LOCUS       sequence-377102-        5977 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Cas12a guide expression in dicots.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 5977)
  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 5977)
  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: pICSL1P6_crU626
FEATURES             Location/Qualifiers
     source          1..5977
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     ncRNA           312..348
                     /product="self-cleaving hammerhead ribozyme (Ferre-D'Amare 
                     and Scott, 2010)"
                     /label=hammerhead ribozyme
                     /ncRNA_class="hammerhead_ribozyme"
     protein_bind    477..498
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     promoter        join(513..518,519..536,537..543)
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    551..567
                     /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             585..596
                     /note="strong bacterial ribosome binding site (Elowitz and 
                     Leibler, 2000)"
     CDS             603..1280
                     /codon_start=1
                     /product="monomeric derivative of DsRed (Campbell et al., 
                     2002)"
                     /label=mRFP1
                     /translation="MASSEDVIKEFMRFKVRMEGSVNGHEFEIEGEGEGRPYEGTQTAK
                     LKVTKGGPLPFAWDILSPQFQYGSKAYVKHPADIPDYLKLSFPEGFKWERVMNFEDGGV
                     VTVTQDSSLQDGEFIYKVKLRGTNFPSDGPVMQKKTMGWEASTERMYPEDGALKGEIKM
                     RLKLKDGGHYDAEVKTTYMAKKPVQLPGAYKTDIKLDITSHNEDYTIVEQYERAEGRHS
                     TGA"
     terminator      1325..1396
                     /label=rrnB T1 terminator
                     /note="transcription terminator T1 from the E. coli rrnB 
                     gene"
     terminator      1412..1439
                     /label=T7Te terminator
                     /note="phage T7 early transcription terminator"
     ncRNA           1453..1520
                     /product="hepatitis delta virus self-cleaving ribozyme 
                     (Ferre-D'Amare and Scott, 2010)"
                     /label=HDV ribozyme
                     /ncRNA_class="ribozyme"
     misc_feature    1667..1691
                     /label=RB T-DNA repeat
                     /note="right border repeat from nopaline C58 T-DNA"
     CDS             complement(1847..2995)
                     /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(3331..3919)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(3411..3430)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(4090..4881,4882..4950))
                     /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     4713..4732
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(4951..5055)
                     /gene="bla"
                     /label=AmpR promoter
     rep_origin      5142..5773
                     /label=oriV
                     /note="incP origin of replication"
     misc_feature    5865..5889
                     /label=LB T-DNA repeat
                     /note="left border repeat from nopaline C58 T-DNA"
ORIGIN
        1 tgtggaattc ctcgagggag tttttgtatc ttgtttcata gtttgtccca ggattagaat
       61 gattaggcat cgaaccttca agaatttgat tgaataaaac atcttcattc ttaagatatg
      121 aagataatct tcaaaaggcc cctgggaatc tgaaagaaga gaagcaggcc catttatatg
      181 ggaaagaaca atagtatttc ttatataggc ccatttaagt tgaaaacaat cttcaaaagt
      241 cccacatcgc ttagataaga aaacgaagct gagtttatat acagctagag tcgaagtagt
      301 gattgaaatt actgatgagt ccgtgaggac gaaacgagta agctcgtcta atttctacta
      361 agtgtagatt gagacccaat acgcaaaccg cctctccccg cgcgttggcc gattcattaa
      421 tgcagctggc acgacaggtt tcccgactgg aaagcgggca gtgagcgcaa cgcaattaat
      481 gtgagttagc tcactcatta ggcaccccag gctttacact ttatgcttcc ggctcgtatg
      541 ttgtgtggaa ttgtgagcgg ataacaattt cacacatact agagaaagag gagaaatact
      601 agatggcttc ctccgaggac gttatcaaag agttcatgcg tttcaaagtt cgtatggaag
      661 gttccgttaa cggtcacgag ttcgaaatcg aaggtgaagg tgaaggtcgt ccgtacgaag
      721 gtacccagac cgctaaactg aaagttacca aaggtggtcc gctgccgttc gcttgggaca
      781 tcctgtcccc gcagttccag tacggttcca aagcttacgt taaacacccg gctgacatcc
      841 cggactacct gaaactgtcc ttcccggaag gtttcaaatg ggaacgtgtt atgaacttcg
      901 aggacggtgg tgttgttacc gttacccagg actcctccct gcaagacggt gagttcatct
      961 acaaagttaa actgcgtggt accaacttcc cgtccgacgg tccggttatg cagaaaaaaa
     1021 ccatgggttg ggaagcttcc accgaacgta tgtacccgga ggacggtgct ctgaaaggtg
     1081 aaatcaaaat gcgtctgaaa ctgaaagacg gtggtcacta cgacgctgaa gttaaaacca
     1141 cctacatggc taaaaaaccg gttcagctgc cgggtgctta caaaaccgac atcaaactgg
     1201 acatcacctc ccacaacgag gactacacca tcgttgaaca gtacgaacgt gctgaaggtc
     1261 gtcactccac cggtgcttaa taacgctgat agtgctagtg tagatcgcta ctagagccag
     1321 gcatcaaata aaacgaaagg ctcagtcgaa agactgggcc tttcgtttta tctgttgttt
     1381 gtcggtgaac gctctctact agagtcacac tggctcacct tcgggtgggc ctttctgcgt
     1441 ttataggtct caggccggca tggtcccagc ctcctcgctg gcgccggctg ggcaacatgc
     1501 ttcggcatgg cgaatgggac tttttttgca aaattttcca gatcgatttc ttcttcctct
     1561 gttcttcggc gttcaatttc tggggttttc tcttcgtttt ctgtaactga aacctaaaat
     1621 tcgctgagct tgtcttctgc acgaagtggt ttaaactatc agtgtttgac aggatatatt
     1681 ggcgggtaaa cctaagagaa aagagcgttt attagaataa tcggatattt aaaagggcgt
     1741 gaaaaggttt atccgttcgt ccatttgtat gtgcatgcca accacagggt tccccagatc
     1801 aggcgctggc tgctgaaccc ccagccggaa ctgaccccac aaggccctag cgtttgcaat
     1861 gcaccaggtc atcattgacc caggcgtgtt ccaccaggcc gctgcctcgc aactcttcgc
     1921 aggcttcgcc gacctgctcg cgccacttct tcacgcgggt ggaatccgat ccgcacatga
     1981 ggcggaaggt ttccagcttg agcgggtacg gctcccggtg cgagctgaaa tagtcgaaca
     2041 tccgtcgggc cgtcggcgac agcttgcggt acttctccca tatgaatttc gtgtagtggt
     2101 cgccagcaaa cagcacgacg atttcctcgt cgatcaggac ctggcaacgg gacgttttct
     2161 tgccacggtc caggacgcgg aagcggtgca gcagcgacac cgattccagg tgcccaacgc
     2221 ggtcggacgt gaagcccatc gccgtcgcct gtaggcgcga caggcattcc tcggccttcg
     2281 tgtaataccg gccattgatc gaccagccca ggtcctggca aagctcgtag aacgtgaagg
     2341 tgatcggctc gccgataggg gtgcgcttcg cgtactccaa cacctgctgc cacaccagtt
     2401 cgtcatcgtc ggcccgcagc tcgacgccgg tgtaggtgat cttcacgtcc ttgttgacgt
     2461 ggaaaatgac cttgttttgc agcgcctcgc gcgggatttt cttgttgcgc gtggtgaaca
     2521 gggcagagcg ggccgtgtcg tttggcatcg ctcgcatcgt gtccggccac ggcgcaatat
     2581 cgaacaagga aagctgcatt tccttgatct gctgcttcgt gtgtttcagc aacgcggcct
     2641 gcttggcctc gctgacctgt tttgccaggt cctcgccggc ggtttttcgc ttcttggtcg
     2701 tcatagttcc tcgcgtgtcg atggtcatcg acttcgccaa acctgccgcc tcctgttcaa
     2761 gacgacgcga acgctccacg gcggccgatg gcgcgggcag ggcaggggga gccagttgca
     2821 cgctgtcgcg ctcgatcttg gccgtagctt gctggaccat cgagccgacg gactggaagg
     2881 tttcgcgggg cgcacgcatg acggtgcggc ttgcgatggt ttcggcatcc tcggcggaaa
     2941 accccgcgtc gatcagttct tgcctgtatg ccttccggtc aaacgtccga ttcattcacc
     3001 ctccttgcgg gattgccccg actcacgccg gggcaatgtg cccttattcc tgatttgacc
     3061 cgcctggtgc cttggtgtcc agataatcca ccttatcggc aatgaagtcg gtcccgtaga
     3121 ccgtctggcc gtccttctcg tacttggtat tccgaatctt gccctgcacg aataccagcg
     3181 accccttgcc caaatacttg ccgtgggcct cggcctgaga gccaaaacac ttgatgcgga
     3241 agaagtcggt gcgctcctgc ttgtcgccgg catcgttgcg ccacatctag gatctgccag
     3301 gaaccgtaaa aaggccgcgt tgctggcgtt tttccatagg ctccgccccc ctgacgagca
     3361 tcacaaaaat cgacgctcaa gtcagaggtg gcgaaacccg acaggactat aaagatacca
     3421 ggcgtttccc cctggaagct ccctcgtgcg ctctcctgtt ccgaccctgc cgcttaccgg
     3481 atacctgtcc gcctttctcc cttcgggaag cgtggcgctt tctcatagct cacgctgtag
     3541 gtatctcagt tcggtgtagg tcgttcgctc caagctgggc tgtgtgcacg aaccccccgt
     3601 tcagcccgac cgctgcgcct tatccggtaa ctatcgtctt gagtccaacc cggtaagaca
     3661 cgacttatcg ccactggcag cagccactgg taacaggatt agcagagcga ggtatgtagg
     3721 cggtgctaca gagttcttga agtggtggcc taactacggc tacactagaa gaacagtatt
     3781 tggtatctgc gctctgctga agccagttac cttcggaaaa agagttggta gctcttgatc
     3841 cggcaaacaa accaccgctg gtagcggtgg tttttttgtt tgcaagcagc agattacgcg
     3901 cagaaaaaaa ggatctcaag aagatccttt gatcttttct acggggtctg acgctcagtg
     3961 gaacgaaaac tcacgttaag ggattttggt catgagatta tcaaaaagga tcttcaccta
     4021 gatcctttta aattaaaaat gaagttttaa atcaatctaa agtatatatg agtaaacttg
     4081 gtctgacagt taccaatgct taatcagtga ggcacctatc tcagcgatct gtctatttcg
     4141 ttcatccata gttgcctgac tccccgtcgt gtagataact acgatacggg agggcttacc
     4201 atctggcccc agtgctgcaa tgataccgcg agaaccacgc tcaccggctc cagatttatc
     4261 agcaataaac cagccagccg gaagggccga gcgcagaagt ggtcctgcaa ctttatccgc
     4321 ctccatccag tctattaatt gttgccggga agctagagta agtagttcgc cagttaatag
     4381 tttgcgcaac gttgttgcca ttgctacagg catcgtggtg tcacgctcgt cgtttggtat
     4441 ggcttcattc agctccggtt cccaacgatc aaggcgagtt acatgatccc ccatgttgtg
     4501 caaaaaagcg gttagctcct tcggtcctcc gatcgttgtc agaagtaagt tggccgcagt
     4561 gttatcactc atggttatgg cagcactgca taattctctt actgtcatgc catccgtaag
     4621 atgcttttct gtgactggtg agtactcaac caagtcattc tgagaatagt gtatgcggcg
     4681 accgagttgc tcttgcccgg cgtcaatacg ggataatacc gcgccacata gcagaacttt
     4741 aaaagtgctc atcattggaa aacgttcttc ggggcgaaaa ctctcaagga tcttaccgct
     4801 gttgagatcc agttcgatgt aacccactcg tgcacccaac tgatcttcag catcttttac
     4861 tttcaccagc gtttctgggt gagcaaaaac aggaaggcaa aatgccgcaa aaaagggaat
     4921 aagggcgaca cggaaatgtt gaatactcat actcttcctt tttcaatatt attgaagcat
     4981 ttatcaaggt tattgtctca tgagcggata catatttgaa tgtatttaga aaaataaaca
     5041 aataggggtt ccgcgcacga attggccagc gctgccattt ttggggtgag gccgttcgcg
     5101 gccgaggggc gcagcccctg gggggatggg aggcccgcgt tagcgggccg ggagggttcg
     5161 agaagggggg gcacccccct tcggcgtgcg cggtcacgcg cacagggcgc agccctggtt
     5221 aaaaacaagg tttataaata ttggtttaaa agcaggttaa aagacaggtt agcggtggcc
     5281 gaaaaacggg cggaaaccct tgcaaatgct ggattttctg cctgtggaca gcccctcaaa
     5341 tgtcaatagg tgcgcccctc atctgtcagc actctgcccc tcaagtgtca aggatcgcgc
     5401 ccctcatctg tcagtagtcg cgcccctcaa gtgtcaatac cgcagggcac ttatccccag
     5461 gcttgtccac atcatctgtg ggaaactcgc gtaaaatcag gcgttttcgc cgatttgcga
     5521 ggctggccag ctccacgtcg ccggccgaaa tcgagcctgc ccctcatctg tcaacgccgc
     5581 gccgggtgag tcggcccctc aagtgtcaac gtccgcccct catctgtcag tgagggccaa
     5641 gttttccgcg aggtatccac aacgccggcg gccgcggtgt ctcgcacacg gcttcgacgg
     5701 cgtttctggc gcgtttgcag ggccatagac ggccgccagc ccagcggcga gggcaaccag
     5761 cccggtgagc gtcgcaaagg agatcctgat ctgactgatg ggctgcctgt atcgagtggt
     5821 gattttgtgc cgagctgccg gtcggggagc tgttggctgg ctggtggcag gatatattgt
     5881 ggtgtaaaca aattgacgct tagacaactt aataacacat tgcggacgtt tttaatgtac
     5941 tggggtggat gcagtgggcc ccactctgtg aagacaa
//
