LOCUS       sequence-377118-        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: pICSL1P1_AtU626Ter26
FEATURES             Location/Qualifiers
     source          1..5812
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     protein_bind    349..370
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     promoter        join(385..390,391..408,409..415)
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    423..439
                     /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             457..468
                     /note="strong bacterial ribosome binding site (Elowitz and 
                     Leibler, 2000)"
     CDS             475..1152
                     /codon_start=1
                     /product="monomeric derivative of DsRed (Campbell et al., 
                     2002)"
                     /label=mRFP1
                     /translation="MASSEDVIKEFMRFKVRMEGSVNGHEFEIEGEGEGRPYEGTQTAK
                     LKVTKGGPLPFAWDILSPQFQYGSKAYVKHPADIPDYLKLSFPEGFKWERVMNFEDGGV
                     VTVTQDSSLQDGEFIYKVKLRGTNFPSDGPVMQKKTMGWEASTERMYPEDGALKGEIKM
                     RLKLKDGGHYDAEVKTTYMAKKPVQLPGAYKTDIKLDITSHNEDYTIVEQYERAEGRHS
                     TGA"
     terminator      1197..1268
                     /label=rrnB T1 terminator
                     /note="transcription terminator T1 from the E. coli rrnB 
                     gene"
     terminator      1284..1311
                     /label=T7Te terminator
                     /note="phage T7 early transcription terminator"
     misc_RNA        1325..1400
                     /label=gRNA scaffold
                     /note="guide RNA scaffold for the Streptococcus pyogenes 
                     CRISPR/Cas9 system"
     misc_feature    1512..1536
                     /label=RB T-DNA repeat
                     /note="right border repeat from nopaline C58 T-DNA"
     CDS             complement(1692..2840)
                     /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(3176..3764)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(3256..3275)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(3935..4726,4727..4795))
                     /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     4558..4577
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(4796..4900)
                     /gene="bla"
                     /label=AmpR promoter
     rep_origin      4987..5618
                     /label=oriV
                     /note="incP origin of replication"
     misc_feature    5710..5734
                     /label=LB T-DNA repeat
                     /note="left border repeat from nopaline C58 T-DNA"
ORIGIN
        1 gtgaagacaa tgccgaattc ggatccggag catcttcatt cttaagatat gaagataatc
       61 ttcaaaaggc ccctgggaat ctgaaagaag agaagcaggc ccatttatat gggaaagaac
      121 aatagtattt cttatatagg cccatttaag ttgaaaacaa tcttcaaaag tcccacatcg
      181 cttagataag aaaacgaagc tgagtttata tacagctaga gtcgaagtag tgattgtgag
      241 accgatatca atacgcaaac cgcctctccc cgcgcgttgg ccgattcatt aatgcagctg
      301 gcacgacagg tttcccgact ggaaagcggg cagtgagcgc aacgcaatta atgtgagtta
      361 gctcactcat taggcacccc aggctttaca ctttatgctt ccggctcgta tgttgtgtgg
      421 aattgtgagc ggataacaat ttcacacata ctagagaaag aggagaaata ctagatggct
      481 tcctccgagg acgttatcaa agagttcatg cgtttcaaag ttcgtatgga aggttccgtt
      541 aacggtcacg agttcgaaat cgaaggtgaa ggtgaaggtc gtccgtacga aggtacccag
      601 accgctaaac tgaaagttac caaaggtggt ccgctgccgt tcgcttggga catcctgtcc
      661 ccgcagttcc agtacggttc caaagcttac gttaaacacc cggctgacat cccggactac
      721 ctgaaactgt ccttcccgga aggtttcaaa tgggaacgtg ttatgaactt cgaggacggt
      781 ggtgttgtta ccgttaccca ggactcctcc ctgcaagacg gtgagttcat ctacaaagtt
      841 aaactgcgtg gtaccaactt cccgtccgac ggtccggtta tgcagaaaaa aaccatgggt
      901 tgggaagctt ccaccgaacg tatgtacccg gaggacggtg ctctgaaagg tgaaatcaaa
      961 atgcgtctga aactgaaaga cggtggtcac tacgacgctg aagttaaaac cacctacatg
     1021 gctaaaaaac cggttcagct gccgggtgct tacaaaaccg acatcaaact ggacatcacc
     1081 tcccacaacg aggactacac catcgttgaa cagtacgaac gtgctgaagg tcgtcactcc
     1141 accggtgctt aataacgctg atagtgctag tgtagatcgc tactagagcc aggcatcaaa
     1201 taaaacgaaa ggctcagtcg aaagactggg cctttcgttt tatctgttgt ttgtcggtga
     1261 acgctctcta ctagagtcac actggctcac cttcgggtgg gcctttctgc gtttataggt
     1321 ctcagtttta gagctagaaa tagcaagtta aaataaggct agtccgttat caacttgaaa
     1381 aagtggcacc gagtcggtgc ttttttttgc aaaattttcc agatcgattt cttcttcctc
     1441 tgttcttcgg cgttcaattt ctggggcgct gcaattgtct tctgcacgaa gtggtttaaa
     1501 ctatcagtgt ttgacaggat atattggcgg gtaaacctaa gagaaaagag cgtttattag
     1561 aataatcgga tatttaaaag ggcgtgaaaa ggtttatccg ttcgtccatt tgtatgtgca
     1621 tgccaaccac agggttcccc agatcaggcg ctggctgctg aacccccagc cggaactgac
     1681 cccacaaggc cctagcgttt gcaatgcacc aggtcatcat tgacccaggc gtgttccacc
     1741 aggccgctgc ctcgcaactc ttcgcaggct tcgccgacct gctcgcgcca cttcttcacg
     1801 cgggtggaat ccgatccgca catgaggcgg aaggtttcca gcttgagcgg gtacggctcc
     1861 cggtgcgagc tgaaatagtc gaacatccgt cgggccgtcg gcgacagctt gcggtacttc
     1921 tcccatatga atttcgtgta gtggtcgcca gcaaacagca cgacgatttc ctcgtcgatc
     1981 aggacctggc aacgggacgt tttcttgcca cggtccagga cgcggaagcg gtgcagcagc
     2041 gacaccgatt ccaggtgccc aacgcggtcg gacgtgaagc ccatcgccgt cgcctgtagg
     2101 cgcgacaggc attcctcggc cttcgtgtaa taccggccat tgatcgacca gcccaggtcc
     2161 tggcaaagct cgtagaacgt gaaggtgatc ggctcgccga taggggtgcg cttcgcgtac
     2221 tccaacacct gctgccacac cagttcgtca tcgtcggccc gcagctcgac gccggtgtag
     2281 gtgatcttca cgtccttgtt gacgtggaaa atgaccttgt tttgcagcgc ctcgcgcggg
     2341 attttcttgt tgcgcgtggt gaacagggca gagcgggccg tgtcgtttgg catcgctcgc
     2401 atcgtgtccg gccacggcgc aatatcgaac aaggaaagct gcatttcctt gatctgctgc
     2461 ttcgtgtgtt tcagcaacgc ggcctgcttg gcctcgctga cctgttttgc caggtcctcg
     2521 ccggcggttt ttcgcttctt ggtcgtcata gttcctcgcg tgtcgatggt catcgacttc
     2581 gccaaacctg ccgcctcctg ttcaagacga cgcgaacgct ccacggcggc cgatggcgcg
     2641 ggcagggcag ggggagccag ttgcacgctg tcgcgctcga tcttggccgt agcttgctgg
     2701 accatcgagc cgacggactg gaaggtttcg cggggcgcac gcatgacggt gcggcttgcg
     2761 atggtttcgg catcctcggc ggaaaacccc gcgtcgatca gttcttgcct gtatgccttc
     2821 cggtcaaacg tccgattcat tcaccctcct tgcgggattg ccccgactca cgccggggca
     2881 atgtgccctt attcctgatt tgacccgcct ggtgccttgg tgtccagata atccacctta
     2941 tcggcaatga agtcggtccc gtagaccgtc tggccgtcct tctcgtactt ggtattccga
     3001 atcttgccct gcacgaatac cagcgacccc ttgcccaaat acttgccgtg ggcctcggcc
     3061 tgagagccaa aacacttgat gcggaagaag tcggtgcgct cctgcttgtc gccggcatcg
     3121 ttgcgccaca tctaggatct gccaggaacc gtaaaaaggc cgcgttgctg gcgtttttcc
     3181 ataggctccg cccccctgac gagcatcaca aaaatcgacg ctcaagtcag aggtggcgaa
     3241 acccgacagg actataaaga taccaggcgt ttccccctgg aagctccctc gtgcgctctc
     3301 ctgttccgac cctgccgctt accggatacc tgtccgcctt tctcccttcg ggaagcgtgg
     3361 cgctttctca tagctcacgc tgtaggtatc tcagttcggt gtaggtcgtt cgctccaagc
     3421 tgggctgtgt gcacgaaccc cccgttcagc ccgaccgctg cgccttatcc ggtaactatc
     3481 gtcttgagtc caacccggta agacacgact tatcgccact ggcagcagcc actggtaaca
     3541 ggattagcag agcgaggtat gtaggcggtg ctacagagtt cttgaagtgg tggcctaact
     3601 acggctacac tagaagaaca gtatttggta tctgcgctct gctgaagcca gttaccttcg
     3661 gaaaaagagt tggtagctct tgatccggca aacaaaccac cgctggtagc ggtggttttt
     3721 ttgtttgcaa gcagcagatt acgcgcagaa aaaaaggatc tcaagaagat cctttgatct
     3781 tttctacggg gtctgacgct cagtggaacg aaaactcacg ttaagggatt ttggtcatga
     3841 gattatcaaa aaggatcttc acctagatcc ttttaaatta aaaatgaagt tttaaatcaa
     3901 tctaaagtat atatgagtaa acttggtctg acagttacca atgcttaatc agtgaggcac
     3961 ctatctcagc gatctgtcta tttcgttcat ccatagttgc ctgactcccc gtcgtgtaga
     4021 taactacgat acgggagggc ttaccatctg gccccagtgc tgcaatgata ccgcgagaac
     4081 cacgctcacc ggctccagat ttatcagcaa taaaccagcc agccggaagg gccgagcgca
     4141 gaagtggtcc tgcaacttta tccgcctcca tccagtctat taattgttgc cgggaagcta
     4201 gagtaagtag ttcgccagtt aatagtttgc gcaacgttgt tgccattgct acaggcatcg
     4261 tggtgtcacg ctcgtcgttt ggtatggctt cattcagctc cggttcccaa cgatcaaggc
     4321 gagttacatg atcccccatg ttgtgcaaaa aagcggttag ctccttcggt cctccgatcg
     4381 ttgtcagaag taagttggcc gcagtgttat cactcatggt tatggcagca ctgcataatt
     4441 ctcttactgt catgccatcc gtaagatgct tttctgtgac tggtgagtac tcaaccaagt
     4501 cattctgaga atagtgtatg cggcgaccga gttgctcttg cccggcgtca atacgggata
     4561 ataccgcgcc acatagcaga actttaaaag tgctcatcat tggaaaacgt tcttcggggc
     4621 gaaaactctc aaggatctta ccgctgttga gatccagttc gatgtaaccc actcgtgcac
     4681 ccaactgatc ttcagcatct tttactttca ccagcgtttc tgggtgagca aaaacaggaa
     4741 ggcaaaatgc cgcaaaaaag ggaataaggg cgacacggaa atgttgaata ctcatactct
     4801 tcctttttca atattattga agcatttatc aaggttattg tctcatgagc ggatacatat
     4861 ttgaatgtat ttagaaaaat aaacaaatag gggttccgcg cacgaattgg ccagcgctgc
     4921 catttttggg gtgaggccgt tcgcggccga ggggcgcagc ccctgggggg atgggaggcc
     4981 cgcgttagcg ggccgggagg gttcgagaag ggggggcacc ccccttcggc gtgcgcggtc
     5041 acgcgcacag ggcgcagccc tggttaaaaa caaggtttat aaatattggt ttaaaagcag
     5101 gttaaaagac aggttagcgg tggccgaaaa acgggcggaa acccttgcaa atgctggatt
     5161 ttctgcctgt ggacagcccc tcaaatgtca ataggtgcgc ccctcatctg tcagcactct
     5221 gcccctcaag tgtcaaggat cgcgcccctc atctgtcagt agtcgcgccc ctcaagtgtc
     5281 aataccgcag ggcacttatc cccaggcttg tccacatcat ctgtgggaaa ctcgcgtaaa
     5341 atcaggcgtt ttcgccgatt tgcgaggctg gccagctcca cgtcgccggc cgaaatcgag
     5401 cctgcccctc atctgtcaac gccgcgccgg gtgagtcggc ccctcaagtg tcaacgtccg
     5461 cccctcatct gtcagtgagg gccaagtttt ccgcgaggta tccacaacgc cggcggccgc
     5521 ggtgtctcgc acacggcttc gacggcgttt ctggcgcgtt tgcagggcca tagacggccg
     5581 ccagcccagc ggcgagggca accagcccgg tgagcgtcgc aaaggagatc ctgatctgac
     5641 tgatgggctg cctgtatcga gtggtgattt tgtgccgagc tgccggtcgg ggagctgttg
     5701 gctggctggt ggcaggatat attgtggtgt aaacaaattg acgcttagac aacttaataa
     5761 cacattgcgg acgtttttaa tgtactgggg tggatgcagt gggccccact ct
//
