LOCUS       sequence-377101-        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: pICSL1P7_crU626
FEATURES             Location/Qualifiers
     source          1..5977
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     ncRNA           303..339
                     /product="self-cleaving hammerhead ribozyme (Ferre-D'Amare 
                     and Scott, 2010)"
                     /label=hammerhead ribozyme
                     /ncRNA_class="hammerhead_ribozyme"
     protein_bind    468..489
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     promoter        join(504..509,510..527,528..534)
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    542..558
                     /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             576..587
                     /note="strong bacterial ribosome binding site (Elowitz and 
                     Leibler, 2000)"
     CDS             594..1271
                     /codon_start=1
                     /product="monomeric derivative of DsRed (Campbell et al., 
                     2002)"
                     /label=mRFP1
                     /translation="MASSEDVIKEFMRFKVRMEGSVNGHEFEIEGEGEGRPYEGTQTAK
                     LKVTKGGPLPFAWDILSPQFQYGSKAYVKHPADIPDYLKLSFPEGFKWERVMNFEDGGV
                     VTVTQDSSLQDGEFIYKVKLRGTNFPSDGPVMQKKTMGWEASTERMYPEDGALKGEIKM
                     RLKLKDGGHYDAEVKTTYMAKKPVQLPGAYKTDIKLDITSHNEDYTIVEQYERAEGRHS
                     TGA"
     terminator      1316..1387
                     /label=rrnB T1 terminator
                     /note="transcription terminator T1 from the E. coli rrnB 
                     gene"
     terminator      1403..1430
                     /label=T7Te terminator
                     /note="phage T7 early transcription terminator"
     ncRNA           1444..1511
                     /product="hepatitis delta virus self-cleaving ribozyme 
                     (Ferre-D'Amare and Scott, 2010)"
                     /label=HDV ribozyme
                     /ncRNA_class="ribozyme"
     misc_feature    1658..1682
                     /label=RB T-DNA repeat
                     /note="right border repeat from nopaline C58 T-DNA"
     CDS             complement(1838..2986)
                     /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(3322..3910)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(3402..3421)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(4081..4872,4873..4941))
                     /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     4704..4723
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(4942..5046)
                     /gene="bla"
                     /label=AmpR promoter
     rep_origin      5133..5764
                     /label=oriV
                     /note="incP origin of replication"
     misc_feature    5856..5880
                     /label=LB T-DNA repeat
                     /note="left border repeat from nopaline C58 T-DNA"
ORIGIN
        1 ccatatggga gtttttgtat cttgtttcat agtttgtccc aggattagaa tgattaggca
       61 tcgaaccttc aagaatttga ttgaataaaa catcttcatt cttaagatat gaagataatc
      121 ttcaaaaggc ccctgggaat ctgaaagaag agaagcaggc ccatttatat gggaaagaac
      181 aatagtattt cttatatagg cccatttaag ttgaaaacaa tcttcaaaag tcccacatcg
      241 cttagataag aaaacgaagc tgagtttata tacagctaga gtcgaagtag tgattgaaat
      301 tactgatgag tccgtgagga cgaaacgagt aagctcgtct aatttctact aagtgtagat
      361 tgagacccaa tacgcaaacc gcctctcccc gcgcgttggc cgattcatta atgcagctgg
      421 cacgacaggt ttcccgactg gaaagcgggc agtgagcgca acgcaattaa tgtgagttag
      481 ctcactcatt aggcacccca ggctttacac tttatgcttc cggctcgtat gttgtgtgga
      541 attgtgagcg gataacaatt tcacacatac tagagaaaga ggagaaatac tagatggctt
      601 cctccgagga cgttatcaaa gagttcatgc gtttcaaagt tcgtatggaa ggttccgtta
      661 acggtcacga gttcgaaatc gaaggtgaag gtgaaggtcg tccgtacgaa ggtacccaga
      721 ccgctaaact gaaagttacc aaaggtggtc cgctgccgtt cgcttgggac atcctgtccc
      781 cgcagttcca gtacggttcc aaagcttacg ttaaacaccc ggctgacatc ccggactacc
      841 tgaaactgtc cttcccggaa ggtttcaaat gggaacgtgt tatgaacttc gaggacggtg
      901 gtgttgttac cgttacccag gactcctccc tgcaagacgg tgagttcatc tacaaagtta
      961 aactgcgtgg taccaacttc ccgtccgacg gtccggttat gcagaaaaaa accatgggtt
     1021 gggaagcttc caccgaacgt atgtacccgg aggacggtgc tctgaaaggt gaaatcaaaa
     1081 tgcgtctgaa actgaaagac ggtggtcact acgacgctga agttaaaacc acctacatgg
     1141 ctaaaaaacc ggttcagctg ccgggtgctt acaaaaccga catcaaactg gacatcacct
     1201 cccacaacga ggactacacc atcgttgaac agtacgaacg tgctgaaggt cgtcactcca
     1261 ccggtgctta ataacgctga tagtgctagt gtagatcgct actagagcca ggcatcaaat
     1321 aaaacgaaag gctcagtcga aagactgggc ctttcgtttt atctgttgtt tgtcggtgaa
     1381 cgctctctac tagagtcaca ctggctcacc ttcgggtggg cctttctgcg tttataggtc
     1441 tcaggccggc atggtcccag cctcctcgct ggcgccggct gggcaacatg cttcggcatg
     1501 gcgaatggga ctttttttgc aaaattttcc agatcgattt cttcttcctc tgttcttcgg
     1561 cgttcaattt ctggggtttt ctcttcgttt tctgtaactg aaacctaaaa ttcgcttgcc
     1621 ttgtcttctg cacgaagtgg tttaaactat cagtgtttga caggatatat tggcgggtaa
     1681 acctaagaga aaagagcgtt tattagaata atcggatatt taaaagggcg tgaaaaggtt
     1741 tatccgttcg tccatttgta tgtgcatgcc aaccacaggg ttccccagat caggcgctgg
     1801 ctgctgaacc cccagccgga actgacccca caaggcccta gcgtttgcaa tgcaccaggt
     1861 catcattgac ccaggcgtgt tccaccaggc cgctgcctcg caactcttcg caggcttcgc
     1921 cgacctgctc gcgccacttc ttcacgcggg tggaatccga tccgcacatg aggcggaagg
     1981 tttccagctt gagcgggtac ggctcccggt gcgagctgaa atagtcgaac atccgtcggg
     2041 ccgtcggcga cagcttgcgg tacttctccc atatgaattt cgtgtagtgg tcgccagcaa
     2101 acagcacgac gatttcctcg tcgatcagga cctggcaacg ggacgttttc ttgccacggt
     2161 ccaggacgcg gaagcggtgc agcagcgaca ccgattccag gtgcccaacg cggtcggacg
     2221 tgaagcccat cgccgtcgcc tgtaggcgcg acaggcattc ctcggccttc gtgtaatacc
     2281 ggccattgat cgaccagccc aggtcctggc aaagctcgta gaacgtgaag gtgatcggct
     2341 cgccgatagg ggtgcgcttc gcgtactcca acacctgctg ccacaccagt tcgtcatcgt
     2401 cggcccgcag ctcgacgccg gtgtaggtga tcttcacgtc cttgttgacg tggaaaatga
     2461 ccttgttttg cagcgcctcg cgcgggattt tcttgttgcg cgtggtgaac agggcagagc
     2521 gggccgtgtc gtttggcatc gctcgcatcg tgtccggcca cggcgcaata tcgaacaagg
     2581 aaagctgcat ttccttgatc tgctgcttcg tgtgtttcag caacgcggcc tgcttggcct
     2641 cgctgacctg ttttgccagg tcctcgccgg cggtttttcg cttcttggtc gtcatagttc
     2701 ctcgcgtgtc gatggtcatc gacttcgcca aacctgccgc ctcctgttca agacgacgcg
     2761 aacgctccac ggcggccgat ggcgcgggca gggcaggggg agccagttgc acgctgtcgc
     2821 gctcgatctt ggccgtagct tgctggacca tcgagccgac ggactggaag gtttcgcggg
     2881 gcgcacgcat gacggtgcgg cttgcgatgg tttcggcatc ctcggcggaa aaccccgcgt
     2941 cgatcagttc ttgcctgtat gccttccggt caaacgtccg attcattcac cctccttgcg
     3001 ggattgcccc gactcacgcc ggggcaatgt gcccttattc ctgatttgac ccgcctggtg
     3061 ccttggtgtc cagataatcc accttatcgg caatgaagtc ggtcccgtag accgtctggc
     3121 cgtccttctc gtacttggta ttccgaatct tgccctgcac gaataccagc gaccccttgc
     3181 ccaaatactt gccgtgggcc tcggcctgag agccaaaaca cttgatgcgg aagaagtcgg
     3241 tgcgctcctg cttgtcgccg gcatcgttgc gccacatcta ggatctgcca ggaaccgtaa
     3301 aaaggccgcg ttgctggcgt ttttccatag gctccgcccc cctgacgagc atcacaaaaa
     3361 tcgacgctca agtcagaggt ggcgaaaccc gacaggacta taaagatacc aggcgtttcc
     3421 ccctggaagc tccctcgtgc gctctcctgt tccgaccctg ccgcttaccg gatacctgtc
     3481 cgcctttctc ccttcgggaa gcgtggcgct ttctcatagc tcacgctgta ggtatctcag
     3541 ttcggtgtag gtcgttcgct ccaagctggg ctgtgtgcac gaaccccccg ttcagcccga
     3601 ccgctgcgcc ttatccggta actatcgtct tgagtccaac ccggtaagac acgacttatc
     3661 gccactggca gcagccactg gtaacaggat tagcagagcg aggtatgtag gcggtgctac
     3721 agagttcttg aagtggtggc ctaactacgg ctacactaga agaacagtat ttggtatctg
     3781 cgctctgctg aagccagtta ccttcggaaa aagagttggt agctcttgat ccggcaaaca
     3841 aaccaccgct ggtagcggtg gtttttttgt ttgcaagcag cagattacgc gcagaaaaaa
     3901 aggatctcaa gaagatcctt tgatcttttc tacggggtct gacgctcagt ggaacgaaaa
     3961 ctcacgttaa gggattttgg tcatgagatt atcaaaaagg atcttcacct agatcctttt
     4021 aaattaaaaa tgaagtttta aatcaatcta aagtatatat gagtaaactt ggtctgacag
     4081 ttaccaatgc ttaatcagtg aggcacctat ctcagcgatc tgtctatttc gttcatccat
     4141 agttgcctga ctccccgtcg tgtagataac tacgatacgg gagggcttac catctggccc
     4201 cagtgctgca atgataccgc gagaaccacg ctcaccggct ccagatttat cagcaataaa
     4261 ccagccagcc ggaagggccg agcgcagaag tggtcctgca actttatccg cctccatcca
     4321 gtctattaat tgttgccggg aagctagagt aagtagttcg ccagttaata gtttgcgcaa
     4381 cgttgttgcc attgctacag gcatcgtggt gtcacgctcg tcgtttggta tggcttcatt
     4441 cagctccggt tcccaacgat caaggcgagt tacatgatcc cccatgttgt gcaaaaaagc
     4501 ggttagctcc ttcggtcctc cgatcgttgt cagaagtaag ttggccgcag tgttatcact
     4561 catggttatg gcagcactgc ataattctct tactgtcatg ccatccgtaa gatgcttttc
     4621 tgtgactggt gagtactcaa ccaagtcatt ctgagaatag tgtatgcggc gaccgagttg
     4681 ctcttgcccg gcgtcaatac gggataatac cgcgccacat agcagaactt taaaagtgct
     4741 catcattgga aaacgttctt cggggcgaaa actctcaagg atcttaccgc tgttgagatc
     4801 cagttcgatg taacccactc gtgcacccaa ctgatcttca gcatctttta ctttcaccag
     4861 cgtttctggg tgagcaaaaa caggaaggca aaatgccgca aaaaagggaa taagggcgac
     4921 acggaaatgt tgaatactca tactcttcct ttttcaatat tattgaagca tttatcaagg
     4981 ttattgtctc atgagcggat acatatttga atgtatttag aaaaataaac aaataggggt
     5041 tccgcgcacg aattggccag cgctgccatt tttggggtga ggccgttcgc ggccgagggg
     5101 cgcagcccct ggggggatgg gaggcccgcg ttagcgggcc gggagggttc gagaaggggg
     5161 ggcacccccc ttcggcgtgc gcggtcacgc gcacagggcg cagccctggt taaaaacaag
     5221 gtttataaat attggtttaa aagcaggtta aaagacaggt tagcggtggc cgaaaaacgg
     5281 gcggaaaccc ttgcaaatgc tggattttct gcctgtggac agcccctcaa atgtcaatag
     5341 gtgcgcccct catctgtcag cactctgccc ctcaagtgtc aaggatcgcg cccctcatct
     5401 gtcagtagtc gcgcccctca agtgtcaata ccgcagggca cttatcccca ggcttgtcca
     5461 catcatctgt gggaaactcg cgtaaaatca ggcgttttcg ccgatttgcg aggctggcca
     5521 gctccacgtc gccggccgaa atcgagcctg cccctcatct gtcaacgccg cgccgggtga
     5581 gtcggcccct caagtgtcaa cgtccgcccc tcatctgtca gtgagggcca agttttccgc
     5641 gaggtatcca caacgccggc ggccgcggtg tctcgcacac ggcttcgacg gcgtttctgg
     5701 cgcgtttgca gggccataga cggccgccag cccagcggcg agggcaacca gcccggtgag
     5761 cgtcgcaaag gagatcctga tctgactgat gggctgcctg tatcgagtgg tgattttgtg
     5821 ccgagctgcc ggtcggggag ctgttggctg gctggtggca ggatatattg tggtgtaaac
     5881 aaattgacgc ttagacaact taataacaca ttgcggacgt ttttaatgta ctggggtgga
     5941 tgcagtgggc cccactctgt gaagacaaga gcgaatt
//
