LOCUS       sequence-371877-        5813 bp    DNA     circular SYN 15-APR-2025
DEFINITION  Cas9 guide expression in dicots.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 5813)
  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 5813)
  AUTHORS   .
  TITLE     Direct Submission
  JOURNAL   Exported Apr 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_AtU626Ter26
FEATURES             Location/Qualifiers
     source          1..5813
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     protein_bind    352..373
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     promoter        join(388..393,394..411,412..418)
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    426..442
                     /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             460..471
                     /note="strong bacterial ribosome binding site (Elowitz and 
                     Leibler, 2000)"
     CDS             478..1155
                     /codon_start=1
                     /product="monomeric derivative of DsRed (Campbell et al., 
                     2002)"
                     /label=mRFP1
                     /translation="MASSEDVIKEFMRFKVRMEGSVNGHEFEIEGEGEGRPYEGTQTAK
                     LKVTKGGPLPFAWDILSPQFQYGSKAYVKHPADIPDYLKLSFPEGFKWERVMNFEDGGV
                     VTVTQDSSLQDGEFIYKVKLRGTNFPSDGPVMQKKTMGWEASTERMYPEDGALKGEIKM
                     RLKLKDGGHYDAEVKTTYMAKKPVQLPGAYKTDIKLDITSHNEDYTIVEQYERAEGRHS
                     TGA"
     terminator      1200..1272
                     /label=rrnB T1 terminator
                     /note="transcription terminator T1 from the E. coli rrnB 
                     gene"
     terminator      1288..1315
                     /label=T7Te terminator
                     /note="phage T7 early transcription terminator"
     misc_RNA        1329..1404
                     /label=gRNA scaffold
                     /note="guide RNA scaffold for the Streptococcus pyogenes 
                     CRISPR/Cas9 system"
     misc_feature    1516..1540
                     /label=RB T-DNA repeat
                     /note="right border repeat from nopaline C58 T-DNA"
     CDS             complement(1696..2844)
                     /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(3180..3768)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(3260..3279)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(3939..4730,4731..4799))
                     /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     4562..4581
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(4800..4904)
                     /gene="bla"
                     /label=AmpR promoter
     rep_origin      4991..5622
                     /label=oriV
                     /note="incP origin of replication"
     misc_feature    5714..5738
                     /label=LB T-DNA repeat
                     /note="left border repeat from nopaline C58 T-DNA"
ORIGIN
        1 tctgtgaaga caagagcgaa ttccatatgg gagcatcttc attcttaaga tatgaagata
       61 atcttcaaaa ggcccctggg aatctgaaag aagagaagca ggcccattta tatgggaaag
      121 aacaatagta tttcttatat aggcccattt aagttgaaaa caatcttcaa aagtcccaca
      181 tcgcttagat aagaaaacga agctgagttt atatacagct agagtcgaag tagtgattgt
      241 gagaccgata tcaatacgca aaccgcctct ccccgcgcgt tggccgattc attaatgcag
      301 ctggcacgac aggtttcccg actggaaagc gggcagtgag cgcaacgcaa ttaatgtgag
      361 ttagctcact cattaggcac cccaggcttt acactttatg cttccggctc gtatgttgtg
      421 tggaattgtg agcggataac aatttcacac atactagaga aagaggagaa atactagatg
      481 gcttcctccg aggacgttat caaagagttc atgcgtttca aagttcgtat ggaaggttcc
      541 gttaacggtc acgagttcga aatcgaaggt gaaggtgaag gtcgtccgta cgaaggtacc
      601 cagaccgcta aactgaaagt taccaaaggt ggtccgctgc cgttcgcttg ggacatcctg
      661 tccccgcagt tccagtacgg ttccaaagct tacgttaaac acccggctga catcccggac
      721 tacctgaaac tgtccttccc ggaaggtttc aaatgggaac gtgttatgaa cttcgaggac
      781 ggtggtgttg ttaccgttac ccaggactcc tccctgcaag acggtgagtt catctacaaa
      841 gttaaactgc gtggtaccaa cttcccgtcc gacggtccgg ttatgcagaa aaaaaccatg
      901 ggttgggaag cttccaccga acgtatgtac ccggaggacg gtgctctgaa aggtgaaatc
      961 aaaatgcgtc tgaaactgaa agacggtggt cactacgacg ctgaagttaa aaccacctac
     1021 atggctaaaa aaccggttca gctgccgggt gcttacaaaa ccgacatcaa actggacatc
     1081 acctcccaca acgaggacta caccatcgtt gaacagtacg aacgtgctga aggtcgtcac
     1141 tccaccggtg cttaataacg ctgatagtgc tagtgtagat cgctactaga gccaggcatc
     1201 aaataaaacg aaaggctcag tcgaaagact gggcctttcg tttttatctg ttgtttgtcg
     1261 gtgaacgctc tctactagag tcacactggc tcaccttcgg gtgggccttt ctgcgtttat
     1321 aggtctcagt tttagagcta gaaatagcaa gttaaaataa ggctagtccg ttatcaactt
     1381 gaaaaagtgg caccgagtcg gtgctttttt ttgcaaaatt ttccagatcg atttcttctt
     1441 cctctgttct tcggcgttca atttctgggg cgcttgcctt gtcttctgca cgaagtggtt
     1501 taaactatca gtgtttgaca ggatatattg gcgggtaaac ctaagagaaa agagcgttta
     1561 ttagaataat cggatattta aaagggcgtg aaaaggttta tccgttcgtc catttgtatg
     1621 tgcatgccaa ccacagggtt ccccagatca ggcgctggct gctgaacccc cagccggaac
     1681 tgaccccaca aggccctagc gtttgcaatg caccaggtca tcattgaccc aggcgtgttc
     1741 caccaggccg ctgcctcgca actcttcgca ggcttcgccg acctgctcgc gccacttctt
     1801 cacgcgggtg gaatccgatc cgcacatgag gcggaaggtt tccagcttga gcgggtacgg
     1861 ctcccggtgc gagctgaaat agtcgaacat ccgtcgggcc gtcggcgaca gcttgcggta
     1921 cttctcccat atgaatttcg tgtagtggtc gccagcaaac agcacgacga tttcctcgtc
     1981 gatcaggacc tggcaacggg acgttttctt gccacggtcc aggacgcgga agcggtgcag
     2041 cagcgacacc gattccaggt gcccaacgcg gtcggacgtg aagcccatcg ccgtcgcctg
     2101 taggcgcgac aggcattcct cggccttcgt gtaataccgg ccattgatcg accagcccag
     2161 gtcctggcaa agctcgtaga acgtgaaggt gatcggctcg ccgatagggg tgcgcttcgc
     2221 gtactccaac acctgctgcc acaccagttc gtcatcgtcg gcccgcagct cgacgccggt
     2281 gtaggtgatc ttcacgtcct tgttgacgtg gaaaatgacc ttgttttgca gcgcctcgcg
     2341 cgggattttc ttgttgcgcg tggtgaacag ggcagagcgg gccgtgtcgt ttggcatcgc
     2401 tcgcatcgtg tccggccacg gcgcaatatc gaacaaggaa agctgcattt ccttgatctg
     2461 ctgcttcgtg tgtttcagca acgcggcctg cttggcctcg ctgacctgtt ttgccaggtc
     2521 ctcgccggcg gtttttcgct tcttggtcgt catagttcct cgcgtgtcga tggtcatcga
     2581 cttcgccaaa cctgccgcct cctgttcaag acgacgcgaa cgctccacgg cggccgatgg
     2641 cgcgggcagg gcagggggag ccagttgcac gctgtcgcgc tcgatcttgg ccgtagcttg
     2701 ctggaccatc gagccgacgg actggaaggt ttcgcggggc gcacgcatga cggtgcggct
     2761 tgcgatggtt tcggcatcct cggcggaaaa ccccgcgtcg atcagttctt gcctgtatgc
     2821 cttccggtca aacgtccgat tcattcaccc tccttgcggg attgccccga ctcacgccgg
     2881 ggcaatgtgc ccttattcct gatttgaccc gcctggtgcc ttggtgtcca gataatccac
     2941 cttatcggca atgaagtcgg tcccgtagac cgtctggccg tccttctcgt acttggtatt
     3001 ccgaatcttg ccctgcacga ataccagcga ccccttgccc aaatacttgc cgtgggcctc
     3061 ggcctgagag ccaaaacact tgatgcggaa gaagtcggtg cgctcctgct tgtcgccggc
     3121 atcgttgcgc cacatctagg atctgccagg aaccgtaaaa aggccgcgtt gctggcgttt
     3181 ttccataggc tccgcccccc tgacgagcat cacaaaaatc gacgctcaag tcagaggtgg
     3241 cgaaacccga caggactata aagataccag gcgtttcccc ctggaagctc cctcgtgcgc
     3301 tctcctgttc cgaccctgcc gcttaccgga tacctgtccg cctttctccc ttcgggaagc
     3361 gtggcgcttt ctcatagctc acgctgtagg tatctcagtt cggtgtaggt cgttcgctcc
     3421 aagctgggct gtgtgcacga accccccgtt cagcccgacc gctgcgcctt atccggtaac
     3481 tatcgtcttg agtccaaccc ggtaagacac gacttatcgc cactggcagc agccactggt
     3541 aacaggatta gcagagcgag gtatgtaggc ggtgctacag agttcttgaa gtggtggcct
     3601 aactacggct acactagaag gacagtattt ggtatctgcg ctctgctgaa gccagttacc
     3661 ttcggaaaaa gagttggtag ctcttgatcc ggcaaacaaa ccaccgctgg tagcggtggt
     3721 ttttttgttt gcaagcagca gattacgcgc agaaaaaaag gatctcaaga agatcctttg
     3781 atcttttcta cggggtctga cgctcagtgg aacgaaaact cacgttaagg gattttggtc
     3841 atgagattat caaaaaggat cttcacctag atccttttaa attaaaaatg aagttttaaa
     3901 tcaatctaaa gtatatatga gtaaacttgg tctgacagtt accaatgctt aatcagtgag
     3961 gcacctatct cagcgatctg tctatttcgt tcatccatag ttgcctgact ccccgtcgtg
     4021 tagataacta cgatacggga gggcttacca tctggcccca gtgctgcaat gataccgcga
     4081 gaaccacgct caccggctcc agatttatca gcaataaacc agccagccgg aagggccgag
     4141 cgcagaagtg gtcctgcaac tttatccgcc tccatccagt ctattaattg ttgccgggaa
     4201 gctagagtaa gtagttcgcc agttaatagt ttgcgcaacg ttgttgccat tgctacaggc
     4261 atcgtggtgt cacgctcgtc gtttggtatg gcttcattca gctccggttc ccaacgatca
     4321 aggcgagtta catgatcccc catgttgtgc aaaaaagcgg ttagctcctt cggtcctccg
     4381 atcgttgtca gaagtaagtt ggccgcagtg ttatcactca tggttatggc agcactgcat
     4441 aattctctta ctgtcatgcc atccgtaaga tgcttttctg tgactggtga gtactcaacc
     4501 aagtcattct gagaatagtg tatgcggcga ccgagttgct cttgcccggc gtcaatacgg
     4561 gataataccg cgccacatag cagaacttta aaagtgctca tcattggaaa acgttcttcg
     4621 gggcgaaaac tctcaaggat cttaccgctg ttgagatcca gttcgatgta acccactcgt
     4681 gcacccaact gatcttcagc atcttttact ttcaccagcg tttctgggtg agcaaaaaca
     4741 ggaaggcaaa atgccgcaaa aaagggaata agggcgacac ggaaatgttg aatactcata
     4801 ctcttccttt ttcaatatta ttgaagcatt tatcagggtt attgtctcat gagcggatac
     4861 atatttgaat gtatttagaa aaataaacaa ataggggttc cgcgcacgaa ttggccagcg
     4921 ctgccatttt tggggtgagg ccgttcgcgg ccgaggggcg cagcccctgg ggggatggga
     4981 ggcccgcgtt agcgggccgg gagggttcga gaaggggggg cacccccctt cggcgtgcgc
     5041 ggtcacgcgc acagggcgca gccctggtta aaaacaaggt ttataaatat tggtttaaaa
     5101 gcaggttaaa agacaggtta gcggtggccg aaaaacgggc ggaaaccctt gcaaatgctg
     5161 gattttctgc ctgtggacag cccctcaaat gtcaataggt gcgcccctca tctgtcagca
     5221 ctctgcccct caagtgtcaa ggatcgcgcc cctcatctgt cagtagtcgc gcccctcaag
     5281 tgtcaatacc gcagggcact tatccccagg cttgtccaca tcatctgtgg gaaactcgcg
     5341 taaaatcagg cgttttcgcc gatttgcgag gctggccagc tccacgtcgc cggccgaaat
     5401 cgagcctgcc cctcatctgt caacgccgcg ccgggtgagt cggcccctca agtgtcaacg
     5461 tccgcccctc atctgtcagt gagggccaag ttttccgcga ggtatccaca acgccggcgg
     5521 ccgcggtgtc tcgcacacgg cttcgacggc gtttctggcg cgtttgcagg gccatagacg
     5581 gccgccagcc cagcggcgag ggcaaccagc ccggtgagcg tcgcaaagga gatcctgatc
     5641 tgactgatgg gctgcctgta tcgagtggtg attttgtgcc gagctgccgg tcggggagct
     5701 gttggctggc tggtggcagg atatattgtg gtgtaaacaa attgacgctt agacaactta
     5761 ataacacatt gcggacgttt ttaatgtact ggggtggatg cagtgggccc cac
//
