LOCUS       sequence-112927-        3167 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 3167)
  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 3167)
  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-AtU3b
FEATURES             Location/Qualifiers
     source          1..3167
                     /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(521..538)
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     complement(521..537)
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(530..552)
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     primer_bind     complement(746..765)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     865..887
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     primer_bind     complement(925..943)
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     promoter        1011..1115
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(1116..1184,1185..1976)
                     /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(1334..1353)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      2147..2735
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     2636..2655
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     2889..2906
                     /label=L4440
                     /note="L4440 vector, forward primer"
     protein_bind    3023..3044
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     promoter        join(3059..3064,3065..3082,3083..3089)
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    3097..3113
                     /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     3102..3124
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     primer_bind     3121..3137
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     3121..3137
                     /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 gatttacttt aaattttttc
      181 ttatgcagcc tgtgatggat aactgaatca aacaaatggc gtctgggttt aagaagatct
      241 gttttggcta tgttggacga aacaagtgaa cttttaggat caacttcagt ttatatatgg
      301 agcttatatc gagcaataag ataagtgggc tttttatgta atttaatggg ctatcgtcca
      361 tagattcact aatacccatg cccagtaccc atgtatgcgt ttcatataag ctcctaattt
      421 ctcccacatc gctcaaatct aaacaaatct tgttgtatat ataacactga gggagcaaca
      481 ttggtcaaga gacctctgaa gataacatac taagcttggc actggccgtc gttttacaac
      541 gtcgtgactg ggaaaaccct ggcgttaccc aacttaatcg ccttgcagca catccccctt
      601 tcgccagctg gcgtaatagc gaagaggccc gcaccgatcg cccttcccaa cagttgcgca
      661 gcctgaatgg cgaatggcgc ctgatgcggt attttctcct tacgcatctg tgcggtattt
      721 cacaccgcat atggtgcact ctcagtacaa tctgctctga tgccgcatag ttaagccagc
      781 cccgacaccc gccaacaccc gctgacgcgc cctgacgggc ttgtctgctc ccggcatccg
      841 cttacagaca agctgtgacc gtctccggga gctgcatgtg tcagaggttt tcaccgtcat
      901 caccgaaacg cgcgagacga aagggcctcg tgatacgcct atttttatag gttaatgtca
      961 tgataataat ggtttcttag acgtcaggtg gcacttttcg gggaaatgtg cgcggaaccc
     1021 ctatttgttt atttttctaa atacattcaa atatgtatcc gctcatgaga caataaccct
     1081 gataaatgct tcaataatat tgaaaaagga agagtatgag tattcaacat ttccgtgtcg
     1141 cccttattcc cttttttgcg gcattttgcc ttcctgtttt tgctcaccca gaaacgctgg
     1201 tgaaagtaaa agatgctgaa gatcagttgg gtgcacgagt gggttacatc gaactggatc
     1261 tcaacagcgg taagatcctt gagagttttc gccccgaaga acgttttcca atgatgagca
     1321 cttttaaagt tctgctatgt ggcgcggtat tatcccgtat tgacgccggg caagagcaac
     1381 tcggtcgccg catacactat tctcagaatg acttggttga gtactcacca gtcacagaaa
     1441 agcatcttac ggatggcatg acagtaagag aattatgcag tgctgccata accatgagtg
     1501 ataacactgc ggccaactta cttctgacaa cgatcggagg accgaaggag ctaaccgctt
     1561 ttttgcacaa catgggggat catgtaactc gccttgatcg ttgggaaccg gagctgaatg
     1621 aagccatacc aaacgacgag cgtgacacca cgatgcctgt agcaatggca acaacgttgc
     1681 gcaaactatt aactggcgaa ctacttactc tagcttcccg gcaacaatta atagactgga
     1741 tggaggcgga taaagttgca ggaccacttc tgcgctcggc ccttccggct ggctggttta
     1801 ttgctgataa atctggagcc ggtgagcgtg ggtctcgcgg tatcattgca gcactggggc
     1861 cagatggtaa gccctcccgt atcgtagtta tctacacgac ggggagtcag gcaactatgg
     1921 atgaacgaaa tagacagatc gctgagatag gtgcctcact gattaagcat tggtaactgt
     1981 cagaccaagt ttactcatat atactttaga ttgatttaaa acttcatttt taatttaaaa
     2041 ggatctaggt gaagatcctt tttgataatc tcatgaccaa aatcccttaa cgtgagtttt
     2101 cgttccactg agcgtcagac cccgtagaaa agatcaaagg atcttcttga gatccttttt
     2161 ttctgcgcgt aatctgctgc ttgcaaacaa aaaaaccacc gctaccagcg gtggtttgtt
     2221 tgccggatca agagctacca actctttttc cgaaggtaac tggcttcagc agagcgcaga
     2281 taccaaatac tgttcttcta gtgtagccgt agttaggcca ccacttcaag aactctgtag
     2341 caccgcctac atacctcgct ctgctaatcc tgttaccagt ggctgctgcc agtggcgata
     2401 agtcgtgtct taccgggttg gactcaagac gatagttacc ggataaggcg cagcggtcgg
     2461 gctgaacggg gggttcgtgc acacagccca gcttggagcg aacgacctac accgaactga
     2521 gatacctaca gcgtgagcta tgagaaagcg ccacgcttcc cgaagggaga aaggcggaca
     2581 ggtatccggt aagcggcagg gtcggaacag gagagcgcac gagggagctt ccagggggaa
     2641 acgcctggta tctttatagt cctgtcgggt ttcgccacct ctgacttgag cgtcgatttt
     2701 tgtgatgctc gtcagggggg cggagcctat ggaaaaacgc cagcaacgcg gcctttttac
     2761 ggttcctggc cttttgctgg ccttttgctc acatgttctt tcctgcgtta tcccctgatt
     2821 ctgtggataa ccgtattacc gcctttgagt gagctgatac cgctcgccgc agccgaacga
     2881 ccgagcgcag cgagtcagtg agcgaggaag cggaagagcg cccaatacgc aaaccgcctc
     2941 tccccgcgcg ttggccgatt cattaatgca gctggcacga caggtttccc gactggaaag
     3001 cgggcagtga gcgcaacgca attaatgtga gttagctcac tcattaggca ccccaggctt
     3061 tacactttat gcttccggct cgtatgttgt gtggaattgt gagcggataa caatttcaca
     3121 caggaaacag ctatgaccat gattacgaat tcgagctcgg tacccgg
//
