LOCUS       sequence-377105-        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: pICSL1P3_crU626
FEATURES             Location/Qualifiers
     source          1..5977
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     ncRNA           344..380
                     /product="self-cleaving hammerhead ribozyme (Ferre-D'Amare 
                     and Scott, 2010)"
                     /label=hammerhead ribozyme
                     /ncRNA_class="hammerhead_ribozyme"
     protein_bind    509..530
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     promoter        join(545..550,551..568,569..575)
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    583..599
                     /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             617..628
                     /note="strong bacterial ribosome binding site (Elowitz and 
                     Leibler, 2000)"
     CDS             635..1312
                     /codon_start=1
                     /product="monomeric derivative of DsRed (Campbell et al., 
                     2002)"
                     /label=mRFP1
                     /translation="MASSEDVIKEFMRFKVRMEGSVNGHEFEIEGEGEGRPYEGTQTAK
                     LKVTKGGPLPFAWDILSPQFQYGSKAYVKHPADIPDYLKLSFPEGFKWERVMNFEDGGV
                     VTVTQDSSLQDGEFIYKVKLRGTNFPSDGPVMQKKTMGWEASTERMYPEDGALKGEIKM
                     RLKLKDGGHYDAEVKTTYMAKKPVQLPGAYKTDIKLDITSHNEDYTIVEQYERAEGRHS
                     TGA"
     terminator      1357..1428
                     /label=rrnB T1 terminator
                     /note="transcription terminator T1 from the E. coli rrnB 
                     gene"
     terminator      1444..1471
                     /label=T7Te terminator
                     /note="phage T7 early transcription terminator"
     ncRNA           1485..1552
                     /product="hepatitis delta virus self-cleaving ribozyme 
                     (Ferre-D'Amare and Scott, 2010)"
                     /label=HDV ribozyme
                     /ncRNA_class="ribozyme"
     misc_feature    1699..1723
                     /label=RB T-DNA repeat
                     /note="right border repeat from nopaline C58 T-DNA"
     CDS             complement(1879..3027)
                     /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(3363..3951)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(3443..3462)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(4122..4913,4914..4982))
                     /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     4745..4764
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(4983..5087)
                     /gene="bla"
                     /label=AmpR promoter
     rep_origin      5174..5805
                     /label=oriV
                     /note="incP origin of replication"
     misc_feature    5897..5921
                     /label=LB T-DNA repeat
                     /note="left border repeat from nopaline C58 T-DNA"
ORIGIN
        1 tggatgcagt gggccccact ctgtgaagac aaactagaat tcgagctcgg agtttttgta
       61 tcttgtttca tagtttgtcc caggattaga atgattaggc atcgaacctt caagaatttg
      121 attgaataaa acatcttcat tcttaagata tgaagataat cttcaaaagg cccctgggaa
      181 tctgaaagaa gagaagcagg cccatttata tgggaaagaa caatagtatt tcttatatag
      241 gcccatttaa gttgaaaaca atcttcaaaa gtcccacatc gcttagataa gaaaacgaag
      301 ctgagtttat atacagctag agtcgaagta gtgattgaaa ttactgatga gtccgtgagg
      361 acgaaacgag taagctcgtc taatttctac taagtgtaga ttgagaccca atacgcaaac
      421 cgcctctccc cgcgcgttgg ccgattcatt aatgcagctg gcacgacagg tttcccgact
      481 ggaaagcggg cagtgagcgc aacgcaatta atgtgagtta gctcactcat taggcacccc
      541 aggctttaca ctttatgctt ccggctcgta tgttgtgtgg aattgtgagc ggataacaat
      601 ttcacacata ctagagaaag aggagaaata ctagatggct tcctccgagg acgttatcaa
      661 agagttcatg cgtttcaaag ttcgtatgga aggttccgtt aacggtcacg agttcgaaat
      721 cgaaggtgaa ggtgaaggtc gtccgtacga aggtacccag accgctaaac tgaaagttac
      781 caaaggtggt ccgctgccgt tcgcttggga catcctgtcc ccgcagttcc agtacggttc
      841 caaagcttac gttaaacacc cggctgacat cccggactac ctgaaactgt ccttcccgga
      901 aggtttcaaa tgggaacgtg ttatgaactt cgaggacggt ggtgttgtta ccgttaccca
      961 ggactcctcc ctgcaagacg gtgagttcat ctacaaagtt aaactgcgtg gtaccaactt
     1021 cccgtccgac ggtccggtta tgcagaaaaa aaccatgggt tgggaagctt ccaccgaacg
     1081 tatgtacccg gaggacggtg ctctgaaagg tgaaatcaaa atgcgtctga aactgaaaga
     1141 cggtggtcac tacgacgctg aagttaaaac cacctacatg gctaaaaaac cggttcagct
     1201 gccgggtgct tacaaaaccg acatcaaact ggacatcacc tcccacaacg aggactacac
     1261 catcgttgaa cagtacgaac gtgctgaagg tcgtcactcc accggtgctt aataacgctg
     1321 atagtgctag tgtagatcgc tactagagcc aggcatcaaa taaaacgaaa ggctcagtcg
     1381 aaagactggg cctttcgttt tatctgttgt ttgtcggtga acgctctcta ctagagtcac
     1441 actggctcac cttcgggtgg gcctttctgc gtttataggt ctcaggccgg catggtccca
     1501 gcctcctcgc tggcgccggc tgggcaacat gcttcggcat ggcgaatggg actttttttg
     1561 caaaattttc cagatcgatt tcttcttcct ctgttcttcg gcgttcaatt tctggggttt
     1621 tctcttcgtt ttctgtaact gaaacctaaa attcgcttta cttgtcttct gcacgaagtg
     1681 gtttaaacta tcagtgtttg acaggatata ttggcgggta aacctaagag aaaagagcgt
     1741 ttattagaat aatcggatat ttaaaagggc gtgaaaaggt ttatccgttc gtccatttgt
     1801 atgtgcatgc caaccacagg gttccccaga tcaggcgctg gctgctgaac ccccagccgg
     1861 aactgacccc acaaggccct agcgtttgca atgcaccagg tcatcattga cccaggcgtg
     1921 ttccaccagg ccgctgcctc gcaactcttc gcaggcttcg ccgacctgct cgcgccactt
     1981 cttcacgcgg gtggaatccg atccgcacat gaggcggaag gtttccagct tgagcgggta
     2041 cggctcccgg tgcgagctga aatagtcgaa catccgtcgg gccgtcggcg acagcttgcg
     2101 gtacttctcc catatgaatt tcgtgtagtg gtcgccagca aacagcacga cgatttcctc
     2161 gtcgatcagg acctggcaac gggacgtttt cttgccacgg tccaggacgc ggaagcggtg
     2221 cagcagcgac accgattcca ggtgcccaac gcggtcggac gtgaagccca tcgccgtcgc
     2281 ctgtaggcgc gacaggcatt cctcggcctt cgtgtaatac cggccattga tcgaccagcc
     2341 caggtcctgg caaagctcgt agaacgtgaa ggtgatcggc tcgccgatag gggtgcgctt
     2401 cgcgtactcc aacacctgct gccacaccag ttcgtcatcg tcggcccgca gctcgacgcc
     2461 ggtgtaggtg atcttcacgt ccttgttgac gtggaaaatg accttgtttt gcagcgcctc
     2521 gcgcgggatt ttcttgttgc gcgtggtgaa cagggcagag cgggccgtgt cgtttggcat
     2581 cgctcgcatc gtgtccggcc acggcgcaat atcgaacaag gaaagctgca tttccttgat
     2641 ctgctgcttc gtgtgtttca gcaacgcggc ctgcttggcc tcgctgacct gttttgccag
     2701 gtcctcgccg gcggtttttc gcttcttggt cgtcatagtt cctcgcgtgt cgatggtcat
     2761 cgacttcgcc aaacctgccg cctcctgttc aagacgacgc gaacgctcca cggcggccga
     2821 tggcgcgggc agggcagggg gagccagttg cacgctgtcg cgctcgatct tggccgtagc
     2881 ttgctggacc atcgagccga cggactggaa ggtttcgcgg ggcgcacgca tgacggtgcg
     2941 gcttgcgatg gtttcggcat cctcggcgga aaaccccgcg tcgatcagtt cttgcctgta
     3001 tgccttccgg tcaaacgtcc gattcattca ccctccttgc gggattgccc cgactcacgc
     3061 cggggcaatg tgcccttatt cctgatttga cccgcctggt gccttggtgt ccagataatc
     3121 caccttatcg gcaatgaagt cggtcccgta gaccgtctgg ccgtccttct cgtacttggt
     3181 attccgaatc ttgccctgca cgaataccag cgaccccttg cccaaatact tgccgtgggc
     3241 ctcggcctga gagccaaaac acttgatgcg gaagaagtcg gtgcgctcct gcttgtcgcc
     3301 ggcatcgttg cgccacatct aggatctgcc aggaaccgta aaaaggccgc gttgctggcg
     3361 tttttccata ggctccgccc ccctgacgag catcacaaaa atcgacgctc aagtcagagg
     3421 tggcgaaacc cgacaggact ataaagatac caggcgtttc cccctggaag ctccctcgtg
     3481 cgctctcctg ttccgaccct gccgcttacc ggatacctgt ccgcctttct cccttcggga
     3541 agcgtggcgc tttctcatag ctcacgctgt aggtatctca gttcggtgta ggtcgttcgc
     3601 tccaagctgg gctgtgtgca cgaacccccc gttcagcccg accgctgcgc cttatccggt
     3661 aactatcgtc ttgagtccaa cccggtaaga cacgacttat cgccactggc agcagccact
     3721 ggtaacagga ttagcagagc gaggtatgta ggcggtgcta cagagttctt gaagtggtgg
     3781 cctaactacg gctacactag aagaacagta tttggtatct gcgctctgct gaagccagtt
     3841 accttcggaa aaagagttgg tagctcttga tccggcaaac aaaccaccgc tggtagcggt
     3901 ggtttttttg tttgcaagca gcagattacg cgcagaaaaa aaggatctca agaagatcct
     3961 ttgatctttt ctacggggtc tgacgctcag tggaacgaaa actcacgtta agggattttg
     4021 gtcatgagat tatcaaaaag gatcttcacc tagatccttt taaattaaaa atgaagtttt
     4081 aaatcaatct aaagtatata tgagtaaact tggtctgaca gttaccaatg cttaatcagt
     4141 gaggcaccta tctcagcgat ctgtctattt cgttcatcca tagttgcctg actccccgtc
     4201 gtgtagataa ctacgatacg ggagggctta ccatctggcc ccagtgctgc aatgataccg
     4261 cgagaaccac gctcaccggc tccagattta tcagcaataa accagccagc cggaagggcc
     4321 gagcgcagaa gtggtcctgc aactttatcc gcctccatcc agtctattaa ttgttgccgg
     4381 gaagctagag taagtagttc gccagttaat agtttgcgca acgttgttgc cattgctaca
     4441 ggcatcgtgg tgtcacgctc gtcgtttggt atggcttcat tcagctccgg ttcccaacga
     4501 tcaaggcgag ttacatgatc ccccatgttg tgcaaaaaag cggttagctc cttcggtcct
     4561 ccgatcgttg tcagaagtaa gttggccgca gtgttatcac tcatggttat ggcagcactg
     4621 cataattctc ttactgtcat gccatccgta agatgctttt ctgtgactgg tgagtactca
     4681 accaagtcat tctgagaata gtgtatgcgg cgaccgagtt gctcttgccc ggcgtcaata
     4741 cgggataata ccgcgccaca tagcagaact ttaaaagtgc tcatcattgg aaaacgttct
     4801 tcggggcgaa aactctcaag gatcttaccg ctgttgagat ccagttcgat gtaacccact
     4861 cgtgcaccca actgatcttc agcatctttt actttcacca gcgtttctgg gtgagcaaaa
     4921 acaggaaggc aaaatgccgc aaaaaaggga ataagggcga cacggaaatg ttgaatactc
     4981 atactcttcc tttttcaata ttattgaagc atttatcaag gttattgtct catgagcgga
     5041 tacatatttg aatgtattta gaaaaataaa caaatagggg ttccgcgcac gaattggcca
     5101 gcgctgccat ttttggggtg aggccgttcg cggccgaggg gcgcagcccc tggggggatg
     5161 ggaggcccgc gttagcgggc cgggagggtt cgagaagggg gggcaccccc cttcggcgtg
     5221 cgcggtcacg cgcacagggc gcagccctgg ttaaaaacaa ggtttataaa tattggttta
     5281 aaagcaggtt aaaagacagg ttagcggtgg ccgaaaaacg ggcggaaacc cttgcaaatg
     5341 ctggattttc tgcctgtgga cagcccctca aatgtcaata ggtgcgcccc tcatctgtca
     5401 gcactctgcc cctcaagtgt caaggatcgc gcccctcatc tgtcagtagt cgcgcccctc
     5461 aagtgtcaat accgcagggc acttatcccc aggcttgtcc acatcatctg tgggaaactc
     5521 gcgtaaaatc aggcgttttc gccgatttgc gaggctggcc agctccacgt cgccggccga
     5581 aatcgagcct gcccctcatc tgtcaacgcc gcgccgggtg agtcggcccc tcaagtgtca
     5641 acgtccgccc ctcatctgtc agtgagggcc aagttttccg cgaggtatcc acaacgccgg
     5701 cggccgcggt gtctcgcaca cggcttcgac ggcgtttctg gcgcgtttgc agggccatag
     5761 acggccgcca gcccagcggc gagggcaacc agcccggtga gcgtcgcaaa ggagatcctg
     5821 atctgactga tgggctgcct gtatcgagtg gtgattttgt gccgagctgc cggtcgggga
     5881 gctgttggct ggctggtggc aggatatatt gtggtgtaaa caaattgacg cttagacaac
     5941 ttaataacac attgcggacg tttttaatgt actgggg
//
