LOCUS       sequence-377111-        5812 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Cas9 guide expression in dicots.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 5812)
  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 5812)
  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: pICSL1P4_AtU626Ter26
FEATURES             Location/Qualifiers
     source          1..5812
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     protein_bind    362..383
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     promoter        join(398..403,404..421,422..428)
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    436..452
                     /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             470..481
                     /note="strong bacterial ribosome binding site (Elowitz and 
                     Leibler, 2000)"
     terminator      1210..1281
                     /label=rrnB T1 terminator
                     /note="transcription terminator T1 from the E. coli rrnB 
                     gene"
     terminator      1297..1324
                     /label=T7Te terminator
                     /note="phage T7 early transcription terminator"
     misc_RNA        1338..1413
                     /label=gRNA scaffold
                     /note="guide RNA scaffold for the Streptococcus pyogenes 
                     CRISPR/Cas9 system"
     misc_feature    1525..1549
                     /label=RB T-DNA repeat
                     /note="right border repeat from nopaline C58 T-DNA"
     CDS             complement(1705..2853)
                     /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(3189..3777)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(3269..3288)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(3948..4739,4740..4808))
                     /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     4571..4590
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(4809..4913)
                     /gene="bla"
                     /label=AmpR promoter
     rep_origin      5000..5631
                     /label=oriV
                     /note="incP origin of replication"
     misc_feature    5723..5747
                     /label=LB T-DNA repeat
                     /note="left border repeat from nopaline C58 T-DNA"
ORIGIN
        1 tgggccccac tctgtgaaga caattacgaa ttcccatggg gagcatcttc attcttaaga
       61 tatgaagata atcttcaaaa ggcccctggg aatctgaaag aagagaagca ggcccattta
      121 tatgggaaag aacaatagta tttcttatat aggcccattt aagttgaaaa caatcttcaa
      181 aagtcccaca tcgcttagat aagaaaacga agctgagttt atatacagct agagtcgaag
      241 tagtgattgt gagaccgata tcaatacgca aaccgcctct ccccgcgcgt tggccgattc
      301 attaatgcag ctggcacgac aggtttcccg actggaaagc gggcagtgag cgcaacgcaa
      361 ttaatgtgag ttagctcact cattaggcac cccaggcttt acactttatg cttccggctc
      421 gtatgttgtg tggaattgtg agcggataac aatttcacac atactagaga aagaggagaa
      481 atactagatg gcttcctccg aggacgttat caaagagttc atgcgtttca aagttcgtat
      541 ggaaggttcc gttaacggtc acgagttcga aatcgaaggt gaaggtgaag gtcgtccgta
      601 cgaaggtacc cagaccgcta aactgaaagt taccaaaggt ggtccgctgc cgttcgcttg
      661 ggacatcctg tccccgcagt tccagtacgg ttccaaagct tacgttaaac acccggctga
      721 catcccggac tacctgaaac tgtccttccc ggaaggtttc aaatgggaac gtgttatgaa
      781 cttcgaggac ggtggtgttg ttaccgttac ccaggactcc tccctgcaag acggtgagtt
      841 catctacaaa gttaaactgc gtggtaccaa cttcccgtcc gacggtccgg ttatgcagaa
      901 aaaaaccatg ggttgggaag cttccaccga acgtatgtac ccggaggacg gtgctctgaa
      961 aggtgaaatc aaaatgcgtc tgaaactgaa agacggtggt cactacgacg ctgaagttaa
     1021 aaccacctac atggctaaaa aaccggttca gctgccgggt gcttacaaaa ccgacatcaa
     1081 actggacatc acctcccaca acaaggacta caccatcgtt gaacagtacg aacgtgctga
     1141 aggtcgtcac tccaccggtg cttaataacg ctgatagtgc tagtgtagat cgctactaga
     1201 gccaggcatc aaataaaatg aaaggctcag tcgaaagact gggcctttcg ttttatctgt
     1261 tgtttgtcgg tgaacgctct ctactagagt cacactggct caccttcggg tgggcctttc
     1321 tgcgtttata ggtctcagtt ttagagctag aaatagcaag ttaaaataag gctagtccgt
     1381 tatcaacttg aaaaagtggc accgagtcgg tgcttttttt tgcaaaattt tccagatcga
     1441 tttcttcttc ctctgttctt cggcgttcaa tttctggggc gctcagattg tcttctgcac
     1501 gaagtggttt aaactatcag tgtttgacag gatatattgg cgggtaaacc taagagaaaa
     1561 gagcgtttat tagaataatc ggatatttaa aagggcgtga aaaggtttat ccgttcgtcc
     1621 atttgtatgt gcatgccaac cacagggttc cccagatcag gcgctggctg ctgaaccccc
     1681 agccggaact gaccccacaa ggccctagcg tttgcaatgc accaggtcat cattgaccca
     1741 ggcgtgttcc accaggccgc tgcctcgcaa ctcttcgcag gcttcgccga cctgctcgcg
     1801 ccacttcttc acgcgggtgg aatccgatcc gcacatgagg cggaaggttt ccagcttgag
     1861 cgggtacggc tcccggtgcg agctgaaata gtcgaacatc cgtcgggccg tcggcgacag
     1921 cttgcggtac ttctcccata tgaatttcgt gtagtggtcg ccagcaaaca gcacgacgat
     1981 ttcctcgtcg atcaggacct ggcaacggga cgttttcttg ccacggtcca ggacgcggaa
     2041 gcggtgcagc agcgacaccg attccaggtg cccaacgcgg tcggacgtga agcccatcgc
     2101 cgtcgcctgt aggcgcgaca ggcattcctc ggccttcgtg taataccggc cattgatcga
     2161 ccagcccagg tcctggcaaa gctcgtagaa cgtgaaggtg atcggctcgc cgataggggt
     2221 gcgcttcgcg tactccaaca cctgctgcca caccagttcg tcatcgtcgg cccgcagctc
     2281 gacgccggtg taggtgatct tcacgtcctt gttgacgtgg aaaatgacct tgttttgcag
     2341 cgcctcgcgc gggattttct tgttgcgcgt ggtgaacagg gcagagcggg ccgtgtcgtt
     2401 tggcatcgct cgcatcgtgt ccggccacgg cgcaatatcg aacaaggaaa gctgcatttc
     2461 cttgatctgc tgcttcgtgt gtttcagcaa cgcggcctgc ttggcctcgc tgacctgttt
     2521 tgccaggtcc tcgccggcgg tttttcgctt cttggtcgtc atagttcctc gcgtgtcgat
     2581 ggtcatcgac ttcgccaaac ctgccgcctc ctgttcaaga cgacgcgaac gctccacggc
     2641 ggccgatggc gcgggcaggg cagggggagc cagttgcacg ctgtcgcgct cgatcttggc
     2701 cgtagcttgc tggaccatcg agccgacgga ctggaaggtt tcgcggggcg cacgcatgac
     2761 ggtgcggctt gcgatggttt cggcatcctc ggcggaaaac cccgcgtcga tcagttcttg
     2821 cctgtatgcc ttccggtcaa acgtccgatt cattcaccct ccttgcggga ttgccccgac
     2881 tcacgccggg gcaatgtgcc cttattcctg atttgacccg cctggtgcct tggtgtccag
     2941 ataatccacc ttatcggcaa tgaagtcggt cccgtagacc gtctggccgt ccttctcgta
     3001 cttggtattc cgaatcttgc cctgcacgaa taccagcgac cccttgccca aatacttgcc
     3061 gtgggcctcg gcctgagagc caaaacactt gatgcggaag aagtcggtgc gctcctgctt
     3121 gtcgccggca tcgttgcgcc acatctagga tctgccagga accgtaaaaa ggccgcgttg
     3181 ctggcgtttt tccataggct ccgcccccct gacgagcatc acaaaaatcg acgctcaagt
     3241 cagaggtggc gaaacccgac aggactataa agataccagg cgtttccccc tggaagctcc
     3301 ctcgtgcgct ctcctgttcc gaccctgccg cttaccggat acctgtccgc ctttctccct
     3361 tcgggaagcg tggcgctttc tcatagctca cgctgtaggt atctcagttc ggtgtaggtc
     3421 gttcgctcca agctgggctg tgtgcacgaa ccccccgttc agcccgaccg ctgcgcctta
     3481 tccggtaact atcgtcttga gtccaacccg gtaagacacg acttatcgcc actggcagca
     3541 gccactggta acaggattag cagagcgagg tatgtaggcg gtgctacaga gttcttgaag
     3601 tggtggccta actacggcta cactagaaga acagtatttg gtatctgcgc tctgctgaag
     3661 ccagttacct tcggaaaaag agttggtagc tcttgatccg gcaaacaaac caccgctggt
     3721 agcggtggtt tttttgtttg caagcagcag attacgcgca gaaaaaaagg atctcaagaa
     3781 gatcctttga tcttttctac ggggtctgac gctcagtgga acgaaaactc acgttaaggg
     3841 attttggtca tgagattatc aaaaaggatc ttcacctaga tccttttaaa ttaaaaatga
     3901 agttttaaat caatctaaag tatatatgag taaacttggt ctgacagtta ccaatgctta
     3961 atcagtgagg cacctatctc agcgatctgt ctatttcgtt catccatagt tgcctgactc
     4021 cccgtcgtgt agataactac gatacgggag ggcttaccat ctggccccag tgctgcaatg
     4081 ataccgcgag aaccacgctc accggctcca gatttatcag caataaacca gccagccgga
     4141 agggccgagc gcagaagtgg tcctgcaact ttatccgcct ccatccagtc tattaattgt
     4201 tgccgggaag ctagagtaag tagttcgcca gttaatagtt tgcgcaacgt tgttgccatt
     4261 gctacaggca tcgtggtgtc acgctcgtcg tttggtatgg cttcattcag ctccggttcc
     4321 caacgatcaa ggcgagttac atgatccccc atgttgtgca aaaaagcggt tagctccttc
     4381 ggtcctccga tcgttgtcag aagtaagttg gccgcagtgt tatcactcat ggttatggca
     4441 gcactgcata attctcttac tgtcatgcca tccgtaagat gcttttctgt gactggtgag
     4501 tactcaacca agtcattctg agaatagtgt atgcggcgac cgagttgctc ttgcccggcg
     4561 tcaatacggg ataataccgc gccacatagc agaactttaa aagtgctcat cattggaaaa
     4621 cgttcttcgg ggcgaaaact ctcaaggatc ttaccgctgt tgagatccag ttcgatgtaa
     4681 cccactcgtg cacccaactg atcttcagca tcttttactt tcaccagcgt ttctgggtga
     4741 gcaaaaacag gaaggcaaaa tgccgcaaaa aagggaataa gggcgacacg gaaatgttga
     4801 atactcatac tcttcctttt tcaatattat tgaagcattt atcaaggtta ttgtctcatg
     4861 agcggataca tatttgaatg tatttagaaa aataaacaaa taggggttcc gcgcacgaat
     4921 tggccagcgc tgccattttt ggggtgaggc cgttcgcggc cgaggggcgc agcccctggg
     4981 gggatgggag gcccgcgtta gcgggccggg agggttcgag aagggggggc accccccttc
     5041 ggcgtgcgcg gtcacgcgca cagggcgcag ccctggttaa aaacaaggtt tataaatatt
     5101 ggtttaaaag caggttaaaa gacaggttag cggtggccga aaaacgggcg gaaacccttg
     5161 caaatgctgg attttctgcc tgtggacagc ccctcaaatg tcaataggtg cgcccctcat
     5221 ctgtcagcac tctgcccctc aagtgtcaag gatcgcgccc ctcatctgtc agtagtcgcg
     5281 cccctcaagt gtcaataccg cagggcactt atccccaggc ttgtccacat catctgtggg
     5341 aaactcgcgt aaaatcaggc gttttcgccg atttgcgagg ctggccagct ccacgtcgcc
     5401 ggccgaaatc gagcctgccc ctcatctgtc aacgccgcgc cgggtgagtc ggcccctcaa
     5461 gtgtcaacgt ccgcccctca tctgtcagtg agggccaagt tttccgcgag gtatccacaa
     5521 cgccggcggc cgcggtgtct cgcacacggc ttcgacggcg tttctggcgc gtttgcaggg
     5581 ccatagacgg ccgccagccc agcggcgagg gcaaccagcc cggtgagcgt cgcaaaggag
     5641 atcctgatct gactgatggg ctgcctgtat cgagtggtga ttttgtgccg agctgccggt
     5701 cggggagctg ttggctggct ggtggcagga tatattgtgg tgtaaacaaa ttgacgctta
     5761 gacaacttaa taacacattg cggacgtttt taatgtactg gggtggatgc ag
//
