LOCUS       sequence-377115-        5812 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Cas9 guide expression in dicots.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 5812)
  AUTHORS   Lawrenson T, Youles M, Chhetry M, Clarke M, Harwood W, Hundleby P
  TITLE     Efficient Targeted Mutagenesis in Brassica Crops Using CRISPR/Cas 
            Systems.
  JOURNAL   Methods Mol Biol. 2023;2653:253-271. doi: 
            10.1007/978-1-0716-3131-7_16.
  PUBMED    36995631
REFERENCE   2  (bases 1 to 5812)
  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: "Methods Mol
            Biol."; date: "2023"; volume: "2653:253-271. doi"; pages: " 
            10.1007/978-1-0716-3131-7_16"
COMMENT     Sequence Label: pICSL1P2_AtU626Ter26
FEATURES             Location/Qualifiers
     source          1..5812
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     protein_bind    351..372
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     promoter        join(387..392,393..410,411..417)
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    425..441
                     /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)."
     RBS             459..470
                     /note="strong bacterial ribosome binding site (Elowitz and 
                     Leibler, 2000)"
     CDS             477..1154
                     /codon_start=1
                     /product="monomeric derivative of DsRed (Campbell et al., 
                     2002)"
                     /label=mRFP1
                     /translation="MASSEDVIKEFMRFKVRMEGSVNGHEFEIEGEGEGRPYEGTQTAK
                     LKVTKGGPLPFAWDILSPQFQYGSKAYVKHPADIPDYLKLSFPEGFKWERVMNFEDGGV
                     VTVTQDSSLQDGEFIYKVKLRGTNFPSDGPVMQKKTMGWEASTERMYPEDGALKGEIKM
                     RLKLKDGGHYDAEVKTTYMAKKPVQLPGAYKTDIKLDITSHNEDYTIVEQYERAEGRHS
                     TGA"
     terminator      1199..1270
                     /label=rrnB T1 terminator
                     /note="transcription terminator T1 from the E. coli rrnB 
                     gene"
     terminator      1286..1313
                     /label=T7Te terminator
                     /note="phage T7 early transcription terminator"
     misc_RNA        1327..1402
                     /label=gRNA scaffold
                     /note="guide RNA scaffold for the Streptococcus pyogenes 
                     CRISPR/Cas9 system"
     misc_feature    1514..1538
                     /label=RB T-DNA repeat
                     /note="right border repeat from nopaline C58 T-DNA"
     CDS             complement(1694..2842)
                     /codon_start=1
                     /product="trans-acting replication protein that binds to 
                     and activates oriV"
                     /label=trfA
                     /translation="MNRTFDRKAYRQELIDAGFSAEDAETIASRTVMRAPRETFQSVGS
                     MVQQATAKIERDSVQLAPPALPAPSAAVERSRRLEQEAAGLAKSMTIDTRGTMTTKKRK
                     TAGEDLAKQVSEAKQAALLKHTKQQIKEMQLSLFDIAPWPDTMRAMPNDTARSALFTTR
                     NKKIPREALQNKVIFHVNKDVKITYTGVELRADDDELVWQQVLEYAKRTPIGEPITFTF
                     YELCQDLGWSINGRYYTKAEECLSRLQATAMGFTSDRVGHLESVSLLHRFRVLDRGKKT
                     SRCQVLIDEEIVVLFAGDHYTKFIWEKYRKLSPTARRMFDYFSSHREPYPLKLETFRLM
                     CGSDSTRVKKWREQVGEACEELRGSGLVEHAWVNDDLVHCKR"
     rep_origin      complement(3178..3766)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(3258..3277)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(3937..4728,4729..4797))
                     /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     4560..4579
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(4798..4902)
                     /gene="bla"
                     /label=AmpR promoter
     rep_origin      4989..5620
                     /label=oriV
                     /note="incP origin of replication"
     misc_feature    5712..5736
                     /label=LB T-DNA repeat
                     /note="left border repeat from nopaline C58 T-DNA"
ORIGIN
        1 ctgtgaagac aagcaagaat tcaagcttgg agcatcttca ttcttaagat atgaagataa
       61 tcttcaaaag gcccctggga atctgaaaga agagaagcag gcccatttat atgggaaaga
      121 acaatagtat ttcttatata ggcccattta agttgaaaac aatcttcaaa agtcccacat
      181 cgcttagata agaaaacgaa gctgagttta tatacagcta gagtcgaagt agtgattgtg
      241 agaccgatat caatacgcaa accgcctctc cccgcgcgtt ggccgattca ttaatgcagc
      301 tggcacgaca ggtttcccga ctggaaagcg ggcagtgagc gcaacgcaat taatgtgagt
      361 tagctcactc attaggcacc ccaggcttta cactttatgc ttccggctcg tatgttgtgt
      421 ggaattgtga gcggataaca atttcacaca tactagagaa agaggagaaa tactagatgg
      481 cttcctccga ggacgttatc aaagagttca tgcgtttcaa agttcgtatg gaaggttccg
      541 ttaacggtca cgagttcgaa atcgaaggtg aaggtgaagg tcgtccgtac gaaggtaccc
      601 agaccgctaa actgaaagtt accaaaggtg gtccgctgcc gttcgcttgg gacatcctgt
      661 ccccgcagtt ccagtacggt tccaaagctt acgttaaaca cccggctgac atcccggact
      721 acctgaaact gtccttcccg gaaggtttca aatgggaacg tgttatgaac ttcgaggacg
      781 gtggtgttgt taccgttacc caggactcct ccctgcaaga cggtgagttc atctacaaag
      841 ttaaactgcg tggtaccaac ttcccgtccg acggtccggt tatgcagaaa aaaaccatgg
      901 gttgggaagc ttccaccgaa cgtatgtacc cggaggacgg tgctctgaaa ggtgaaatca
      961 aaatgcgtct gaaactgaaa gacggtggtc actacgacgc tgaagttaaa accacctaca
     1021 tggctaaaaa accggttcag ctgccgggtg cttacaaaac cgacatcaaa ctggacatca
     1081 cctcccacaa cgaggactac accatcgttg aacagtacga acgtgctgaa ggtcgtcact
     1141 ccaccggtgc ttaataacgc tgatagtgct agtgtagatc gctactagag ccaggcatca
     1201 aataaaacga aaggctcagt cgaaagactg ggcctttcgt tttatctgtt gtttgtcggt
     1261 gaacgctctc tactagagtc acactggctc accttcgggt gggcctttct gcgtttatag
     1321 gtctcagttt tagagctaga aatagcaagt taaaataagg ctagtccgtt atcaacttga
     1381 aaaagtggca ccgagtcggt gctttttttt gcaaaatttt ccagatcgat ttcttcttcc
     1441 tctgttcttc ggcgttcaat ttctggggcg ctactattgt cttctgcacg aagtggttta
     1501 aactatcagt gtttgacagg atatattggc gggtaaacct aagagaaaag agcgtttatt
     1561 agaataatcg gatatttaaa agggcgtgaa aaggtttatc cgttcgtcca tttgtatgtg
     1621 catgccaacc acagggttcc ccagatcagg cgctggctgc tgaaccccca gccggaactg
     1681 accccacaag gccctagcgt ttgcaatgca ccaggtcatc attgacccag gcgtgttcca
     1741 ccaggccgct gcctcgcaac tcttcgcagg cttcgccgac ctgctcgcgc cacttcttca
     1801 cgcgggtgga atccgatccg cacatgaggc ggaaggtttc cagcttgagc gggtacggct
     1861 cccggtgcga gctgaaatag tcgaacatcc gtcgggccgt cggcgacagc ttgcggtact
     1921 tctcccatat gaatttcgtg tagtggtcgc cagcaaacag cacgacgatt tcctcgtcga
     1981 tcaggacctg gcaacgggac gttttcttgc cacggtccag gacgcggaag cggtgcagca
     2041 gcgacaccga ttccaggtgc ccaacgcggt cggacgtgaa gcccatcgcc gtcgcctgta
     2101 ggcgcgacag gcattcctcg gccttcgtgt aataccggcc attgatcgac cagcccaggt
     2161 cctggcaaag ctcgtagaac gtgaaggtga tcggctcgcc gataggggtg cgcttcgcgt
     2221 actccaacac ctgctgccac accagttcgt catcgtcggc ccgcagctcg acgccggtgt
     2281 aggtgatctt cacgtccttg ttgacgtgga aaatgacctt gttttgcagc gcctcgcgcg
     2341 ggattttctt gttgcgcgtg gtgaacaggg cagagcgggc cgtgtcgttt ggcatcgctc
     2401 gcatcgtgtc cggccacggc gcaatatcga acaaggaaag ctgcatttcc ttgatctgct
     2461 gcttcgtgtg tttcagcaac gcggcctgct tggcctcgct gacctgtttt gccaggtcct
     2521 cgccggcggt ttttcgcttc ttggtcgtca tagttcctcg cgtgtcgatg gtcatcgact
     2581 tcgccaaacc tgccgcctcc tgttcaagac gacgcgaacg ctccacggcg gccgatggcg
     2641 cgggcagggc agggggagcc agttgcacgc tgtcgcgctc gatcttggcc gtagcttgct
     2701 ggaccatcga gccgacggac tggaaggttt cgcggggcgc acgcatgacg gtgcggcttg
     2761 cgatggtttc ggcatcctcg gcggaaaacc ccgcgtcgat cagttcttgc ctgtatgcct
     2821 tccggtcaaa cgtccgattc attcaccctc cttgcgggat tgccccgact cacgccgggg
     2881 caatgtgccc ttattcctga tttgacccgc ctggtgcctt ggtgtccaga taatccacct
     2941 tatcggcaat gaagtcggtc ccgtagaccg tctggccgtc cttctcgtac ttggtattcc
     3001 gaatcttgcc ctgcacgaat accagcgacc ccttgcccaa atacttgccg tgggcctcgg
     3061 cctgagagcc aaaacacttg atgcggaaga agtcggtgcg ctcctgcttg tcgccggcat
     3121 cgttgcgcca catctaggat ctgccaggaa ccgtaaaaag gccgcgttgc tggcgttttt
     3181 ccataggctc cgcccccctg acgagcatca caaaaatcga cgctcaagtc agaggtggcg
     3241 aaacccgaca ggactataaa gataccaggc gtttccccct ggaagctccc tcgtgcgctc
     3301 tcctgttccg accctgccgc ttaccggata cctgtccgcc tttctccctt cgggaagcgt
     3361 ggcgctttct catagctcac gctgtaggta tctcagttcg gtgtaggtcg ttcgctccaa
     3421 gctgggctgt gtgcacgaac cccccgttca gcccgaccgc tgcgccttat ccggtaacta
     3481 tcgtcttgag tccaacccgg taagacacga cttatcgcca ctggcagcag ccactggtaa
     3541 caggattagc agagcgaggt atgtaggcgg tgctacagag ttcttgaagt ggtggcctaa
     3601 ctacggctac actagaagaa cagtatttgg tatctgcgct ctgctgaagc cagttacctt
     3661 cggaaaaaga gttggtagct cttgatccgg caaacaaacc accgctggta gcggtggttt
     3721 ttttgtttgc aagcagcaga ttacgcgcag aaaaaaagga tctcaagaag atcctttgat
     3781 cttttctacg gggtctgacg ctcagtggaa cgaaaactca cgttaaggga ttttggtcat
     3841 gagattatca aaaaggatct tcacctagat ccttttaaat taaaaatgaa gttttaaatc
     3901 aatctaaagt atatatgagt aaacttggtc tgacagttac caatgcttaa tcagtgaggc
     3961 acctatctca gcgatctgtc tatttcgttc atccatagtt gcctgactcc ccgtcgtgta
     4021 gataactacg atacgggagg gcttaccatc tggccccagt gctgcaatga taccgcgaga
     4081 accacgctca ccggctccag atttatcagc aataaaccag ccagccggaa gggccgagcg
     4141 cagaagtggt cctgcaactt tatccgcctc catccagtct attaattgtt gccgggaagc
     4201 tagagtaagt agttcgccag ttaatagttt gcgcaacgtt gttgccattg ctacaggcat
     4261 cgtggtgtca cgctcgtcgt ttggtatggc ttcattcagc tccggttccc aacgatcaag
     4321 gcgagttaca tgatccccca tgttgtgcaa aaaagcggtt agctccttcg gtcctccgat
     4381 cgttgtcaga agtaagttgg ccgcagtgtt atcactcatg gttatggcag cactgcataa
     4441 ttctcttact gtcatgccat ccgtaagatg cttttctgtg actggtgagt actcaaccaa
     4501 gtcattctga gaatagtgta tgcggcgacc gagttgctct tgcccggcgt caatacggga
     4561 taataccgcg ccacatagca gaactttaaa agtgctcatc attggaaaac gttcttcggg
     4621 gcgaaaactc tcaaggatct taccgctgtt gagatccagt tcgatgtaac ccactcgtgc
     4681 acccaactga tcttcagcat cttttacttt caccagcgtt tctgggtgag caaaaacagg
     4741 aaggcaaaat gccgcaaaaa agggaataag ggcgacacgg aaatgttgaa tactcatact
     4801 cttccttttt caatattatt gaagcattta tcaaggttat tgtctcatga gcggatacat
     4861 atttgaatgt atttagaaaa ataaacaaat aggggttccg cgcacgaatt ggccagcgct
     4921 gccatttttg gggtgaggcc gttcgcggcc gaggggcgca gcccctgggg ggatgggagg
     4981 cccgcgttag cgggccggga gggttcgaga agggggggca ccccccttcg gcgtgcgcgg
     5041 tcacgcgcac agggcgcagc cctggttaaa aacaaggttt ataaatattg gtttaaaagc
     5101 aggttaaaag acaggttagc ggtggccgaa aaacgggcgg aaacccttgc aaatgctgga
     5161 ttttctgcct gtggacagcc cctcaaatgt caataggtgc gcccctcatc tgtcagcact
     5221 ctgcccctca agtgtcaagg atcgcgcccc tcatctgtca gtagtcgcgc ccctcaagtg
     5281 tcaataccgc agggcactta tccccaggct tgtccacatc atctgtggga aactcgcgta
     5341 aaatcaggcg ttttcgccga tttgcgaggc tggccagctc cacgtcgccg gccgaaatcg
     5401 agcctgcccc tcatctgtca acgccgcgcc gggtgagtcg gcccctcaag tgtcaacgtc
     5461 cgcccctcat ctgtcagtga gggccaagtt ttccgcgagg tatccacaac gccggcggcc
     5521 gcggtgtctc gcacacggct tcgacggcgt ttctggcgcg tttgcagggc catagacggc
     5581 cgccagccca gcggcgaggg caaccagccc ggtgagcgtc gcaaaggaga tcctgatctg
     5641 actgatgggc tgcctgtatc gagtggtgat tttgtgccga gctgccggtc ggggagctgt
     5701 tggctggctg gtggcaggat atattgtggt gtaaacaaat tgacgcttag acaacttaat
     5761 aacacattgc ggacgttttt aatgtactgg ggtggatgca gtgggcccca ct
//
