LOCUS       sequence-377110-        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: pICSL1P5_AtU626Ter26
FEATURES             Location/Qualifiers
     source          1..5812
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     protein_bind    364..385
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     promoter        join(400..405,406..423,424..430)
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    438..454
                     /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             472..483
                     /note="strong bacterial ribosome binding site (Elowitz and 
                     Leibler, 2000)"
     CDS             490..1167
                     /codon_start=1
                     /product="monomeric derivative of DsRed (Campbell et al., 
                     2002)"
                     /label=mRFP1
                     /translation="MASSEDVIKEFMRFKVRMEGSVNGHEFEIEGEGEGRPYEGTQTAK
                     LKVTKGGPLPFAWDILSPQFQYGSKAYVKHPADIPDYLKLSFPEGFKWERVMNFEDGGV
                     VTVTQDSSLQDGEFIYKVKLRGTNFPSDGPVMQKKTMGWEASTERMYPEDGALKGEIKM
                     RLKLKDGGHYDAEVKTTYMAKKPVQLPGAYKTDIKLDITSHNEDYTIVEQYERAEGRHS
                     TGA"
     terminator      1212..1283
                     /label=rrnB T1 terminator
                     /note="transcription terminator T1 from the E. coli rrnB 
                     gene"
     terminator      1299..1326
                     /label=T7Te terminator
                     /note="phage T7 early transcription terminator"
     misc_RNA        1340..1415
                     /label=gRNA scaffold
                     /note="guide RNA scaffold for the Streptococcus pyogenes 
                     CRISPR/Cas9 system"
     misc_feature    1527..1551
                     /label=RB T-DNA repeat
                     /note="right border repeat from nopaline C58 T-DNA"
     CDS             complement(1707..2855)
                     /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(3191..3779)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(3271..3290)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(3950..4741,4742..4810))
                     /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     4573..4592
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(4811..4915)
                     /gene="bla"
                     /label=AmpR promoter
     rep_origin      5002..5633
                     /label=oriV
                     /note="incP origin of replication"
     misc_feature    5725..5749
                     /label=LB T-DNA repeat
                     /note="left border repeat from nopaline C58 T-DNA"
ORIGIN
        1 agtgggcccc actctgtgaa gacaacagag aattcgcatg cggagcatct tcattcttaa
       61 gatatgaaga taatcttcaa aaggcccctg ggaatctgaa agaagagaag caggcccatt
      121 tatatgggaa agaacaatag tatttcttat ataggcccat ttaagttgaa aacaatcttc
      181 aaaagtccca catcgcttag ataagaaaac gaagctgagt ttatatacag ctagagtcga
      241 agtagtgatt gtgagaccga tatcaatacg caaaccgcct ctccccgcgc gttggccgat
      301 tcattaatgc agctggcacg acaggtttcc cgactggaaa gcgggcagtg agcgcaacgc
      361 aattaatgtg agttagctca ctcattaggc accccaggct ttacacttta tgcttccggc
      421 tcgtatgttg tgtggaattg tgagcggata acaatttcac acatactaga gaaagaggag
      481 aaatactaga tggcttcctc cgaggacgtt atcaaagagt tcatgcgttt caaagttcgt
      541 atggaaggtt ccgttaacgg tcacgagttc gaaatcgaag gtgaaggtga aggtcgtccg
      601 tacgaaggta cccagaccgc taaactgaaa gttaccaaag gtggtccgct gccgttcgct
      661 tgggacatcc tgtccccgca gttccagtac ggttccaaag cttacgttaa acacccggct
      721 gacatcccgg actacctgaa actgtccttc ccggaaggtt tcaaatggga acgtgttatg
      781 aacttcgagg acggtggtgt tgttaccgtt acccaggact cctccctgca agacggtgag
      841 ttcatctaca aagttaaact gcgtggtacc aacttcccgt ccgacggtcc ggttatgcag
      901 aaaaaaacca tgggttggga agcttccacc gaacgtatgt acccggagga cggtgctctg
      961 aaaggtgaaa tcaaaatgcg tctgaaactg aaagacggtg gtcactacga cgctgaagtt
     1021 aaaaccacct acatggctaa aaaaccggtt cagctgccgg gtgcttacaa aaccgacatc
     1081 aaactggaca tcacctccca caacgaggac tacaccatcg ttgaacagta cgaacgtgct
     1141 gaaggtcgtc actccaccgg tgcttaataa cgctgatagt gctagtgtag atcgctacta
     1201 gagccaggca tcaaataaaa cgaaaggctc agtcgaaaga ctgggccttt cgttttatct
     1261 gttgtttgtc ggtgaacgct ctctactaga gtcacactgg ctcaccttcg ggtgggcctt
     1321 tctgcgttta taggtctcag ttttagagct agaaatagca agttaaaata aggctagtcc
     1381 gttatcaact tgaaaaagtg gcaccgagtc ggtgcttttt tttgcaaaat tttccagatc
     1441 gatttcttct tcctctgttc ttcggcgttc aatttctggg gcgcttgtgt tgtcttctgc
     1501 acgaagtggt ttaaactatc agtgtttgac aggatatatt ggcgggtaaa cctaagagaa
     1561 aagagcgttt attagaataa tcggatattt aaaagggcgt gaaaaggttt atccgttcgt
     1621 ccatttgtat gtgcatgcca accacagggt tccccagatc aggcgctggc tgctgaaccc
     1681 ccagccggaa ctgaccccac aaggccctag cgtttgcaat gcaccaggtc atcattgacc
     1741 caggcgtgtt ccaccaggcc gctgcctcgc aactcttcgc aggcttcgcc gacctgctcg
     1801 cgccacttct tcacgcgggt ggaatccgat ccgcacatga ggcggaaggt ttccagcttg
     1861 agcgggtacg gctcccggtg cgagctgaaa tagtcgaaca tccgtcgggc cgtcggcgac
     1921 agcttgcggt acttctccca tatgaatttc gtgtagtggt cgccagcaaa cagcacgacg
     1981 atttcctcgt cgatcaggac ctggcaacgg gacgttttct tgccacggtc caggacgcgg
     2041 aagcggtgca gcagcgacac cgattccagg tgcccaacgc ggtcggacgt gaagcccatc
     2101 gccgtcgcct gtaggcgcga caggcattcc tcggccttcg tgtaataccg gccattgatc
     2161 gaccagccca ggtcctggca aagctcgtag aacgtgaagg tgatcggctc gccgataggg
     2221 gtgcgcttcg cgtactccaa cacctgctgc cacaccagtt cgtcatcgtc ggcccgcagc
     2281 tcgacgccgg tgtaggtgat cttcacgtcc ttgttgacgt ggaaaatgac cttgttttgc
     2341 agcgcctcgc gcgggatttt cttgttgcgc gtggtgaaca gggcagagcg ggccgtgtcg
     2401 tttggcatcg ctcgcatcgt gtccggccac ggcgcaatat cgaacaagga aagctgcatt
     2461 tccttgatct gctgcttcgt gtgtttcagc aacgcggcct gcttggcctc gctgacctgt
     2521 tttgccaggt cctcgccggc ggtttttcgc ttcttggtcg tcatagttcc tcgcgtgtcg
     2581 atggtcatcg acttcgccaa acctgccgcc tcctgttcaa gacgacgcga acgctccacg
     2641 gcggccgatg gcgcgggcag ggcaggggga gccagttgca cgctgtcgcg ctcgatcttg
     2701 gccgtagctt gctggaccat cgagccgacg gactggaagg tttcgcgggg cgcacgcatg
     2761 acggtgcggc ttgcgatggt ttcggcatcc tcggcggaaa accccgcgtc gatcagttct
     2821 tgcctgtatg ccttccggtc aaacgtccga ttcattcacc ctccttgcgg gattgccccg
     2881 actcacgccg gggcaatgtg cccttattcc tgatttgacc cgcctggtgc cttggtgtcc
     2941 agataatcca ccttatcggc aatgaagtcg gtcccgtaga ccgtctggcc gtccttctcg
     3001 tacttggtat tccgaatctt gccctgcacg aataccagcg accccttgcc caaatacttg
     3061 ccgtgggcct cggcctgaga gccaaaacac ttgatgcgga agaagtcggt gcgctcctgc
     3121 ttgtcgccgg catcgttgcg ccacatctag gatctgccag gaaccgtaaa aaggccgcgt
     3181 tgctggcgtt tttccatagg ctccgccccc ctgacgagca tcacaaaaat cgacgctcaa
     3241 gtcagaggtg gcgaaacccg acaggactat aaagatacca ggcgtttccc cctggaagct
     3301 ccctcgtgcg ctctcctgtt ccgaccctgc cgcttaccgg atacctgtcc gcctttctcc
     3361 cttcgggaag cgtggcgctt tctcatagct cacgctgtag gtatctcagt tcggtgtagg
     3421 tcgttcgctc caagctgggc tgtgtgcacg aaccccccgt tcagcccgac cgctgcgcct
     3481 tatccggtaa ctatcgtctt gagtccaacc cggtaagaca cgacttatcg ccactggcag
     3541 cagccactgg taacaggatt agcagagcga ggtatgtagg cggtgctaca gagttcttga
     3601 agtggtggcc taactacggc tacactagaa gaacagtatt tggtatctgc gctctgctga
     3661 agccagttac cttcggaaaa agagttggta gctcttgatc cggcaaacaa accaccgctg
     3721 gtagcggtgg tttttttgtt tgcaagcagc agattacgcg cagaaaaaaa ggatctcaag
     3781 aagatccttt gatcttttct acggggtctg acgctcagtg gaacgaaaac tcacgttaag
     3841 ggattttggt catgagatta tcaaaaagga tcttcaccta gatcctttta aattaaaaat
     3901 gaagttttaa atcaatctaa agtatatatg agtaaacttg gtctgacagt taccaatgct
     3961 taatcagtga ggcacctatc tcagcgatct gtctatttcg ttcatccata gttgcctgac
     4021 tccccgtcgt gtagataact acgatacggg agggcttacc atctggcccc agtgctgcaa
     4081 tgataccgcg agaaccacgc tcaccggctc cagatttatc agcaataaac cagccagccg
     4141 gaagggccga gcgcagaagt ggtcctgcaa ctttatccgc ctccatccag tctattaatt
     4201 gttgccggga agctagagta agtagttcgc cagttaatag tttgcgcaac gttgttgcca
     4261 ttgctacagg catcgtggtg tcacgctcgt cgtttggtat ggcttcattc agctccggtt
     4321 cccaacgatc aaggcgagtt acatgatccc ccatgttgtg caaaaaagcg gttagctcct
     4381 tcggtcctcc gatcgttgtc agaagtaagt tggccgcagt gttatcactc atggttatgg
     4441 cagcactgca taattctctt actgtcatgc catccgtaag atgcttttct gtgactggtg
     4501 agtactcaac caagtcattc tgagaatagt gtatgcggcg accgagttgc tcttgcccgg
     4561 cgtcaatacg ggataatacc gcgccacata gcagaacttt aaaagtgctc atcattggaa
     4621 aacgttcttc ggggcgaaaa ctctcaagga tcttaccgct gttgagatcc agttcgatgt
     4681 aacccactcg tgcacccaac tgatcttcag catcttttac tttcaccagc gtttctgggt
     4741 gagcaaaaac aggaaggcaa aatgccgcaa aaaagggaat aagggcgaca cggaaatgtt
     4801 gaatactcat actcttcctt tttcaatatt attgaagcat ttatcaaggt tattgtctca
     4861 tgagcggata catatttgaa tgtatttaga aaaataaaca aataggggtt ccgcgcacga
     4921 attggccagc gctgccattt ttggggtgag gccgttcgcg gccgaggggc gcagcccctg
     4981 gggggatggg aggcccgcgt tagcgggccg ggagggttcg agaagggggg gcacccccct
     5041 tcggcgtgcg cggtcacgcg cacagggcgc agccctggtt aaaaacaagg tttataaata
     5101 ttggtttaaa agcaggttaa aagacaggtt agcggtggcc gaaaaacggg cggaaaccct
     5161 tgcaaatgct ggattttctg cctgtggaca gcccctcaaa tgtcaatagg tgcgcccctc
     5221 atctgtcagc actctgcccc tcaagtgtca aggatcgcgc ccctcatctg tcagtagtcg
     5281 cgcccctcaa gtgtcaatac cgcagggcac ttatccccag gcttgtccac atcatctgtg
     5341 ggaaactcgc gtaaaatcag gcgttttcgc cgatttgcga ggctggccag ctccacgtcg
     5401 ccggccgaaa tcgagcctgc ccctcatctg tcaacgccgc gccgggtgag tcggcccctc
     5461 aagtgtcaac gtccgcccct catctgtcag tgagggccaa gttttccgcg aggtatccac
     5521 aacgccggcg gccgcggtgt ctcgcacacg gcttcgacgg cgtttctggc gcgtttgcag
     5581 ggccatagac ggccgccagc ccagcggcga gggcaaccag cccggtgagc gtcgcaaagg
     5641 agatcctgat ctgactgatg ggctgcctgt atcgagtggt gattttgtgc cgagctgccg
     5701 gtcggggagc tgttggctgg ctggtggcag gatatattgt ggtgtaaaca aattgacgct
     5761 tagacaactt aataacacat tgcggacgtt tttaatgtac tggggtggat gc
//
