LOCUS       sequence-377104-        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: pICSL1P4_crU626
FEATURES             Location/Qualifiers
     source          1..5977
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     ncRNA           329..365
                     /product="self-cleaving hammerhead ribozyme (Ferre-D'Amare 
                     and Scott, 2010)"
                     /label=hammerhead ribozyme
                     /ncRNA_class="hammerhead_ribozyme"
     protein_bind    494..515
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     promoter        join(530..535,536..553,554..560)
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    568..584
                     /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             602..613
                     /note="strong bacterial ribosome binding site (Elowitz and 
                     Leibler, 2000)"
     CDS             620..1297
                     /codon_start=1
                     /product="monomeric derivative of DsRed (Campbell et al., 
                     2002)"
                     /label=mRFP1
                     /translation="MASSEDVIKEFMRFKVRMEGSVNGHEFEIEGEGEGRPYEGTQTAK
                     LKVTKGGPLPFAWDILSPQFQYGSKAYVKHPADIPDYLKLSFPEGFKWERVMNFEDGGV
                     VTVTQDSSLQDGEFIYKVKLRGTNFPSDGPVMQKKTMGWEASTERMYPEDGALKGEIKM
                     RLKLKDGGHYDAEVKTTYMAKKPVQLPGAYKTDIKLDITSHNEDYTIVEQYERAEGRHS
                     TGA"
     terminator      1342..1413
                     /label=rrnB T1 terminator
                     /note="transcription terminator T1 from the E. coli rrnB 
                     gene"
     terminator      1429..1456
                     /label=T7Te terminator
                     /note="phage T7 early transcription terminator"
     ncRNA           1470..1537
                     /product="hepatitis delta virus self-cleaving ribozyme 
                     (Ferre-D'Amare and Scott, 2010)"
                     /label=HDV ribozyme
                     /ncRNA_class="ribozyme"
     misc_feature    1684..1708
                     /label=RB T-DNA repeat
                     /note="right border repeat from nopaline C58 T-DNA"
     CDS             complement(1864..3012)
                     /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(3348..3936)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(3428..3447)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(4107..4898,4899..4967))
                     /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     4730..4749
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(4968..5072)
                     /gene="bla"
                     /label=AmpR promoter
     rep_origin      5159..5790
                     /label=oriV
                     /note="incP origin of replication"
     misc_feature    5882..5906
                     /label=LB T-DNA repeat
                     /note="left border repeat from nopaline C58 T-DNA"
ORIGIN
        1 ccactctgtg aagacaatta cgaattccca tggggagttt ttgtatcttg tttcatagtt
       61 tgtcccagga ttagaatgat taggcatcga accttcaaga atttgattga ataaaacatc
      121 ttcattctta agatatgaag ataatcttca aaaggcccct gggaatctga aagaagagaa
      181 gcaggcccat ttatatggga aagaacaata gtatttctta tataggccca tttaagttga
      241 aaacaatctt caaaagtccc acatcgctta gataagaaaa cgaagctgag tttatataca
      301 gctagagtcg aagtagtgat tgaaattact gatgagtccg tgaggacgaa acgagtaagc
      361 tcgtctaatt tctactaagt gtagattgag acccaatacg caaaccgcct ctccccgcgc
      421 gttggccgat tcattaatgc agctggcacg acaggtttcc cgactggaaa gcgggcagtg
      481 agcgcaacgc aattaatgtg agttagctca ctcattaggc accccaggct ttacacttta
      541 tgcttccggc tcgtatgttg tgtggaattg tgagcggata acaatttcac acatactaga
      601 gaaagaggag aaatactaga tggcttcctc cgaggacgtt atcaaagagt tcatgcgttt
      661 caaagttcgt atggaaggtt ccgttaacgg tcacgagttc gaaatcgaag gtgaaggtga
      721 aggtcgtccg tacgaaggta cccagaccgc taaactgaaa gttaccaaag gtggtccgct
      781 gccgttcgct tgggacatcc tgtccccgca gttccagtac ggttccaaag cttacgttaa
      841 acacccggct gacatcccgg actacctgaa actgtccttc ccggaaggtt tcaaatggga
      901 acgtgttatg aacttcgagg acggtggtgt tgttaccgtt acccaggact cctccctgca
      961 agacggtgag ttcatctaca aagttaaact gcgtggtacc aacttcccgt ccgacggtcc
     1021 ggttatgcag aaaaaaacca tgggttggga agcttccacc gaacgtatgt acccggagga
     1081 cggtgctctg aaaggtgaaa tcaaaatgcg tctgaaactg aaagacggtg gtcactacga
     1141 cgctgaagtt aaaaccacct acatggctaa aaaaccggtt cagctgccgg gtgcttacaa
     1201 aaccgacatc aaactggaca tcacctccca caacgaggac tacaccatcg ttgaacagta
     1261 cgaacgtgct gaaggtcgtc actccaccgg tgcttaataa cgctgatagt gctagtgtag
     1321 atcgctacta gagccaggca tcaaataaaa cgaaaggctc agtcgaaaga ctgggccttt
     1381 cgttttatct gttgtttgtc ggtgaacgct ctctactaga gtcacactgg ctcaccttcg
     1441 ggtgggcctt tctgcgttta taggtctcag gccggcatgg tcccagcctc ctcgctggcg
     1501 ccggctgggc aacatgcttc ggcatggcga atgggacttt ttttgcaaaa ttttccagat
     1561 cgatttcttc ttcctctgtt cttcggcgtt caatttctgg ggttttctct tcgttttctg
     1621 taactgaaac ctaaaattcg ctcagattgt cttctgcacg aagtggttta aactatcagt
     1681 gtttgacagg atatattggc gggtaaacct aagagaaaag agcgtttatt agaataatcg
     1741 gatatttaaa agggcgtgaa aaggtttatc cgttcgtcca tttgtatgtg catgccaacc
     1801 acagggttcc ccagatcagg cgctggctgc tgaaccccca gccggaactg accccacaag
     1861 gccctagcgt ttgcaatgca ccaggtcatc attgacccag gcgtgttcca ccaggccgct
     1921 gcctcgcaac tcttcgcagg cttcgccgac ctgctcgcgc cacttcttca cgcgggtgga
     1981 atccgatccg cacatgaggc ggaaggtttc cagcttgagc gggtacggct cccggtgcga
     2041 gctgaaatag tcgaacatcc gtcgggccgt cggcgacagc ttgcggtact tctcccatat
     2101 gaatttcgtg tagtggtcgc cagcaaacag cacgacgatt tcctcgtcga tcaggacctg
     2161 gcaacgggac gttttcttgc cacggtccag gacgcggaag cggtgcagca gcgacaccga
     2221 ttccaggtgc ccaacgcggt cggacgtgaa gcccatcgcc gtcgcctgta ggcgcgacag
     2281 gcattcctcg gccttcgtgt aataccggcc attgatcgac cagcccaggt cctggcaaag
     2341 ctcgtagaac gtgaaggtga tcggctcgcc gataggggtg cgcttcgcgt actccaacac
     2401 ctgctgccac accagttcgt catcgtcggc ccgcagctcg acgccggtgt aggtgatctt
     2461 cacgtccttg ttgacgtgga aaatgacctt gttttgcagc gcctcgcgcg ggattttctt
     2521 gttgcgcgtg gtgaacaggg cagagcgggc cgtgtcgttt ggcatcgctc gcatcgtgtc
     2581 cggccacggc gcaatatcga acaaggaaag ctgcatttcc ttgatctgct gcttcgtgtg
     2641 tttcagcaac gcggcctgct tggcctcgct gacctgtttt gccaggtcct cgccggcggt
     2701 ttttcgcttc ttggtcgtca tagttcctcg cgtgtcgatg gtcatcgact tcgccaaacc
     2761 tgccgcctcc tgttcaagac gacgcgaacg ctccacggcg gccgatggcg cgggcagggc
     2821 agggggagcc agttgcacgc tgtcgcgctc gatcttggcc gtagcttgct ggaccatcga
     2881 gccgacggac tggaaggttt cgcggggcgc acgcatgacg gtgcggcttg cgatggtttc
     2941 ggcatcctcg gcggaaaacc ccgcgtcgat cagttcttgc ctgtatgcct tccggtcaaa
     3001 cgtccgattc attcaccctc cttgcgggat tgccccgact cacgccgggg caatgtgccc
     3061 ttattcctga tttgacccgc ctggtgcctt ggtgtccaga taatccacct tatcggcaat
     3121 gaagtcggtc ccgtagaccg tctggccgtc cttctcgtac ttggtattcc gaatcttgcc
     3181 ctgcacgaat accagcgacc ccttgcccaa atacttgccg tgggcctcgg cctgagagcc
     3241 aaaacacttg atgcggaaga agtcggtgcg ctcctgcttg tcgccggcat cgttgcgcca
     3301 catctaggat ctgccaggaa ccgtaaaaag gccgcgttgc tggcgttttt ccataggctc
     3361 cgcccccctg acgagcatca caaaaatcga cgctcaagtc agaggtggcg aaacccgaca
     3421 ggactataaa gataccaggc gtttccccct ggaagctccc tcgtgcgctc tcctgttccg
     3481 accctgccgc ttaccggata cctgtccgcc tttctccctt cgggaagcgt ggcgctttct
     3541 catagctcac gctgtaggta tctcagttcg gtgtaggtcg ttcgctccaa gctgggctgt
     3601 gtgcacgaac cccccgttca gcccgaccgc tgcgccttat ccggtaacta tcgtcttgag
     3661 tccaacccgg taagacacga cttatcgcca ctggcagcag ccactggtaa caggattagc
     3721 agagcgaggt atgtaggcgg tgctacagag ttcttgaagt ggtggcctaa ctacggctac
     3781 actagaagaa cagtatttgg tatctgcgct ctgctgaagc cagttacctt cggaaaaaga
     3841 gttggtagct cttgatccgg caaacaaacc accgctggta gcggtggttt ttttgtttgc
     3901 aagcagcaga ttacgcgcag aaaaaaagga tctcaagaag atcctttgat cttttctacg
     3961 gggtctgacg ctcagtggaa cgaaaactca cgttaaggga ttttggtcat gagattatca
     4021 aaaaggatct tcacctagat ccttttaaat taaaaatgaa gttttaaatc aatctaaagt
     4081 atatatgagt aaacttggtc tgacagttac caatgcttaa tcagtgaggc acctatctca
     4141 gcgatctgtc tatttcgttc atccatagtt gcctgactcc ccgtcgtgta gataactacg
     4201 atacgggagg gcttaccatc tggccccagt gctgcaatga taccgcgaga accacgctca
     4261 ccggctccag atttatcagc aataaaccag ccagccggaa gggccgagcg cagaagtggt
     4321 cctgcaactt tatccgcctc catccagtct attaattgtt gccgggaagc tagagtaagt
     4381 agttcgccag ttaatagttt gcgcaacgtt gttgccattg ctacaggcat cgtggtgtca
     4441 cgctcgtcgt ttggtatggc ttcattcagc tccggttccc aacgatcaag gcgagttaca
     4501 tgatccccca tgttgtgcaa aaaagcggtt agctccttcg gtcctccgat cgttgtcaga
     4561 agtaagttgg ccgcagtgtt atcactcatg gttatggcag cactgcataa ttctcttact
     4621 gtcatgccat ccgtaagatg cttttctgtg actggtgagt actcaaccaa gtcattctga
     4681 gaatagtgta tgcggcgacc gagttgctct tgcccggcgt caatacggga taataccgcg
     4741 ccacatagca gaactttaaa agtgctcatc attggaaaac gttcttcggg gcgaaaactc
     4801 tcaaggatct taccgctgtt gagatccagt tcgatgtaac ccactcgtgc acccaactga
     4861 tcttcagcat cttttacttt caccagcgtt tctgggtgag caaaaacagg aaggcaaaat
     4921 gccgcaaaaa agggaataag ggcgacacgg aaatgttgaa tactcatact cttccttttt
     4981 caatattatt gaagcattta tcaaggttat tgtctcatga gcggatacat atttgaatgt
     5041 atttagaaaa ataaacaaat aggggttccg cgcacgaatt ggccagcgct gccatttttg
     5101 gggtgaggcc gttcgcggcc gaggggcgca gcccctgggg ggatgggagg cccgcgttag
     5161 cgggccggga gggttcgaga agggggggca ccccccttcg gcgtgcgcgg tcacgcgcac
     5221 agggcgcagc cctggttaaa aacaaggttt ataaatattg gtttaaaagc aggttaaaag
     5281 acaggttagc ggtggccgaa aaacgggcgg aaacccttgc aaatgctgga ttttctgcct
     5341 gtggacagcc cctcaaatgt caataggtgc gcccctcatc tgtcagcact ctgcccctca
     5401 agtgtcaagg atcgcgcccc tcatctgtca gtagtcgcgc ccctcaagtg tcaataccgc
     5461 agggcactta tccccaggct tgtccacatc atctgtggga aactcgcgta aaatcaggcg
     5521 ttttcgccga tttgcgaggc tggccagctc cacgtcgccg gccgaaatcg agcctgcccc
     5581 tcatctgtca acgccgcgcc gggtgagtcg gcccctcaag tgtcaacgtc cgcccctcat
     5641 ctgtcagtga gggccaagtt ttccgcgagg tatccacaac gccggcggcc gcggtgtctc
     5701 gcacacggct tcgacggcgt ttctggcgcg tttgcagggc catagacggc cgccagccca
     5761 gcggcgaggg caaccagccc ggtgagcgtc gcaaaggaga tcctgatctg actgatgggc
     5821 tgcctgtatc gagtggtgat tttgtgccga gctgccggtc ggggagctgt tggctggctg
     5881 gtggcaggat atattgtggt gtaaacaaat tgacgcttag acaacttaat aacacattgc
     5941 ggacgttttt aatgtactgg ggtggatgca gtgggcc
//
