LOCUS       sequence-377106-        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: pICSL1P2_crU626
FEATURES             Location/Qualifiers
     source          1..5977
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     ncRNA           340..376
                     /product="self-cleaving hammerhead ribozyme (Ferre-D'Amare 
                     and Scott, 2010)"
                     /label=hammerhead ribozyme
                     /ncRNA_class="hammerhead_ribozyme"
     protein_bind    505..526
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     promoter        join(541..546,547..564,565..571)
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    579..595
                     /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             613..624
                     /note="strong bacterial ribosome binding site (Elowitz and 
                     Leibler, 2000)"
     CDS             631..1308
                     /codon_start=1
                     /product="monomeric derivative of DsRed (Campbell et al., 
                     2002)"
                     /label=mRFP1
                     /translation="MASSEDVIKEFMRFKVRMEGSVNGHEFEIEGEGEGRPYEGTQTAK
                     LKVTKGGPLPFAWDILSPQFQYGSKAYVKHPADIPDYLKLSFPEGFKWERVMNFEDGGV
                     VTVTQDSSLQDGEFIYKVKLRGTNFPSDGPVMQKKTMGWEASTERMYPEDGALKGEIKM
                     RLKLKDGGHYDAEVKTTYMAKKPVQLPGAYKTDIKLDITSHNEDYTIVEQYERAEGRHS
                     TGA"
     terminator      1353..1424
                     /label=rrnB T1 terminator
                     /note="transcription terminator T1 from the E. coli rrnB 
                     gene"
     terminator      1440..1467
                     /label=T7Te terminator
                     /note="phage T7 early transcription terminator"
     ncRNA           1481..1548
                     /product="hepatitis delta virus self-cleaving ribozyme 
                     (Ferre-D'Amare and Scott, 2010)"
                     /label=HDV ribozyme
                     /ncRNA_class="ribozyme"
     misc_feature    1695..1719
                     /label=RB T-DNA repeat
                     /note="right border repeat from nopaline C58 T-DNA"
     CDS             complement(1875..3023)
                     /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(3359..3947)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(3439..3458)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(4118..4909,4910..4978))
                     /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     4741..4760
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(4979..5083)
                     /gene="bla"
                     /label=AmpR promoter
     rep_origin      5170..5801
                     /label=oriV
                     /note="incP origin of replication"
     misc_feature    5893..5917
                     /label=LB T-DNA repeat
                     /note="left border repeat from nopaline C58 T-DNA"
ORIGIN
        1 tgcagtgggc cccactctgt gaagacaagc aagaattcaa gcttggagtt tttgtatctt
       61 gtttcatagt ttgtcccagg attagaatga ttaggcatcg aaccttcaag aatttgattg
      121 aataaaacat cttcattctt aagatatgaa gataatcttc aaaaggcccc tgggaatctg
      181 aaagaagaga agcaggccca tttatatggg aaagaacaat agtatttctt atataggccc
      241 atttaagttg aaaacaatct tcaaaagtcc cacatcgctt agataagaaa acgaagctga
      301 gtttatatac agctagagtc gaagtagtga ttgaaattac tgatgagtcc gtgaggacga
      361 aacgagtaag ctcgtctaat ttctactaag tgtagattga gacccaatac gcaaaccgcc
      421 tctccccgcg cgttggccga ttcattaatg cagctggcac gacaggtttc ccgactggaa
      481 agcgggcagt gagcgcaacg caattaatgt gagttagctc actcattagg caccccaggc
      541 tttacacttt atgcttccgg ctcgtatgtt gtgtggaatt gtgagcggat aacaatttca
      601 cacatactag agaaagagga gaaatactag atggcttcct ccgaggacgt tatcaaagag
      661 ttcatgcgtt tcaaagttcg tatggaaggt tccgttaacg gtcacgagtt cgaaatcgaa
      721 ggtgaaggtg aaggtcgtcc gtacgaaggt acccagaccg ctaaactgaa agttaccaaa
      781 ggtggtccgc tgccgttcgc ttgggacatc ctgtccccgc agttccagta cggttccaaa
      841 gcttacgtta aacacccggc tgacatcccg gactacctga aactgtcctt cccggaaggt
      901 ttcaaatggg aacgtgttat gaacttcgag gacggtggtg ttgttaccgt tacccaggac
      961 tcctccctgc aagacggtga gttcatctac aaagttaaac tgcgtggtac caacttcccg
     1021 tccgacggtc cggttatgca gaaaaaaacc atgggttggg aagcttccac cgaacgtatg
     1081 tacccggagg acggtgctct gaaaggtgaa atcaaaatgc gtctgaaact gaaagacggt
     1141 ggtcactacg acgctgaagt taaaaccacc tacatggcta aaaaaccggt tcagctgccg
     1201 ggtgcttaca aaaccgacat caaactggac atcacctccc acaacgagga ctacaccatc
     1261 gttgaacagt acgaacgtgc tgaaggtcgt cactccaccg gtgcttaata acgctgatag
     1321 tgctagtgta gatcgctact agagccaggc atcaaataaa acgaaaggct cagtcgaaag
     1381 actgggcctt tcgttttatc tgttgtttgt cggtgaacgc tctctactag agtcacactg
     1441 gctcaccttc gggtgggcct ttctgcgttt ataggtctca ggccggcatg gtcccagcct
     1501 cctcgctggc gccggctggg caacatgctt cggcatggcg aatgggactt tttttgcaaa
     1561 attttccaga tcgatttctt cttcctctgt tcttcggcgt tcaatttctg gggttttctc
     1621 ttcgttttct gtaactgaaa cctaaaattc gctactattg tcttctgcac gaagtggttt
     1681 aaactatcag tgtttgacag gatatattgg cgggtaaacc taagagaaaa gagcgtttat
     1741 tagaataatc ggatatttaa aagggcgtga aaaggtttat ccgttcgtcc atttgtatgt
     1801 gcatgccaac cacagggttc cccagatcag gcgctggctg ctgaaccccc agccggaact
     1861 gaccccacaa ggccctagcg tttgcaatgc accaggtcat cattgaccca ggcgtgttcc
     1921 accaggccgc tgcctcgcaa ctcttcgcag gcttcgccga cctgctcgcg ccacttcttc
     1981 acgcgggtgg aatccgatcc gcacatgagg cggaaggttt ccagcttgag cgggtacggc
     2041 tcccggtgcg agctgaaata gtcgaacatc cgtcgggccg tcggcgacag cttgcggtac
     2101 ttctcccata tgaatttcgt gtagtggtcg ccagcaaaca gcacgacgat ttcctcgtcg
     2161 atcaggacct ggcaacggga cgttttcttg ccacggtcca ggacgcggaa gcggtgcagc
     2221 agcgacaccg attccaggtg cccaacgcgg tcggacgtga agcccatcgc cgtcgcctgt
     2281 aggcgcgaca ggcattcctc ggccttcgtg taataccggc cattgatcga ccagcccagg
     2341 tcctggcaaa gctcgtagaa cgtgaaggtg atcggctcgc cgataggggt gcgcttcgcg
     2401 tactccaaca cctgctgcca caccagttcg tcatcgtcgg cccgcagctc gacgccggtg
     2461 taggtgatct tcacgtcctt gttgacgtgg aaaatgacct tgttttgcag cgcctcgcgc
     2521 gggattttct tgttgcgcgt ggtgaacagg gcagagcggg ccgtgtcgtt tggcatcgct
     2581 cgcatcgtgt ccggccacgg cgcaatatcg aacaaggaaa gctgcatttc cttgatctgc
     2641 tgcttcgtgt gtttcagcaa cgcggcctgc ttggcctcgc tgacctgttt tgccaggtcc
     2701 tcgccggcgg tttttcgctt cttggtcgtc atagttcctc gcgtgtcgat ggtcatcgac
     2761 ttcgccaaac ctgccgcctc ctgttcaaga cgacgcgaac gctccacggc ggccgatggc
     2821 gcgggcaggg cagggggagc cagttgcacg ctgtcgcgct cgatcttggc cgtagcttgc
     2881 tggaccatcg agccgacgga ctggaaggtt tcgcggggcg cacgcatgac ggtgcggctt
     2941 gcgatggttt cggcatcctc ggcggaaaac cccgcgtcga tcagttcttg cctgtatgcc
     3001 ttccggtcaa acgtccgatt cattcaccct ccttgcggga ttgccccgac tcacgccggg
     3061 gcaatgtgcc cttattcctg atttgacccg cctggtgcct tggtgtccag ataatccacc
     3121 ttatcggcaa tgaagtcggt cccgtagacc gtctggccgt ccttctcgta cttggtattc
     3181 cgaatcttgc cctgcacgaa taccagcgac cccttgccca aatacttgcc gtgggcctcg
     3241 gcctgagagc caaaacactt gatgcggaag aagtcggtgc gctcctgctt gtcgccggca
     3301 tcgttgcgcc acatctagga tctgccagga accgtaaaaa ggccgcgttg ctggcgtttt
     3361 tccataggct ccgcccccct gacgagcatc acaaaaatcg acgctcaagt cagaggtggc
     3421 gaaacccgac aggactataa agataccagg cgtttccccc tggaagctcc ctcgtgcgct
     3481 ctcctgttcc gaccctgccg cttaccggat acctgtccgc ctttctccct tcgggaagcg
     3541 tggcgctttc tcatagctca cgctgtaggt atctcagttc ggtgtaggtc gttcgctcca
     3601 agctgggctg tgtgcacgaa ccccccgttc agcccgaccg ctgcgcctta tccggtaact
     3661 atcgtcttga gtccaacccg gtaagacacg acttatcgcc actggcagca gccactggta
     3721 acaggattag cagagcgagg tatgtaggcg gtgctacaga gttcttgaag tggtggccta
     3781 actacggcta cactagaaga acagtatttg gtatctgcgc tctgctgaag ccagttacct
     3841 tcggaaaaag agttggtagc tcttgatccg gcaaacaaac caccgctggt agcggtggtt
     3901 tttttgtttg caagcagcag attacgcgca gaaaaaaagg atctcaagaa gatcctttga
     3961 tcttttctac ggggtctgac gctcagtgga acgaaaactc acgttaaggg attttggtca
     4021 tgagattatc aaaaaggatc ttcacctaga tccttttaaa ttaaaaatga agttttaaat
     4081 caatctaaag tatatatgag taaacttggt ctgacagtta ccaatgctta atcagtgagg
     4141 cacctatctc agcgatctgt ctatttcgtt catccatagt tgcctgactc cccgtcgtgt
     4201 agataactac gatacgggag ggcttaccat ctggccccag tgctgcaatg ataccgcgag
     4261 aaccacgctc accggctcca gatttatcag caataaacca gccagccgga agggccgagc
     4321 gcagaagtgg tcctgcaact ttatccgcct ccatccagtc tattaattgt tgccgggaag
     4381 ctagagtaag tagttcgcca gttaatagtt tgcgcaacgt tgttgccatt gctacaggca
     4441 tcgtggtgtc acgctcgtcg tttggtatgg cttcattcag ctccggttcc caacgatcaa
     4501 ggcgagttac atgatccccc atgttgtgca aaaaagcggt tagctccttc ggtcctccga
     4561 tcgttgtcag aagtaagttg gccgcagtgt tatcactcat ggttatggca gcactgcata
     4621 attctcttac tgtcatgcca tccgtaagat gcttttctgt gactggtgag tactcaacca
     4681 agtcattctg agaatagtgt atgcggcgac cgagttgctc ttgcccggcg tcaatacggg
     4741 ataataccgc gccacatagc agaactttaa aagtgctcat cattggaaaa cgttcttcgg
     4801 ggcgaaaact ctcaaggatc ttaccgctgt tgagatccag ttcgatgtaa cccactcgtg
     4861 cacccaactg atcttcagca tcttttactt tcaccagcgt ttctgggtga gcaaaaacag
     4921 gaaggcaaaa tgccgcaaaa aagggaataa gggcgacacg gaaatgttga atactcatac
     4981 tcttcctttt tcaatattat tgaagcattt atcaaggtta ttgtctcatg agcggataca
     5041 tatttgaatg tatttagaaa aataaacaaa taggggttcc gcgcacgaat tggccagcgc
     5101 tgccattttt ggggtgaggc cgttcgcggc cgaggggcgc agcccctggg gggatgggag
     5161 gcccgcgtta gcgggccggg agggttcgag aagggggggc accccccttc ggcgtgcgcg
     5221 gtcacgcgca cagggcgcag ccctggttaa aaacaaggtt tataaatatt ggtttaaaag
     5281 caggttaaaa gacaggttag cggtggccga aaaacgggcg gaaacccttg caaatgctgg
     5341 attttctgcc tgtggacagc ccctcaaatg tcaataggtg cgcccctcat ctgtcagcac
     5401 tctgcccctc aagtgtcaag gatcgcgccc ctcatctgtc agtagtcgcg cccctcaagt
     5461 gtcaataccg cagggcactt atccccaggc ttgtccacat catctgtggg aaactcgcgt
     5521 aaaatcaggc gttttcgccg atttgcgagg ctggccagct ccacgtcgcc ggccgaaatc
     5581 gagcctgccc ctcatctgtc aacgccgcgc cgggtgagtc ggcccctcaa gtgtcaacgt
     5641 ccgcccctca tctgtcagtg agggccaagt tttccgcgag gtatccacaa cgccggcggc
     5701 cgcggtgtct cgcacacggc ttcgacggcg tttctggcgc gtttgcaggg ccatagacgg
     5761 ccgccagccc agcggcgagg gcaaccagcc cggtgagcgt cgcaaaggag atcctgatct
     5821 gactgatggg ctgcctgtat cgagtggtga ttttgtgccg agctgccggt cggggagctg
     5881 ttggctggct ggtggcagga tatattgtgg tgtaaacaaa ttgacgctta gacaacttaa
     5941 taacacattg cggacgtttt taatgtactg gggtgga
//
