LOCUS       sequence-112930-        3146 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 3146)
  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 3146)
  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-AtU3d/LacZ
FEATURES             Location/Qualifiers
     source          1..3146
                     /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(500..516)
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(725..744)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     844..866
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     primer_bind     complement(904..922)
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     promoter        990..1094
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(1095..1163,1164..1955)
                     /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(1313..1332)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      2126..2714
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     2615..2634
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     2868..2885
                     /label=L4440
                     /note="L4440 vector, forward primer"
     protein_bind    3002..3023
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     promoter        join(3038..3043,3044..3061,3062..3068)
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    3076..3092
                     /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     3081..3103
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     primer_bind     3100..3116
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     3100..3116
                     /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 ctaaataagc ttatgatttc ttttttctta cgaattttgc gtcccacatc ggtaagcgag
      421 tgaagaaata actgctttat atatggctac aaagcaccat tggtcaagag acctctgaag
      481 ataacatact aagcttggca ctggccgtcg ttttacaacg tcgtgactgg gaaaaccctg
      541 gcgttaccca acttaatcgc cttgcagcac atcccccttt cgccagctgg cgtaatagcg
      601 aagaggcccg caccgatcgc ccttcccaac agttgcgcag cctgaatggc gaatggcgcc
      661 tgatgcggta ttttctcctt acgcatctgt gcggtatttc acaccgcata tggtgcactc
      721 tcagtacaat ctgctctgat gccgcatagt taagccagcc ccgacacccg ccaacacccg
      781 ctgacgcgcc ctgacgggct tgtctgctcc cggcatccgc ttacagacaa gctgtgaccg
      841 tctccgggag ctgcatgtgt cagaggtttt caccgtcatc accgaaacgc gcgagacgaa
      901 agggcctcgt gatacgccta tttttatagg ttaatgtcat gataataatg gtttcttaga
      961 cgtcaggtgg cacttttcgg ggaaatgtgc gcggaacccc tatttgttta tttttctaaa
     1021 tacattcaaa tatgtatccg ctcatgagac aataaccctg ataaatgctt caataatatt
     1081 gaaaaaggaa gagtatgagt attcaacatt tccgtgtcgc ccttattccc ttttttgcgg
     1141 cattttgcct tcctgttttt gctcacccag aaacgctggt gaaagtaaaa gatgctgaag
     1201 atcagttggg tgcacgagtg ggttacatcg aactggatct caacagcggt aagatccttg
     1261 agagttttcg ccccgaagaa cgttttccaa tgatgagcac ttttaaagtt ctgctatgtg
     1321 gcgcggtatt atcccgtatt gacgccgggc aagagcaact cggtcgccgc atacactatt
     1381 ctcagaatga cttggttgag tactcaccag tcacagaaaa gcatcttacg gatggcatga
     1441 cagtaagaga attatgcagt gctgccataa ccatgagtga taacactgcg gccaacttac
     1501 ttctgacaac gatcggagga ccgaaggagc taaccgcttt tttgcacaac atgggggatc
     1561 atgtaactcg ccttgatcgt tgggaaccgg agctgaatga agccatacca aacgacgagc
     1621 gtgacaccac gatgcctgta gcaatggcaa caacgttgcg caaactatta actggcgaac
     1681 tacttactct agcttcccgg caacaattaa tagactggat ggaggcggat aaagttgcag
     1741 gaccacttct gcgctcggcc cttccggctg gctggtttat tgctgataaa tctggagccg
     1801 gtgagcgtgg gtctcgcggt atcattgcag cactggggcc agatggtaag ccctcccgta
     1861 tcgtagttat ctacacgacg gggagtcagg caactatgga tgaacgaaat agacagatcg
     1921 ctgagatagg tgcctcactg attaagcatt ggtaactgtc agaccaagtt tactcatata
     1981 tactttagat tgatttaaaa cttcattttt aatttaaaag gatctaggtg aagatccttt
     2041 ttgataatct catgaccaaa atcccttaac gtgagttttc gttccactga gcgtcagacc
     2101 ccgtagaaaa gatcaaagga tcttcttgag atcctttttt tctgcgcgta atctgctgct
     2161 tgcaaacaaa aaaaccaccg ctaccagcgg tggtttgttt gccggatcaa gagctaccaa
     2221 ctctttttcc gaaggtaact ggcttcagca gagcgcagat accaaatact gttcttctag
     2281 tgtagccgta gttaggccac cacttcaaga actctgtagc accgcctaca tacctcgctc
     2341 tgctaatcct gttaccagtg gctgctgcca gtggcgataa gtcgtgtctt accgggttgg
     2401 actcaagacg atagttaccg gataaggcgc agcggtcggg ctgaacgggg ggttcgtgca
     2461 cacagcccag cttggagcga acgacctaca ccgaactgag atacctacag cgtgagctat
     2521 gagaaagcgc cacgcttccc gaagggagaa aggcggacag gtatccggta agcggcaggg
     2581 tcggaacagg agagcgcacg agggagcttc cagggggaaa cgcctggtat ctttatagtc
     2641 ctgtcgggtt tcgccacctc tgacttgagc gtcgattttt gtgatgctcg tcaggggggc
     2701 ggagcctatg gaaaaacgcc agcaacgcgg cctttttacg gttcctggcc ttttgctggc
     2761 cttttgctca catgttcttt cctgcgttat cccctgattc tgtggataac cgtattaccg
     2821 cctttgagtg agctgatacc gctcgccgca gccgaacgac cgagcgcagc gagtcagtga
     2881 gcgaggaagc ggaagagcgc ccaatacgca aaccgcctct ccccgcgcgt tggccgattc
     2941 attaatgcag ctggcacgac aggtttcccg actggaaagc gggcagtgag cgcaacgcaa
     3001 ttaatgtgag ttagctcact cattaggcac cccaggcttt acactttatg cttccggctc
     3061 gtatgttgtg tggaattgtg agcggataac aatttcacac aggaaacagc tatgaccatg
     3121 attacgaatt cgagctcggt acccgg
//
