LOCUS       sequence-273087-        6140 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Broccoli Stripe.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1088 to 1122)
  AUTHORS   Santos-Moreno J, Tasiudi E, Stelling J, Schaerli Y
  TITLE     Multistable and dynamic CRISPRi-based synthetic circuits.
  JOURNAL   Nat Commun. 2020 Jun 2;11(1):2746. doi: 10.1038/s41467-020-16574-1.
  PUBMED    32488086
REFERENCE   2  (bases 1 to 6140)
  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: "Nat 
            Commun."; date: "2020-06-2"; volume: "11(1):2746. doi"; pages: " 
            10.1038/s41467-020-16574-1"
COMMENT     Sequence Label: pJ2042.2_Brocc
FEATURES             Location/Qualifiers
     source          1..6140
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     promoter        27..311
                     /gene="araBAD"
                     /label=araBAD promoter
                     /note="promoter of the L-arabinose operon of E. coli; the 
                     araC regulatory gene is transcribed in the opposite 
                     direction (Guzman et al., 1995)"
     primer_bind     234..253
                     /label=pBAD Forward
                     /note="For vectors with E. coli araBAD promoter, forward 
                     primer"
     protein_bind    372..391
                     /label=Csy4 Site
                     /bound_moiety="Pseudomonas aeruginosa Csy4 (Cas6f)"
                     /note="cleavage site for the Csy4 endoribonuclease (Cermak 
                     et al. 2017)"
     protein_bind    494..513
                     /label=Csy4 Site
                     /bound_moiety="Pseudomonas aeruginosa Csy4 (Cas6f)"
                     /note="cleavage site for the Csy4 endoribonuclease (Cermak 
                     et al. 2017)"
     protein_bind    574..593
                     /label=Csy4 Site
                     /bound_moiety="Pseudomonas aeruginosa Csy4 (Cas6f)"
                     /note="cleavage site for the Csy4 endoribonuclease (Cermak 
                     et al. 2017)"
     protein_bind    700..719
                     /label=Csy4 Site
                     /bound_moiety="Pseudomonas aeruginosa Csy4 (Cas6f)"
                     /note="cleavage site for the Csy4 endoribonuclease (Cermak 
                     et al. 2017)"
     promoter        1088..1122
                     /label=﻿BBa_J23100
                     /note="Anderson constitutive promoter 
                     (https://parts.igem.org/Promoters/Catalog/Anderson). 
                     Measured strength relative to BBa_J223100: 1.0
                     "
                     /citation=[1]
     protein_bind    1272..1291
                     /label=Csy4 Site
                     /bound_moiety="Pseudomonas aeruginosa Csy4 (Cas6f)"
                     /note="cleavage site for the Csy4 endoribonuclease (Cermak 
                     et al. 2017)"
     protein_bind    1526..1545
                     /label=Csy4 Site
                     /bound_moiety="Pseudomonas aeruginosa Csy4 (Cas6f)"
                     /note="cleavage site for the Csy4 endoribonuclease (Cermak 
                     et al. 2017)"
     protein_bind    1780..1799
                     /label=Csy4 Site
                     /bound_moiety="Pseudomonas aeruginosa Csy4 (Cas6f)"
                     /note="cleavage site for the Csy4 endoribonuclease (Cermak 
                     et al. 2017)"
     protein_bind    1860..1879
                     /label=Csy4 Site
                     /bound_moiety="Pseudomonas aeruginosa Csy4 (Cas6f)"
                     /note="cleavage site for the Csy4 endoribonuclease (Cermak 
                     et al. 2017)"
     protein_bind    2114..2133
                     /label=Csy4 Site
                     /bound_moiety="Pseudomonas aeruginosa Csy4 (Cas6f)"
                     /note="cleavage site for the Csy4 endoribonuclease (Cermak 
                     et al. 2017)"
     protein_bind    2368..2387
                     /label=Csy4 Site
                     /bound_moiety="Pseudomonas aeruginosa Csy4 (Cas6f)"
                     /note="cleavage site for the Csy4 endoribonuclease (Cermak 
                     et al. 2017)"
     promoter        2693..2727
                     /label=BBa_J23105
                     /note="Anderson constitutive promoter 
                     (https://parts.igem.org/Promoters/Catalog/Anderson). 
                     Measured strength relative to BBa_J223100: 0.24"
     promoter        2693..2727
                     /label=BBa_J23107
                     /note="Anderson constitutive promoter 
                     (https://parts.igem.org/Promoters/Catalog/Anderson). 
                     Measured strength relative to BBa_J223100: 0.36"
     promoter        2693..2727
                     /label=BBa_J23101
                     /note="Anderson constitutive promoter 
                     (https://parts.igem.org/Promoters/Catalog/Anderson). 
                     Measured strength relative to BBa_J223100: 0.7"
     protein_bind    2871..2890
                     /label=Csy4 Site
                     /bound_moiety="Pseudomonas aeruginosa Csy4 (Cas6f)"
                     /note="cleavage site for the Csy4 endoribonuclease (Cermak 
                     et al. 2017)"
     protein_bind    2997..3016
                     /label=Csy4 Site
                     /bound_moiety="Pseudomonas aeruginosa Csy4 (Cas6f)"
                     /note="cleavage site for the Csy4 endoribonuclease (Cermak 
                     et al. 2017)"
     terminator      3336..3363
                     /label=rrnB T2 terminator
                     /note="transcription terminator T2 from the E. coli rrnB 
                     gene"
     CDS             complement(3551..4366)
                     /codon_start=1
                     /product="aminoglycoside phosphotransferase"
                     /label=KanR
                     /note="confers resistance to kanamycin in bacteria or G418 
                     (Geneticin(R)) in eukaryotes"
                     /translation="MSHIQRETSCSRPRLNSNMDADLYGYKWARDNVGQSGATIYRLYG
                     KPDAPELFLKHGKGSVANDVTDEMVRLNWLTEFMPLPTIKHFIRTPDDAWLLTTAIPGK
                     TAFQVLEEYPDSGENIVDALAVFLRRLHSIPVCNCPFNSDRVFRLAQAQSRMNNGLVDA
                     SDFDDERNGWPVEQVWKEMHKLLPFSPDSVVTHGDFSLDNLIFDEGKLIGCIDVGRVGI
                     ADRYQDLAILWNCLGEFSPSLQKRLFQKYGIDNPDMNKLQFHLMLDEFF"
     CDS             complement(3551..4366)
                     /codon_start=1
                     /gene="aph(3')-Ia"
                     /product="aminoglycoside phosphotransferase"
                     /label=KanR
                     /note="confers resistance to kanamycin in bacteria or G418 
                     (Geneticin(R)) in eukaryotes"
                     /translation="MSHIQRETSCSRPRLNSNMDADLYGYKWARDNVGQSGATIYRLYG
                     KPDAPELFLKHGKGSVANDVTDEMVRLNWLTEFMPLPTIKHFIRTPDDAWLLTTAIPGK
                     TAFQVLEEYPDSGENIVDALAVFLRRLHSIPVCNCPFNSDRVFRLAQAQSRMNNGLVDA
                     SDFDDERNGWPVEQVWKEMHKLLPFSPDSVVTHGDFSLDNLIFDEGKLIGCIDVGRVGI
                     ADRYQDLAILWNCLGEFSPSLQKRLFQKYGIDNPDMNKLQFHLMLDEFF"
     primer_bind     4274..4293
                     /label=Kan-R
                     /note="Kanamycin resistance gene, reverse primer"
     promoter        complement(4367..4458)
                     /gene="bla"
                     /label=AmpR promoter
     rep_origin      complement(4476..5111)
                     /direction=LEFT
                     /label=ColA ori
                     /note="Plasmids containing the ColA origin of replication 
                     can be propagated in E. coli cells that contain additional 
                     plasmids with compatible origins."
     primer_bind     complement(4621..4640)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(5262..6140)
                     /codon_start=1
                     /gene="araC"
                     /product="L-arabinose regulatory protein"
                     /label=araC
                     /translation="MAEAQNDPLLPGYSFNAHLVAGLTPIEANGYLDFFIDRPLGMKGY
                     ILNLTIRGQGVVKNQGREFVCRPGDILLFPPGEIHHYGRHPEAREWYHQWVYFRPRAYW
                     HEWLNWPSIFANTGFFRPDEAHQPHFSDLFGQIINAGQGEGRYSELLAINLLEQLLLRR
                     MEAINESLHPPMDNRVREACQYISDHLADSNFDIASVAQHVCLSPSRLSHLFRQQLGIS
                     VLSWREDQRISQAKLLLSTTRMPIATVGRNVGFDDQLYFSRVFKKCTGASPSEFRAGCE
                     EKVNDVAVKLS"
ORIGIN
        1 acttttcata ctcccgccat tcagagaaga aaccaattgt ccatattgca tcagacattg
       61 ccgtcactgc gtcttttact ggctcttctc gctaaccaaa ccggtaaccc cgcttattaa
      121 aagcattctg taacaaagcg ggaccaaagc catgacaaaa acgcgtaaca aaagtgtcta
      181 taatcacggc agaaaagtcc acattgatta tttgcacggc gtcacacttt gctatgccat
      241 agcattttta tccataagat tagcggttcc tacctgacgc tttttatcgc aactctctac
      301 tgtttctcca taccgaattc ataggataga ttctggaaac tttaccgtcc gagctccagc
      361 ctgcggtccg ggttcactgc cgtataggca gaagctagac tctagtggtt tcagagctat
      421 gctggaaaca gcatagcaag ttgaaataag gctagtccgt tatcaacttg aaaaagtggc
      481 accgagtcgg tgcgttcact gccgtatagg cagtcgctgg gacgcccggg gactacactt
      541 acgaaactat tgattgctca gcctgcggtc cgggttcact gccgtatagg cagatcagtg
      601 tgtactaagt actgtttcag agctatgctg gaaacagcat agcaagttga aataaggcta
      661 gtccgttatc aacttgaaaa agtggcaccg agtcggtgcg ttcactgccg tataggcagt
      721 cgctgggacg cccgctcgag caataaacag ttgatagggc ttctccgtta ccatggttca
      781 gccaaaaaac ttaagaccgc cggtcttgtc cactaccttg cagtaatgcg gtggacagga
      841 tcggcggttt tcttttctct tctcaattct tctgacctgt aacgaataat agatagtaaa
      901 gtagtctccg attgagtttt ctctgccgag tcccacccag ttctgtgatt tcagtaagtt
      961 ggtaattgat acactgttgc gagaactgct gcctggtagt agataggttg ttattgagta
     1021 agaaggtaaa gtgaacgaaa tccctgaaac tgagactgta gaaaataagc ttcagcctgc
     1081 ggtccggttg acggctagct cagtcctagg tacagtgcta gctcgctggg acgcccgaga
     1141 tagccgttac acaggtgaca cttatttcag cctgcggtcc ggccacacta gagtctagct
     1201 tgagatcgct gggacgcccg ggatccaaga gatttctaca cgattgagca ctgtctcagc
     1261 ctgcggtccg ggttcactgc cgtataggca gttgccatgt gtatgtggga gacggtcggg
     1321 tccatctgag acggtcgggt ccagatattc gtatctgtcg agtagagtgt gggctcagat
     1381 gtcgagtaga gtgtgggctc ccacatactc tgatgatcca gacggtcggg tccatctgag
     1441 acggtcgggt ccagatattc gtatctgtcg agtagagtgt gggctcagat gtcgagtaga
     1501 gtgtgggctg gatcattcat ggcaagttca ctgccgtata ggcagttgcc atgtgtatgt
     1561 gggagacggt cgggtccatc tgagacggtc gggtccagat attcgtatct gtcgagtaga
     1621 gtgtgggctc agatgtcgag tagagtgtgg gctcccacat actctgatga tccagacggt
     1681 cgggtccatc tgagacggtc gggtccagat attcgtatct gtcgagtaga gtgtgggctc
     1741 agatgtcgag tagagtgtgg gctggatcat tcatggcaag ttcactgccg tataggcagt
     1801 cgctgggacg cccgagagcc gaatcgcact tatttacagt agttcagcct gcggtccggg
     1861 ttcactgccg tataggcagt tgccatgtgt atgtgggaga cggtcgggtc catctgagac
     1921 ggtcgggtcc agatattcgt atctgtcgag tagagtgtgg gctcagatgt cgagtagagt
     1981 gtgggctccc acatactctg atgatccaga cggtcgggtc catctgagac ggtcgggtcc
     2041 agatattcgt atctgtcgag tagagtgtgg gctcagatgt cgagtagagt gtgggctgga
     2101 tcattcatgg caagttcact gccgtatagg cagttgccat gtgtatgtgg gagacggtcg
     2161 ggtccatctg agacggtcgg gtccagatat tcgtatctgt cgagtagagt gtgggctcag
     2221 atgtcgagta gagtgtgggc tcccacatac tctgatgatc cagacggtcg ggtccatctg
     2281 agacggtcgg gtccagatat tcgtatctgt cgagtagagt gtgggctcag atgtcgagta
     2341 gagtgtgggc tggatcattc atggcaagtt cactgccgta taggcagtcg ctgggacgcc
     2401 cgcctgcagg ctcggtacca aattccagaa aagaggcctc ccgaaagggg ggcctttttt
     2461 cgttttggtc ctaatagata aaggataggt ctggtagtgt tgttcgttct cgcaggtaaa
     2521 tcaataatac tcagcagttc cgtagacttt tcagtgggac agggtagcga taacagatag
     2581 attgtaataa gacacagtag gtgctcgtag ttgcgtgaag agaaccgctc aggaaatcca
     2641 gtcagaagta ttggtaatcg ttgaaaactc agtcgaccag cctgcggtcc ggtttacggc
     2701 tagctcagtc ctaggtatta tgctagctcg ctgggacgcc cgagtgacga ctgcgaagta
     2761 acctctattt atcagcctgc ggtccggcca agtacttagt acacactgat tcgctgggac
     2821 gcccggtagt gcttatcaga cccaatactg ttgaacagcc tgcggtccgg gttcactgcc
     2881 gtataggcag tctcaagcta gactctagtg gtttcagagc tatgctggaa acagcatagc
     2941 aagttgaaat aaggctagtc cgttatcaac ttgaaaaagt ggcaccgagt cggtgcgttc
     3001 actgccgtat aggcagtcgc tgggacgccc ggacgtccta ttacactcgt cgttggaaac
     3061 tgaagatgcg gccgcggaaa cacagaaaaa agcccgcacc tgacagtgcg ggcttttttt
     3121 ttcgaccaaa ggtagcgaac gacgagtcac tgttgaggat aaatactttc tctactaggc
     3181 gcctgttaca caggtcctca gcggcgcgcc tttgtcggtg aacgctctcc tgagtaggac
     3241 aaatccgccg ggagcggatt tgaacgttgt gaagcaacgg cccggagggt ggcgggcagg
     3301 acgcccgcca taaactgcca ggcatcaaac taagcagaag gccatcctga cggatggcct
     3361 ttttgcgttt cagatctacc ggtaaaccag caatagacat aagcggctat ttaacgaccc
     3421 tgccctgaac cgacgacaag ctgacgaccg ggtctccgca agtggcactt ttcggggaaa
     3481 tgtgcgcgga acccctattt gtttattttt ctaaatacat tcaaatatgt atccgctcat
     3541 gaattaattc ttagaaaaac tcatcgagca tcaaatgaaa ctgcaattta ttcatatcag
     3601 gattatcaat accatatttt tgaaaaagcc gtttctgtaa tgaaggagaa aactcaccga
     3661 ggcagttcca taggatggca agatcctggt atcggtctgc gattccgact cgtccaacat
     3721 caatacaacc tattaatttc ccctcgtcaa aaataaggtt atcaagtgag aaatcaccat
     3781 gagtgacgac tgaatccggt gagaatggca aaagtttatg catttctttc cagacttgtt
     3841 caacaggcca gccattacgc tcgtcatcaa aatcactcgc atcaaccaaa ccgttattca
     3901 ttcgtgattg cgcctgagcg agacgaaata cgcggtcgct gttaaaagga caattacaaa
     3961 caggaatcga atgcaaccgg cgcaggaaca ctgccagcgc atcaacaata ttttcacctg
     4021 aatcaggata ttcttctaat acctggaatg ctgttttccc ggggatcgca gtggtgagta
     4081 accatgcatc atcaggagta cggataaaat gcttgatggt cggaagaggc ataaattccg
     4141 tcagccagtt tagtctgacc atctcatctg taacatcatt ggcaacgcta cctttgccat
     4201 gtttcagaaa caactctggc gcatcgggct tcccatacaa tcgatagatt gtcgcacctg
     4261 attgcccgac attatcgcga gcccatttat acccatataa atcagcatcc atgttggaat
     4321 ttaatcgcgg cctagagcaa gacgtttccc gttgaatatg gctcatactc ttcctttttc
     4381 aatattattg aagcatttat cagggttatt gtctcatgag cggatacata tttgaatgta
     4441 tttagaaaaa taaacaaata ggcatgctag cgcagaaacg tcctagaaga tgccaggagg
     4501 atacttagca gagagacaat aaggccggag cgaagccgtt tttccatagg ctccgccccc
     4561 ctgacgaaca tcacgaaatc tgacgctcaa atcagtggtg gcgaaacccg acaggactat
     4621 aaagatacca ggcgtttccc cctgatggct ccctcttgcg ctctcctgtt cccgtcctgc
     4681 ggcgtccgtg ttgtggtgga ggctttaccc aaatcaccac gtcccgttcc gtgtagacag
     4741 ttcgctccaa gctgggctgt gtgcaagaac cccccgttca gcccgactgc tgcgccttat
     4801 ccggtaacta tcatcttgag tccaacccgg aaagacacga caaaacgcca ctggcagcag
     4861 ccattggtaa ctgagaatta gtggatttag atatcgagag tcttgaagtg gtggcctaac
     4921 agaggctaca ctgaaaggac agtatttggt atctgcgctc cactaaagcc agttaccagg
     4981 ttaagcagtt ccccaactga cttaaccttc gatcaaaccg cctccccagg cggttttttc
     5041 gtttacagag caggagatta cgacgatcgt aaaaggatct caagaagatc ctttacggat
     5101 tcccgacacc atcactctag atttcagtgc aatttatctc ttcaaatgta gcacctgaag
     5161 tcagccccat acgatataag ttgtaattct catgttagtc atgccccgcg cccaccggaa
     5221 ggagctgact gggttgctcc tagggtctga ttcgttacca attatgacaa cttgacggct
     5281 acatcattca ctttttcttc acaaccggca cggaactcgc tcgggctggc cccggtgcat
     5341 tttttaaata cccgcgagaa atagagttga tcgtcaaaac caacattgcg accgacggtg
     5401 gcgataggca tccgggtggt gctcaaaagc agcttcgcct ggctgatacg ttggtcctcg
     5461 cgccagctta agacgctaat ccctaactgc tggcggaaaa gatgtgacag acgcgacggc
     5521 gacaagcaaa catgctgtgc gacgctggcg atatcaaaat tgctgtctgc caggtgatcg
     5581 ctgatgtact gacaagcctc gcgtacccga ttatccatcg gtggatggag cgactcgtta
     5641 atcgcttcca tgcgccgcag taacaattgc tcaagcagat ttatcgccag cagctccgaa
     5701 tagcgccctt ccccttgccc ggcgttaatg atttgcccaa acaggtcgct gaaatgcggc
     5761 tggtgcgctt catccgggcg aaagaacccc gtattggcaa atattgacgg ccagttaagc
     5821 cattcatgcc agtaggcgcg cggacgaaag taaacccact ggtgatacca ttcgcgagcc
     5881 tccggatgac gaccgtagtg atgaatctct cctggcggga acagcaaaat atcacccggt
     5941 cggcaaacaa attctcgtcc ctgatttttc accaccccct gaccgcgaat ggtgagattg
     6001 agaatataac ctttcattcc cagcggtcgg tcgataaaaa aatcgagata accgttggcc
     6061 tcaatcggcg ttaaacccgc caccagatgg gcattaaacg agtatcccgg cagcagggga
     6121 tcattttgcg cttcagccat
//
