LOCUS       sequence-377103-        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: pICSL1P5_crU626
FEATURES             Location/Qualifiers
     source          1..5977
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     ncRNA           337..373
                     /product="self-cleaving hammerhead ribozyme (Ferre-D'Amare 
                     and Scott, 2010)"
                     /label=hammerhead ribozyme
                     /ncRNA_class="hammerhead_ribozyme"
     protein_bind    502..523
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     promoter        join(538..543,544..561,562..568)
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    576..592
                     /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             610..621
                     /note="strong bacterial ribosome binding site (Elowitz and 
                     Leibler, 2000)"
     CDS             628..1305
                     /codon_start=1
                     /product="monomeric derivative of DsRed (Campbell et al., 
                     2002)"
                     /label=mRFP1
                     /translation="MASSEDVIKEFMRFKVRMEGSVNGHEFEIEGEGEGRPYEGTQTAK
                     LKVTKGGPLPFAWDILSPQFQYGSKAYVKHPADIPDYLKLSFPEGFKWERVMNFEDGGV
                     VTVTQDSSLQDGEFIYKVKLRGTNFPSDGPVMQKKTMGWEASTERMYPEDGALKGEIKM
                     RLKLKDGGHYDAEVKTTYMAKKPVQLPGAYKTDIKLDITSHNEDYTIVEQYERAEGRHS
                     TGA"
     terminator      1350..1421
                     /label=rrnB T1 terminator
                     /note="transcription terminator T1 from the E. coli rrnB 
                     gene"
     terminator      1437..1464
                     /label=T7Te terminator
                     /note="phage T7 early transcription terminator"
     ncRNA           1478..1545
                     /product="hepatitis delta virus self-cleaving ribozyme 
                     (Ferre-D'Amare and Scott, 2010)"
                     /label=HDV ribozyme
                     /ncRNA_class="ribozyme"
     misc_feature    1692..1716
                     /label=RB T-DNA repeat
                     /note="right border repeat from nopaline C58 T-DNA"
     CDS             complement(1872..3020)
                     /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(3356..3944)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(3436..3455)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(4115..4906,4907..4975))
                     /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     4738..4757
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(4976..5080)
                     /gene="bla"
                     /label=AmpR promoter
     rep_origin      5167..5798
                     /label=oriV
                     /note="incP origin of replication"
     misc_feature    5890..5914
                     /label=LB T-DNA repeat
                     /note="left border repeat from nopaline C58 T-DNA"
ORIGIN
        1 agtgggcccc actctgtgaa gacaacagag aattcgcatg cggagttttt gtatcttgtt
       61 tcatagtttg tcccaggatt agaatgatta ggcatcgaac cttcaagaat ttgattgaat
      121 aaaacatctt cattcttaag atatgaagat aatcttcaaa aggcccctgg gaatctgaaa
      181 gaagagaagc aggcccattt atatgggaaa gaacaatagt atttcttata taggcccatt
      241 taagttgaaa acaatcttca aaagtcccac atcgcttaga taagaaaacg aagctgagtt
      301 tatatacagc tagagtcgaa gtagtgattg aaattactga tgagtccgtg aggacgaaac
      361 gagtaagctc gtctaatttc tactaagtgt agattgagac ccaatacgca aaccgcctct
      421 ccccgcgcgt tggccgattc attaatgcag ctggcacgac aggtttcccg actggaaagc
      481 gggcagtgag cgcaacgcaa ttaatgtgag ttagctcact cattaggcac cccaggcttt
      541 acactttatg cttccggctc gtatgttgtg tggaattgtg agcggataac aatttcacac
      601 atactagaga aagaggagaa atactagatg gcttcctccg aggacgttat caaagagttc
      661 atgcgtttca aagttcgtat ggaaggttcc gttaacggtc acgagttcga aatcgaaggt
      721 gaaggtgaag gtcgtccgta cgaaggtacc cagaccgcta aactgaaagt taccaaaggt
      781 ggtccgctgc cgttcgcttg ggacatcctg tccccgcagt tccagtacgg ttccaaagct
      841 tacgttaaac acccggctga catcccggac tacctgaaac tgtccttccc ggaaggtttc
      901 aaatgggaac gtgttatgaa cttcgaggac ggtggtgttg ttaccgttac ccaggactcc
      961 tccctgcaag acggtgagtt catctacaaa gttaaactgc gtggtaccaa cttcccgtcc
     1021 gacggtccgg ttatgcagaa aaaaaccatg ggttgggaag cttccaccga acgtatgtac
     1081 ccggaggacg gtgctctgaa aggtgaaatc aaaatgcgtc tgaaactgaa agacggtggt
     1141 cactacgacg ctgaagttaa aaccacctac atggctaaaa aaccggttca gctgccgggt
     1201 gcttacaaaa ccgacatcaa actggacatc acctcccaca acgaggacta caccatcgtt
     1261 gaacagtacg aacgtgctga aggtcgtcac tccaccggtg cttaataacg ctgatagtgc
     1321 tagtgtagat cgctactaga gccaggcatc aaataaaacg aaaggctcag tcgaaagact
     1381 gggcctttcg ttttatctgt tgtttgtcgg tgaacgctct ctactagagt cacactggct
     1441 caccttcggg tgggcctttc tgcgtttata ggtctcaggc cggcatggtc ccagcctcct
     1501 cgctggcgcc ggctgggcaa catgcttcgg catggcgaat gggacttttt ttgcaaaatt
     1561 ttccagatcg atttcttctt cctctgttct tcggcgttca atttctgggg ttttctcttc
     1621 gttttctgta actgaaacct aaaattcgct tgtgttgtct tctgcacgaa gtggtttaaa
     1681 ctatcagtgt ttgacaggat atattggcgg gtaaacctaa gagaaaagag cgtttattag
     1741 aataatcgga tatttaaaag ggcgtgaaaa ggtttatccg ttcgtccatt tgtatgtgca
     1801 tgccaaccac agggttcccc agatcaggcg ctggctgctg aacccccagc cggaactgac
     1861 cccacaaggc cctagcgttt gcaatgcacc aggtcatcat tgacccaggc gtgttccacc
     1921 aggccgctgc ctcgcaactc ttcgcaggct tcgccgacct gctcgcgcca cttcttcacg
     1981 cgggtggaat ccgatccgca catgaggcgg aaggtttcca gcttgagcgg gtacggctcc
     2041 cggtgcgagc tgaaatagtc gaacatccgt cgggccgtcg gcgacagctt gcggtacttc
     2101 tcccatatga atttcgtgta gtggtcgcca gcaaacagca cgacgatttc ctcgtcgatc
     2161 aggacctggc aacgggacgt tttcttgcca cggtccagga cgcggaagcg gtgcagcagc
     2221 gacaccgatt ccaggtgccc aacgcggtcg gacgtgaagc ccatcgccgt cgcctgtagg
     2281 cgcgacaggc attcctcggc cttcgtgtaa taccggccat tgatcgacca gcccaggtcc
     2341 tggcaaagct cgtagaacgt gaaggtgatc ggctcgccga taggggtgcg cttcgcgtac
     2401 tccaacacct gctgccacac cagttcgtca tcgtcggccc gcagctcgac gccggtgtag
     2461 gtgatcttca cgtccttgtt gacgtggaaa atgaccttgt tttgcagcgc ctcgcgcggg
     2521 attttcttgt tgcgcgtggt gaacagggca gagcgggccg tgtcgtttgg catcgctcgc
     2581 atcgtgtccg gccacggcgc aatatcgaac aaggaaagct gcatttcctt gatctgctgc
     2641 ttcgtgtgtt tcagcaacgc ggcctgcttg gcctcgctga cctgttttgc caggtcctcg
     2701 ccggcggttt ttcgcttctt ggtcgtcata gttcctcgcg tgtcgatggt catcgacttc
     2761 gccaaacctg ccgcctcctg ttcaagacga cgcgaacgct ccacggcggc cgatggcgcg
     2821 ggcagggcag ggggagccag ttgcacgctg tcgcgctcga tcttggccgt agcttgctgg
     2881 accatcgagc cgacggactg gaaggtttcg cggggcgcac gcatgacggt gcggcttgcg
     2941 atggtttcgg catcctcggc ggaaaacccc gcgtcgatca gttcttgcct gtatgccttc
     3001 cggtcaaacg tccgattcat tcaccctcct tgcgggattg ccccgactca cgccggggca
     3061 atgtgccctt attcctgatt tgacccgcct ggtgccttgg tgtccagata atccacctta
     3121 tcggcaatga agtcggtccc gtagaccgtc tggccgtcct tctcgtactt ggtattccga
     3181 atcttgccct gcacgaatac cagcgacccc ttgcccaaat acttgccgtg ggcctcggcc
     3241 tgagagccaa aacacttgat gcggaagaag tcggtgcgct cctgcttgtc gccggcatcg
     3301 ttgcgccaca tctaggatct gccaggaacc gtaaaaaggc cgcgttgctg gcgtttttcc
     3361 ataggctccg cccccctgac gagcatcaca aaaatcgacg ctcaagtcag aggtggcgaa
     3421 acccgacagg actataaaga taccaggcgt ttccccctgg aagctccctc gtgcgctctc
     3481 ctgttccgac cctgccgctt accggatacc tgtccgcctt tctcccttcg ggaagcgtgg
     3541 cgctttctca tagctcacgc tgtaggtatc tcagttcggt gtaggtcgtt cgctccaagc
     3601 tgggctgtgt gcacgaaccc cccgttcagc ccgaccgctg cgccttatcc ggtaactatc
     3661 gtcttgagtc caacccggta agacacgact tatcgccact ggcagcagcc actggtaaca
     3721 ggattagcag agcgaggtat gtaggcggtg ctacagagtt cttgaagtgg tggcctaact
     3781 acggctacac tagaagaaca gtatttggta tctgcgctct gctgaagcca gttaccttcg
     3841 gaaaaagagt tggtagctct tgatccggca aacaaaccac cgctggtagc ggtggttttt
     3901 ttgtttgcaa gcagcagatt acgcgcagaa aaaaaggatc tcaagaagat cctttgatct
     3961 tttctacggg gtctgacgct cagtggaacg aaaactcacg ttaagggatt ttggtcatga
     4021 gattatcaaa aaggatcttc acctagatcc ttttaaatta aaaatgaagt tttaaatcaa
     4081 tctaaagtat atatgagtaa acttggtctg acagttacca atgcttaatc agtgaggcac
     4141 ctatctcagc gatctgtcta tttcgttcat ccatagttgc ctgactcccc gtcgtgtaga
     4201 taactacgat acgggagggc ttaccatctg gccccagtgc tgcaatgata ccgcgagaac
     4261 cacgctcacc ggctccagat ttatcagcaa taaaccagcc agccggaagg gccgagcgca
     4321 gaagtggtcc tgcaacttta tccgcctcca tccagtctat taattgttgc cgggaagcta
     4381 gagtaagtag ttcgccagtt aatagtttgc gcaacgttgt tgccattgct acaggcatcg
     4441 tggtgtcacg ctcgtcgttt ggtatggctt cattcagctc cggttcccaa cgatcaaggc
     4501 gagttacatg atcccccatg ttgtgcaaaa aagcggttag ctccttcggt cctccgatcg
     4561 ttgtcagaag taagttggcc gcagtgttat cactcatggt tatggcagca ctgcataatt
     4621 ctcttactgt catgccatcc gtaagatgct tttctgtgac tggtgagtac tcaaccaagt
     4681 cattctgaga atagtgtatg cggcgaccga gttgctcttg cccggcgtca atacgggata
     4741 ataccgcgcc acatagcaga actttaaaag tgctcatcat tggaaaacgt tcttcggggc
     4801 gaaaactctc aaggatctta ccgctgttga gatccagttc gatgtaaccc actcgtgcac
     4861 ccaactgatc ttcagcatct tttactttca ccagcgtttc tgggtgagca aaaacaggaa
     4921 ggcaaaatgc cgcaaaaaag ggaataaggg cgacacggaa atgttgaata ctcatactct
     4981 tcctttttca atattattga agcatttatc aaggttattg tctcatgagc ggatacatat
     5041 ttgaatgtat ttagaaaaat aaacaaatag gggttccgcg cacgaattgg ccagcgctgc
     5101 catttttggg gtgaggccgt tcgcggccga ggggcgcagc ccctgggggg atgggaggcc
     5161 cgcgttagcg ggccgggagg gttcgagaag ggggggcacc ccccttcggc gtgcgcggtc
     5221 acgcgcacag ggcgcagccc tggttaaaaa caaggtttat aaatattggt ttaaaagcag
     5281 gttaaaagac aggttagcgg tggccgaaaa acgggcggaa acccttgcaa atgctggatt
     5341 ttctgcctgt ggacagcccc tcaaatgtca ataggtgcgc ccctcatctg tcagcactct
     5401 gcccctcaag tgtcaaggat cgcgcccctc atctgtcagt agtcgcgccc ctcaagtgtc
     5461 aataccgcag ggcacttatc cccaggcttg tccacatcat ctgtgggaaa ctcgcgtaaa
     5521 atcaggcgtt ttcgccgatt tgcgaggctg gccagctcca cgtcgccggc cgaaatcgag
     5581 cctgcccctc atctgtcaac gccgcgccgg gtgagtcggc ccctcaagtg tcaacgtccg
     5641 cccctcatct gtcagtgagg gccaagtttt ccgcgaggta tccacaacgc cggcggccgc
     5701 ggtgtctcgc acacggcttc gacggcgttt ctggcgcgtt tgcagggcca tagacggccg
     5761 ccagcccagc ggcgagggca accagcccgg tgagcgtcgc aaaggagatc ctgatctgac
     5821 tgatgggctg cctgtatcga gtggtgattt tgtgccgagc tgccggtcgg ggagctgttg
     5881 gctggctggt ggcaggatat attgtggtgt aaacaaattg acgcttagac aacttaataa
     5941 cacattgcgg acgtttttaa tgtactgggg tggatgc
//
