LOCUS       sequence-377107-        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: pICSL1P1_crU626
FEATURES             Location/Qualifiers
     source          1..5977
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     ncRNA           324..360
                     /product="self-cleaving hammerhead ribozyme (Ferre-D'Amare 
                     and Scott, 2010)"
                     /label=hammerhead ribozyme
                     /ncRNA_class="hammerhead_ribozyme"
     protein_bind    489..510
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     promoter        join(525..530,531..548,549..555)
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    563..579
                     /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             597..608
                     /note="strong bacterial ribosome binding site (Elowitz and 
                     Leibler, 2000)"
     CDS             615..1292
                     /codon_start=1
                     /product="monomeric derivative of DsRed (Campbell et al., 
                     2002)"
                     /label=mRFP1
                     /translation="MASSEDVIKEFMRFKVRMEGSVNGHEFEIEGEGEGRPYEGTQTAK
                     LKVTKGGPLPFAWDILSPQFQYGSKAYVKHPADIPDYLKLSFPEGFKWERVMNFEDGGV
                     VTVTQDSSLQDGEFIYKVKLRGTNFPSDGPVMQKKTMGWEASTERMYPEDGALKGEIKM
                     RLKLKDGGHYDAEVKTTYMAKKPVQLPGAYKTDIKLDITSHNEDYTIVEQYERAEGRHS
                     TGA"
     terminator      1337..1408
                     /label=rrnB T1 terminator
                     /note="transcription terminator T1 from the E. coli rrnB 
                     gene"
     terminator      1424..1451
                     /label=T7Te terminator
                     /note="phage T7 early transcription terminator"
     ncRNA           1465..1532
                     /product="hepatitis delta virus self-cleaving ribozyme 
                     (Ferre-D'Amare and Scott, 2010)"
                     /label=HDV ribozyme
                     /ncRNA_class="ribozyme"
     misc_feature    1679..1703
                     /label=RB T-DNA repeat
                     /note="right border repeat from nopaline C58 T-DNA"
     CDS             complement(1859..3007)
                     /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(3343..3931)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(3423..3442)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(4102..4893,4894..4962))
                     /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     4725..4744
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(4963..5067)
                     /gene="bla"
                     /label=AmpR promoter
     rep_origin      5154..5785
                     /label=oriV
                     /note="incP origin of replication"
     misc_feature    5877..5901
                     /label=LB T-DNA repeat
                     /note="left border repeat from nopaline C58 T-DNA"
ORIGIN
        1 ctgtgaagac aatgccgaat tcggatccgg agtttttgta tcttgtttca tagtttgtcc
       61 caggattaga atgattaggc atcgaacctt caagaatttg attgaataaa acatcttcat
      121 tcttaagata tgaagataat cttcaaaagg cccctgggaa tctgaaagaa gagaagcagg
      181 cccatttata tgggaaagaa caatagtatt tcttatatag gcccatttaa gttgaaaaca
      241 atcttcaaaa gtcccacatc gcttagataa gaaaacgaag ctgagtttat atacagctag
      301 agtcgaagta gtgattgaaa ttactgatga gtccgtgagg acgaaacgag taagctcgtc
      361 taatttctac taagtgtaga ttgagaccca atacgcaaac cgcctctccc cgcgcgttgg
      421 ccgattcatt aatgcagctg gcacgacagg tttcccgact ggaaagcggg cagtgagcgc
      481 aacgcaatta atgtgagtta gctcactcat taggcacccc aggctttaca ctttatgctt
      541 ccggctcgta tgttgtgtgg aattgtgagc ggataacaat ttcacacata ctagagaaag
      601 aggagaaata ctagatggct tcctccgagg acgttatcaa agagttcatg cgtttcaaag
      661 ttcgtatgga aggttccgtt aacggtcacg agttcgaaat cgaaggtgaa ggtgaaggtc
      721 gtccgtacga aggtacccag accgctaaac tgaaagttac caaaggtggt ccgctgccgt
      781 tcgcttggga catcctgtcc ccgcagttcc agtacggttc caaagcttac gttaaacacc
      841 cggctgacat cccggactac ctgaaactgt ccttcccgga aggtttcaaa tgggaacgtg
      901 ttatgaactt cgaggacggt ggtgttgtta ccgttaccca ggactcctcc ctgcaagacg
      961 gtgagttcat ctacaaagtt aaactgcgtg gtaccaactt cccgtccgac ggtccggtta
     1021 tgcagaaaaa aaccatgggt tgggaagctt ccaccgaacg tatgtacccg gaggacggtg
     1081 ctctgaaagg tgaaatcaaa atgcgtctga aactgaaaga cggtggtcac tacgacgctg
     1141 aagttaaaac cacctacatg gctaaaaaac cggttcagct gccgggtgct tacaaaaccg
     1201 acatcaaact ggacatcacc tcccacaacg aggactacac catcgttgaa cagtacgaac
     1261 gtgctgaagg tcgtcactcc accggtgctt aataacgctg atagtgctag tgtagatcgc
     1321 tactagagcc aggcatcaaa taaaacgaaa ggctcagtcg aaagactggg cctttcgttt
     1381 tatctgttgt ttgtcggtga acgctctcta ctagagtcac actggctcac cttcgggtgg
     1441 gcctttctgc gtttataggt ctcaggccgg catggtccca gcctcctcgc tggcgccggc
     1501 tgggcaacat gcttcggcat ggcgaatggg actttttttg caaaattttc cagatcgatt
     1561 tcttcttcct ctgttcttcg gcgttcaatt tctggggttt tctcttcgtt ttctgtaact
     1621 gaaacctaaa attcgctgca attgtcttct gcacgaagtg gtttaaacta tcagtgtttg
     1681 acaggatata ttggcgggta aacctaagag aaaagagcgt ttattagaat aatcggatat
     1741 ttaaaagggc gtgaaaaggt ttatccgttc gtccatttgt atgtgcatgc caaccacagg
     1801 gttccccaga tcaggcgctg gctgctgaac ccccagccgg aactgacccc acaaggccct
     1861 agcgtttgca atgcaccagg tcatcattga cccaggcgtg ttccaccagg ccgctgcctc
     1921 gcaactcttc gcaggcttcg ccgacctgct cgcgccactt cttcacgcgg gtggaatccg
     1981 atccgcacat gaggcggaag gtttccagct tgagcgggta cggctcccgg tgcgagctga
     2041 aatagtcgaa catccgtcgg gccgtcggcg acagcttgcg gtacttctcc catatgaatt
     2101 tcgtgtagtg gtcgccagca aacagcacga cgatttcctc gtcgatcagg acctggcaac
     2161 gggacgtttt cttgccacgg tccaggacgc ggaagcggtg cagcagcgac accgattcca
     2221 ggtgcccaac gcggtcggac gtgaagccca tcgccgtcgc ctgtaggcgc gacaggcatt
     2281 cctcggcctt cgtgtaatac cggccattga tcgaccagcc caggtcctgg caaagctcgt
     2341 agaacgtgaa ggtgatcggc tcgccgatag gggtgcgctt cgcgtactcc aacacctgct
     2401 gccacaccag ttcgtcatcg tcggcccgca gctcgacgcc ggtgtaggtg atcttcacgt
     2461 ccttgttgac gtggaaaatg accttgtttt gcagcgcctc gcgcgggatt ttcttgttgc
     2521 gcgtggtgaa cagggcagag cgggccgtgt cgtttggcat cgctcgcatc gtgtccggcc
     2581 acggcgcaat atcgaacaag gaaagctgca tttccttgat ctgctgcttc gtgtgtttca
     2641 gcaacgcggc ctgcttggcc tcgctgacct gttttgccag gtcctcgccg gcggtttttc
     2701 gcttcttggt cgtcatagtt cctcgcgtgt cgatggtcat cgacttcgcc aaacctgccg
     2761 cctcctgttc aagacgacgc gaacgctcca cggcggccga tggcgcgggc agggcagggg
     2821 gagccagttg cacgctgtcg cgctcgatct tggccgtagc ttgctggacc atcgagccga
     2881 cggactggaa ggtttcgcgg ggcgcacgca tgacggtgcg gcttgcgatg gtttcggcat
     2941 cctcggcgga aaaccccgcg tcgatcagtt cttgcctgta tgccttccgg tcaaacgtcc
     3001 gattcattca ccctccttgc gggattgccc cgactcacgc cggggcaatg tgcccttatt
     3061 cctgatttga cccgcctggt gccttggtgt ccagataatc caccttatcg gcaatgaagt
     3121 cggtcccgta gaccgtctgg ccgtccttct cgtacttggt attccgaatc ttgccctgca
     3181 cgaataccag cgaccccttg cccaaatact tgccgtgggc ctcggcctga gagccaaaac
     3241 acttgatgcg gaagaagtcg gtgcgctcct gcttgtcgcc ggcatcgttg cgccacatct
     3301 aggatctgcc aggaaccgta aaaaggccgc gttgctggcg tttttccata ggctccgccc
     3361 ccctgacgag catcacaaaa atcgacgctc aagtcagagg tggcgaaacc cgacaggact
     3421 ataaagatac caggcgtttc cccctggaag ctccctcgtg cgctctcctg ttccgaccct
     3481 gccgcttacc ggatacctgt ccgcctttct cccttcggga agcgtggcgc tttctcatag
     3541 ctcacgctgt aggtatctca gttcggtgta ggtcgttcgc tccaagctgg gctgtgtgca
     3601 cgaacccccc gttcagcccg accgctgcgc cttatccggt aactatcgtc ttgagtccaa
     3661 cccggtaaga cacgacttat cgccactggc agcagccact ggtaacagga ttagcagagc
     3721 gaggtatgta ggcggtgcta cagagttctt gaagtggtgg cctaactacg gctacactag
     3781 aagaacagta tttggtatct gcgctctgct gaagccagtt accttcggaa aaagagttgg
     3841 tagctcttga tccggcaaac aaaccaccgc tggtagcggt ggtttttttg tttgcaagca
     3901 gcagattacg cgcagaaaaa aaggatctca agaagatcct ttgatctttt ctacggggtc
     3961 tgacgctcag tggaacgaaa actcacgtta agggattttg gtcatgagat tatcaaaaag
     4021 gatcttcacc tagatccttt taaattaaaa atgaagtttt aaatcaatct aaagtatata
     4081 tgagtaaact tggtctgaca gttaccaatg cttaatcagt gaggcaccta tctcagcgat
     4141 ctgtctattt cgttcatcca tagttgcctg actccccgtc gtgtagataa ctacgatacg
     4201 ggagggctta ccatctggcc ccagtgctgc aatgataccg cgagaaccac gctcaccggc
     4261 tccagattta tcagcaataa accagccagc cggaagggcc gagcgcagaa gtggtcctgc
     4321 aactttatcc gcctccatcc agtctattaa ttgttgccgg gaagctagag taagtagttc
     4381 gccagttaat agtttgcgca acgttgttgc cattgctaca ggcatcgtgg tgtcacgctc
     4441 gtcgtttggt atggcttcat tcagctccgg ttcccaacga tcaaggcgag ttacatgatc
     4501 ccccatgttg tgcaaaaaag cggttagctc cttcggtcct ccgatcgttg tcagaagtaa
     4561 gttggccgca gtgttatcac tcatggttat ggcagcactg cataattctc ttactgtcat
     4621 gccatccgta agatgctttt ctgtgactgg tgagtactca accaagtcat tctgagaata
     4681 gtgtatgcgg cgaccgagtt gctcttgccc ggcgtcaata cgggataata ccgcgccaca
     4741 tagcagaact ttaaaagtgc tcatcattgg aaaacgttct tcggggcgaa aactctcaag
     4801 gatcttaccg ctgttgagat ccagttcgat gtaacccact cgtgcaccca actgatcttc
     4861 agcatctttt actttcacca gcgtttctgg gtgagcaaaa acaggaaggc aaaatgccgc
     4921 aaaaaaggga ataagggcga cacggaaatg ttgaatactc atactcttcc tttttcaata
     4981 ttattgaagc atttatcaag gttattgtct catgagcgga tacatatttg aatgtattta
     5041 gaaaaataaa caaatagggg ttccgcgcac gaattggcca gcgctgccat ttttggggtg
     5101 aggccgttcg cggccgaggg gcgcagcccc tggggggatg ggaggcccgc gttagcgggc
     5161 cgggagggtt cgagaagggg gggcaccccc cttcggcgtg cgcggtcacg cgcacagggc
     5221 gcagccctgg ttaaaaacaa ggtttataaa tattggttta aaagcaggtt aaaagacagg
     5281 ttagcggtgg ccgaaaaacg ggcggaaacc cttgcaaatg ctggattttc tgcctgtgga
     5341 cagcccctca aatgtcaata ggtgcgcccc tcatctgtca gcactctgcc cctcaagtgt
     5401 caaggatcgc gcccctcatc tgtcagtagt cgcgcccctc aagtgtcaat accgcagggc
     5461 acttatcccc aggcttgtcc acatcatctg tgggaaactc gcgtaaaatc aggcgttttc
     5521 gccgatttgc gaggctggcc agctccacgt cgccggccga aatcgagcct gcccctcatc
     5581 tgtcaacgcc gcgccgggtg agtcggcccc tcaagtgtca acgtccgccc ctcatctgtc
     5641 agtgagggcc aagttttccg cgaggtatcc acaacgccgg cggccgcggt gtctcgcaca
     5701 cggcttcgac ggcgtttctg gcgcgtttgc agggccatag acggccgcca gcccagcggc
     5761 gagggcaacc agcccggtga gcgtcgcaaa ggagatcctg atctgactga tgggctgcct
     5821 gtatcgagtg gtgattttgt gccgagctgc cggtcgggga gctgttggct ggctggtggc
     5881 aggatatatt gtggtgtaaa caaattgacg cttagacaac ttaataacac attgcggacg
     5941 tttttaatgt actggggtgg atgcagtggg ccccact
//
