggccagat
     1861 ggtaagccct cccgtatcgt agttatctac acgacgggga gtcaggcaac tatggatgaa
     1921 cgaaatagac agatcgctga gataggtgcc tcactgatta agcattggta actgtcagac
     1981 caagtttact catatatact ttagattgat ttaaaacttc atttttaatt taaaaggatc
     2041 taggtgaaga tcctttttga taatctcatg accaaaatcc cttaacgtga gttttcgttc
     2101 cactgagcgt cagaccccgt agaaaagatc aaaggatctt cttgagatcc tttttttctg
     2161 cgcgtaatct gctgcttgca aacaaaaaaa ccaccgctac cagcggtggt ttgtttgccg
     2221 gatcaagagc taccaactct ttttccgaag gtaactggct tcagcagagc gcagatacca
     2281 aatactgttc ttctagtgta gccgtagtta ggccaccact tcaagaactc tgtagcaccg
     2341 cctacatacc tcgctctgct aatcctgtta ccagtggctg ctgccagtgg cgataagtcg
     2401 tgtcttaccg ggttggactc aagacgatag ttaccggata aggcgcagcg gtcgggctga
     2461 acggggggtt cgtgcacaca gcccagcttg gagcgaacga cctacaccga actgagatac
     2521 ctacagcgtg agctatgaga aagcgccacg cttcccgaag ggagaaaggc ggacaggtat
     2581 ccggtaagcg gcagggtcgg aacaggagag cgcacgaggg agcttccagg gggaaacgcc
     2641 tggtatcttt atagtcctgt cgggtttcgc cacctctgac ttgagcgtcg atttttgtga
     2701 tgctcgtcag gggggcggag cctatggaaa aacgccagca acgcggcctt tttacggttc
     2761 ctggcctttt gctggccttt tgctcacatg ttctttcctg cgttatcccc tgattctgtg
     2821 gataaccgta ttaccgcctt tgagtgagct gataccgctc gccgcagccg aacgaccgag
     2881 cgcagcgagt cagtgagcga ggaagcggaa gagcgcccaa tacgcaaacc gcctctcccc
     2941 gcgcgttggc cgattcatta atgcagctgg cacgacaggt ttcccgactg gaaagcgggc
     3001 agtgagcgca acgcaattaa tgtgagttag ctcactcatt aggcacccca ggctttacac
     3061 tttatgcttc cggctcgtat gttgtgtgga attgtgagcg gataacaatt tcacacagga
     3121 aacagctatg accatgatta cgaattcgag ctcggtaccc gg
//
