LOCUS       sequence-112928-        3369 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 3369)
  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 3369)
  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/LacZ
FEATURES             Location/Qualifiers
     source          1..3369
                     /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(723..739)
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(948..967)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     1067..1089
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     primer_bind     complement(1127..1145)
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     promoter        1213..1317
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(1318..1386,1387..2178)
                     /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(1536..1555)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      2349..2937
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     2838..2857
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     3091..3108
                     /label=L4440
                     /note="L4440 vector, forward primer"
     protein_bind    3225..3246
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     promoter        join(3261..3266,3267..3284,3285..3291)
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    3299..3315
                     /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     3304..3326
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     primer_bind     3323..3339
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     3323..3339
                     /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 ctaatttact ttaaattttt tcttatgcag cctgtgatgg ataactgaat caaacaaatg
      421 gcgtctgggt ttaagaagat ctgttttggc tatgttggac gaaacaagtg aacttttagg
      481 atcaacttca gtttatatat ggagcttata tcgagcaata agataagtgg gctttttatg
      541 taatttaatg ggctatcgtc catagattca ctaataccca tgcccagtac ccatgtatgc
      601 gtttcatata agctcctaat ttctcccaca tcgctcaaat ctaaacaaat cttgttgtat
      661 atataacact gagggagcaa cattggtcaa gagacctctg aagataacat actaagcttg
      721 gcactggccg tcgttttaca acgtcgtgac tgggaaaacc ctggcgttac ccaacttaat
      781 cgccttgcag cacatccccc tttcgccagc tggcgtaata gcgaagaggc ccgcaccgat
      841 cgcccttccc aacagttgcg cagcctgaat ggcgaatggc gcctgatgcg gtattttctc
      901 cttacgcatc tgtgcggtat ttcacaccgc atatggtgca ctctcagtac aatctgctct
      961 gatgccgcat agttaagcca gccccgacac ccgccaacac ccgctgacgc gccctgacgg
     1021 gcttgtctgc tcccggcatc cgcttacaga caagctgtga ccgtctccgg gagctgcatg
     1081 tgtcagaggt tttcaccgtc atcaccgaaa cgcgcgagac gaaagggcct cgtgatacgc
     1141 ctatttttat aggttaatgt catgataata atggtttctt agacgtcagg tggcactttt
     1201 cggggaaatg tgcgcggaac ccctatttgt ttatttttct aaatacattc aaatatgtat
     1261 ccgctcatga gacaataacc ctgataaatg cttcaataat attgaaaaag gaagagtatg
     1321 agtattcaac atttccgtgt cgcccttatt cccttttttg cggcattttg ccttcctgtt
     1381 tttgctcacc cagaaacgct ggtgaaagta aaagatgctg aagatcagtt gggtgcacga
     1441 gtgggttaca tcgaactgga tctcaacagc ggtaagatcc ttgagagttt tcgccccgaa
     1501 gaacgttttc caatgatgag cacttttaaa gttctgctat gtggcgcggt attatcccgt
     1561 attgacgccg ggcaagagca actcggtcgc cgcatacact attctcagaa tgacttggtt
     1621 gagtactcac cagtcacaga aaagcatctt acggatggca tgacagtaag agaattatgc
     1681 agtgctgcca taaccatgag tgataacact gcggccaact tacttctgac aacgatcgga
     1741 ggaccgaagg agctaaccgc ttttttgcac aacatggggg atcatgtaac tcgccttgat
     1801 cgttgggaac cggagctgaa tgaagccata ccaaacgacg agcgtgacac cacgatgcct
     1861 gtagcaatgg caacaacgtt gcgcaaacta ttaactggcg aactacttac tctagcttcc
     1921 cggcaacaat taatagactg gatggaggcg gataaagttg caggaccact tctgcgctcg
     1981 gcccttccgg ctggctggtt tattgctgat aaatctggag ccggtgagcg tgggtctcgc
     2041 ggtatcattg cagcactggg gccagatggt aagccctccc gtatcgtagt tatctacacg
     2101 acggggagtc aggcaactat ggatgaacga aatagacaga tcgctgagat aggtgcctca
     2161 ctgattaagc attggtaact gtcagaccaa gtttactcat atatacttta gattgattta
     2221 aaacttcatt tttaatttaa aaggatctag gtgaagatcc tttttgataa tctcatgacc
     2281 aaaatccctt aacgtgagtt ttcgttccac tgagcgtcag accccgtaga aaagatcaaa
     2341 ggatcttctt gagatccttt ttttctgcgc gtaatctgct gcttgcaaac aaaaaaacca
     2401 ccgctaccag cggtggtttg tttgccggat caagagctac caactctttt tccgaaggta
     2461 actggcttca gcagagcgca gataccaaat actgttcttc tagtgtagcc gtagttaggc
     2521 caccacttca agaactctgt agcaccgcct acatacctcg ctctgctaat cctgttacca
     2581 gtggctgctg ccagtggcga taagtcgtgt cttaccgggt tggactcaag acgatagtta
     2641 ccggataagg cgcagcggtc gggctgaacg gggggttcgt gcacacagcc cagcttggag
     2701 cgaacgacct acaccgaact gagataccta cagcgtgagc tatgagaaag cgccacgctt
     2761 cccgaaggga gaaaggcgga caggtatccg gtaagcggca gggtcggaac aggagagcgc
     2821 acgagggagc ttccaggggg aaacgcctgg tatctttata gtcctgtcgg gtttcgccac
     2881 ctctgacttg agcgtcgatt tttgtgatgc tcgtcagggg ggcggagcct atggaaaaac
     2941 gccagcaacg cggccttttt acggttcctg gccttttgct ggccttttgc tcacatgttc
     3001 tttcctgcgt tatcccctga ttctgtggat aaccgtatta ccgcctttga gtgagctgat
     3061 accgctcgcc gcagccgaac gaccgagcgc agcgagtcag tgagcgagga agcggaagag
     3121 cgcccaatac gcaaaccgcc tctccccgcg cgttggccga ttcattaatg cagctggcac
     3181 gacaggtttc ccgactggaa agcgggcagt gagcgcaacg caattaatgt gagttagctc
     3241 actcattagg caccccaggc tttacacttt atgcttccgg ctcgtatgtt gtgtggaatt
     3301 gtgagcggat aacaatttca cacaggaaac agctatgacc atgattacga attcgagctc
     3361 ggtacccgg
//
