LOCUS       sequence-311063-        6900 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  CRISPRlator control ("open ring").
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 6900)
  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 6900)
  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: pJ2072.2_c1
FEATURES             Location/Qualifiers
     source          1..6900
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     promoter        99..133
                     /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    277..296
                     /label=Csy4 Site
                     /bound_moiety="Pseudomonas aeruginosa Csy4 (Cas6f)"
                     /note="cleavage site for the Csy4 endoribonuclease (Cermak 
                     et al. 2017)"
     protein_bind    403..422
                     /label=Csy4 Site
                     /bound_moiety="Pseudomonas aeruginosa Csy4 (Cas6f)"
                     /note="cleavage site for the Csy4 endoribonuclease (Cermak 
                     et al. 2017)"
     protein_bind    489..508
                     /label=Csy4 Site
                     /bound_moiety="Pseudomonas aeruginosa Csy4 (Cas6f)"
                     /note="cleavage site for the Csy4 endoribonuclease (Cermak 
                     et al. 2017)"
     RBS             513..535
                     /note="efficient ribosome binding site from bacteriophage 
                     T7 gene 10 (Olins and Rangwala, 1989)"
     CDS             543..1250
                     /codon_start=1
                     /product="monomeric derivative of DsRed fluorescent protein
                     (Shaner et al., 2004)"
                     /label=mCherry
                     /note="mammalian codon-optimized"
                     /translation="MVSKGEEDNMAIIKEFMRFKVHMEGSVNGHEFEIEGEGEGRPYEG
                     TQTAKLKVTKGGPLPFAWDILSPQFMYGSKAYVKHPADIPDYLKLSFPEGFKWERVMNF
                     EDGGVVTVTQDSSLQDGEFIYKVKLRGTNFPSDGPVMQKKTMGWEASSERMYPEDGALK
                     GEIKQRLKLKDGGHYDAEVKTTYKAKKPVQLPGAYNVNIKLDITSHNEDYTIVEQYERA
                     EGRHSTGGMDELYK"
     primer_bind     complement(688..706)
                     /label=mCherry-R
                     /note="mCherry, reverse primer"
     primer_bind     956..975
                     /label=mCherry-F
                     /note="mCherry, forward primer"
     promoter        1970..2004
                     /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    2148..2167
                     /label=Csy4 Site
                     /bound_moiety="Pseudomonas aeruginosa Csy4 (Cas6f)"
                     /note="cleavage site for the Csy4 endoribonuclease (Cermak 
                     et al. 2017)"
     protein_bind    2274..2293
                     /label=Csy4 Site
                     /bound_moiety="Pseudomonas aeruginosa Csy4 (Cas6f)"
                     /note="cleavage site for the Csy4 endoribonuclease (Cermak 
                     et al. 2017)"
     protein_bind    2360..2379
                     /label=Csy4 Site
                     /bound_moiety="Pseudomonas aeruginosa Csy4 (Cas6f)"
                     /note="cleavage site for the Csy4 endoribonuclease (Cermak 
                     et al. 2017)"
     RBS             2384..2406
                     /note="efficient ribosome binding site from bacteriophage 
                     T7 gene 10 (Olins and Rangwala, 1989)"
     CDS             2414..3127
                     /codon_start=1
                     /product="yeast-enhanced green fluorescent protein"
                     /label=yeGFP
                     /note="codons optimized for translation in S. cerevisiae 
                     and C. albicans"
                     /translation="MSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTLK
                     FICTTGKLPVPWPTLVTTLGYGLMCFARYPDHMKQHDFFKSAMPEGYVQERTIFFKDDG
                     NYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIKV
                     NFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSYQSKLSKDPNEKRDHMVLLE
                     FVTAAGITHGMDELYK"
     promoter        3499..3533
                     /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    3677..3696
                     /label=Csy4 Site
                     /bound_moiety="Pseudomonas aeruginosa Csy4 (Cas6f)"
                     /note="cleavage site for the Csy4 endoribonuclease (Cermak 
                     et al. 2017)"
     protein_bind    3803..3822
                     /label=Csy4 Site
                     /bound_moiety="Pseudomonas aeruginosa Csy4 (Cas6f)"
                     /note="cleavage site for the Csy4 endoribonuclease (Cermak 
                     et al. 2017)"
     protein_bind    3889..3908
                     /label=Csy4 Site
                     /bound_moiety="Pseudomonas aeruginosa Csy4 (Cas6f)"
                     /note="cleavage site for the Csy4 endoribonuclease (Cermak 
                     et al. 2017)"
     RBS             3913..3935
                     /note="efficient ribosome binding site from bacteriophage 
                     T7 gene 10 (Olins and Rangwala, 1989)"
     primer_bind     4806..4826
                     /label=GFP-F
                     /note="GFP, forward primer. Does NOT anneal to EGFP"
     terminator      5156..5183
                     /label=rrnB T2 terminator
                     /note="transcription terminator T2 from the E. coli rrnB 
                     gene"
     CDS             complement(5371..6186)
                     /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(5371..6186)
                     /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     6094..6113
                     /label=Kan-R
                     /note="Kanamycin resistance gene, reverse primer"
     promoter        complement(6187..6278)
                     /gene="bla"
                     /label=AmpR promoter
     rep_origin      complement(join(6296..6900,1..31))
                     /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(6441..6460)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
ORIGIN
        1 caagaagatc ctttacggat tcccgacacc atcactctag agaattcata ggatagattc
       61 tggaaacttt accgtccgag ctccagcctg cggtccggtt gacggctagc tcagtcctag
      121 gtacagtgct agctcgctgg gacgcccgag agccgaatcg cacttattta cagtagttca
      181 gcctgcggtc cggccataca tactctaaga tgtgtctcgc tgggacgccc ggggactaca
      241 cttacgaaac tattgattgc tcagcctgcg gtccgggttc actgccgtat aggcagatca
      301 gtgtgtacta agtactgttt cagagctatg ctggaaacag catagcaagt tgaaataagg
      361 ctagtccgtt atcaacttga aaaagtggca ccgagtcggt gcgttcactg ccgtataggc
      421 agtcgctggg acgcccgctc gagcaataaa cagttgatag ggcttctccg ttacagcctg
      481 cggtccgggt tcactgccgt ataggcagta attttgttta actttaagaa ggagatatac
      541 atatggtgag caagggcgag gaggataaca tggccatcat caaggagttc atgcgcttca
      601 aggtgcacat ggagggctcc gtgaacggcc acgagttcga gatcgagggc gagggcgagg
      661 gccgccccta cgagggcacc cagaccgcca agctgaaggt gaccaagggt ggccccctgc
      721 ccttcgcctg ggacatcctg tcccctcagt tcatgtacgg ctccaaggcc tacgtgaagc
      781 accccgccga catccccgac tacttgaagc tgtccttccc cgagggcttc aagtgggagc
      841 gcgtgatgaa cttcgaggac ggcggcgtgg tgaccgtgac ccaggactcc tctctgcagg
      901 acggcgagtt catctacaag gtgaagctgc gcggcaccaa cttcccctcc gacggccccg
      961 taatgcagaa gaagactatg ggctgggagg cctcctccga gcggatgtac cccgaggacg
     1021 gcgcgctgaa gggcgagatc aagcagaggc tgaagctgaa ggacggcggc cactacgacg
     1081 ctgaagtcaa gaccacctac aaggccaaga agcccgtgca gctgcccggc gcgtacaacg
     1141 tcaacatcaa gttggacatc acctcccaca acgaggacta caccatcgtg gaacagtacg
     1201 aacgcgccga gggccgccac tccaccggcg gcatggacga gctgtacaag atgtcccgcc
     1261 gtaatactga cgccatcaca atccacagca tcctggattg gattgaagac aacttggagt
     1321 cgccgttgag tttagaaaaa gttagtgaac gtagtggtta ctcaaagtgg caccttcagc
     1381 gcatgtttaa gaaggaaacg ggtcattcat tgggtcaata tattcgttct cgcaagatga
     1441 ctgaaattgc ccagaaattg aaagagtcta atgaacctat tttgtacctg gcggagcgtt
     1501 acggctttga aagtcagcaa acccttacac gtaccttcaa gaattacttt gacgttccac
     1561 cacacaaata tcgtatgacc aacatgcagg gtgagtcacg ttttttgcat ccgttgaatc
     1621 attacaattc ctaataatcg ctgggacgcc cgccatggtt cagccaaaaa acttaagacc
     1681 gccggtcttg tccactacct tgcagtaatg cggtggacag gatcggcggt tttcttttct
     1741 cttctcaatt cttctgacct gtaacgaata atagatagta aagtagtctc cgattgagtt
     1801 ttctctgccg agtcccaccc agttctgtga tttcagtaag ttggtaattg atacactgtt
     1861 gcgagaactg ctgcctggta gtagataggt tgttattgag taagaaggta aagtgaacga
     1921 aatccctgaa actgagactg tagaaaataa gcttcagcct gcggtccggt tgacggctag
     1981 ctcagtccta ggtacagtgc tagctcgctg ggacgcccga gatagccgtt acacaggtga
     2041 cacttatttc agcctgcggt ccggccacac tagagtctag cttgagatcg ctgggacgcc
     2101 cgtgaacagt tgctctgatt gaaaccacga ttcagcctgc ggtccgggtt cactgccgta
     2161 taggcagtga ctgagctagt gtactctgtt tcagagctat gctggaaaca gcatagcaag
     2221 ttgaaataag gctagtccgt tatcaacttg aaaaagtggc accgagtcgg tgcgttcact
     2281 gccgtatagg cagtcgctgg gacgcccggg atccaagaga tttctacacg attgagcact
     2341 gtctcagcct gcggtccggg ttcactgccg tataggcagt aattttgttt aactttaaga
     2401 aggagatata catatgtcta aaggtgaaga attattcact ggtgttgtcc caattttggt
     2461 tgaattagat ggtgatgtta atggtcacaa attttctgtc tccggtgaag gtgaaggtga
     2521 tgctacttac ggtaaattga ccttaaaatt tatttgtact actggtaaat tgccagttcc
     2581 gtggccaacc ttagtcacta ctttaggtta tggtttgatg tgttttgcta gatacccaga
     2641 tcatatgaaa caacatgact ttttcaagtc tgccatgcca gaaggttatg ttcaagaaag
     2701 aactattttt ttcaaagatg acggtaacta caagaccaga gctgaagtca agtttgaagg
     2761 tgatacctta gttaatagaa tcgaattaaa aggtattgat tttaaagaag atggtaacat
     2821 tttaggtcac aaattggaat acaactataa ctctcacaat gtttacatca tggctgacaa
     2881 acaaaagaat ggtatcaaag ttaacttcaa aattagacac aacattgaag atggttctgt
     2941 tcaattagct gaccattatc aacaaaatac tccaattggt gatggtccag tcttgttacc
     3001 agacaaccat tacttatcct atcaatctaa attatccaaa gatccaaacg aaaagagaga
     3061 ccacatggtc ttgttagaat ttgttactgc tgctggtatt acccacggta tggatgaatt
     3121 gtacaaaatg tcccgccgta atactgacgc catcacaatc cacagcatcc tggattggat
     3181 tgaagactaa taatcgctgg gacgcccgcc tgcaggctcg gtaccaaatt ccagaaaaga
     3241 ggcctcccga aaggggggcc ttttttcgtt ttggtcctaa tagataaagg ataggtctgg
     3301 tagtgttgtt cgttctcgca ggtaaatcaa taatactcag cagttccgta gacttttcag
     3361 tgggacaggg tagcgataac agatagattg taataagaca cagtaggtgc tcgtagttgc
     3421 gtgaagagaa ccgctcagga aatccagtca gaagtattgg taatcgttga aaactcagtc
     3481 gaccagcctg cggtccggtt gacggctagc tcagtcctag gtacagtgct agctcgctgg
     3541 gacgcccgag tgacgactgc gaagtaacct ctatttatca gcctgcggtc cggccaagta
     3601 cttagtacac actgattcgc tgggacgccc ggtagtgctt atcagaccca atactgttga
     3661 acagcctgcg gtccgggttc actgccgtat aggcagtctc aagctagact ctagtggttt
     3721 cagagctatg ctggaaacag catagcaagt tgaaataagg ctagtccgtt atcaacttga
     3781 aaaagtggca ccgagtcggt gcgttcactg ccgtataggc agtcgctggg acgcccggac
     3841 gtcctattac actcgtcgtt ggaaactgaa gatcagcctg cggtccgggt tcactgccgt
     3901 ataggcagta attttgttta actttaagaa ggagatatac atatgaatca gtcattcatc
     3961 tcggacatct tatatgccga catcgaatcg aaggctaagg aacttacagt caattccaac
     4021 aatactgtcc agccggtcgc gcttatgcgc ttaggagttt tcgttcccaa accttccaag
     4081 agcaaaggag aaagtaagga aattgacgcc accaaagcct tctctcaact ggagattgct
     4141 aaagcagagg gcatgagtaa aggagaagaa cttttcactg gagttgtccc aattcttgtt
     4201 gaattagatg gtgatgttaa tgggcacaaa ttttctgtca gtggagaggg tgaaggtgat
     4261 gcaacatacg gaaaacttac ccttaaattt atttgcacta ctggaaaact acctgttccg
     4321 tggccaacac ttgtcactac tttgacttgg ggtgttcaat gctttgctag atacccagat
     4381 catatgaaac agcatgactt tttcaagagt gccatgcccg aaggttatgt acaggaaaga
     4441 actatatttt tcaaagatga cgggaactac aagacacgtg ctgaagtcaa gtttgaaggt
     4501 gatacccttg ttaatagaat cgagttaaaa ggtattgatt ttaaagaaga tggaaacatt
     4561 cttggacaca aattggaata caacgctatt tcagataatg tatacatcac tgcagacaaa
     4621 caaaagaatg gaatcaaagc taatttcaaa attagacaca acattgaaga tggaagcgtt
     4681 caactagcag accattatca acaaaatact ccaattggcg atggccctgt ccttttacca
     4741 gacaaccatt acctgtccac acaatctaaa ctttcgaaag atcccaacga aaagagagac
     4801 cacatggtcc ttcttgagtt tgtaacagct gctgggatta cactaggcat ggatgaacta
     4861 tacaaataat aatcgctggg acgcccggcg gccgcggaaa cacagaaaaa agcccgcacc
     4921 tgacagtgcg ggcttttttt ttcgaccaaa ggtagcgaac gacgagtcac tgttgaggat
     4981 aaatactttc tctactaggc gcctgttaca caggtcctca gcggcgcgcc tttgtcggtg
     5041 aacgctctcc tgagtaggac aaatccgccg ggagcggatt tgaacgttgt gaagcaacgg
     5101 cccggagggt ggcgggcagg acgcccgcca taaactgcca ggcatcaaac taagcagaag
     5161 gccatcctga cggatggcct ttttgcgttt cagatctacc ggtaaaccag caatagacat
     5221 aagcggctat ttaacgaccc tgccctgaac cgacgacaag ctgacgaccg ggtctccgca
     5281 agtggcactt ttcggggaaa tgtgcgcgga acccctattt gtttattttt ctaaatacat
     5341 tcaaatatgt atccgctcat gaattaattc ttagaaaaac tcatcgagca tcaaatgaaa
     5401 ctgcaattta ttcatatcag gattatcaat accatatttt tgaaaaagcc gtttctgtaa
     5461 tgaaggagaa aactcaccga ggcagttcca taggatggca agatcctggt atcggtctgc
     5521 gattccgact cgtccaacat caatacaacc tattaatttc ccctcgtcaa aaataaggtt
     5581 atcaagtgag aaatcaccat gagtgacgac tgaatccggt gagaatggca aaagtttatg
     5641 catttctttc cagacttgtt caacaggcca gccattacgc tcgtcatcaa aatcactcgc
     5701 atcaaccaaa ccgttattca ttcgtgattg cgcctgagcg agacgaaata cgcggtcgct
     5761 gttaaaagga caattacaaa caggaatcga atgcaaccgg cgcaggaaca ctgccagcgc
     5821 atcaacaata ttttcacctg aatcaggata ttcttctaat acctggaatg ctgttttccc
     5881 ggggatcgca gtggtgagta accatgcatc atcaggagta cggataaaat gcttgatggt
     5941 cggaagaggc ataaattccg tcagccagtt tagtctgacc atctcatctg taacatcatt
     6001 ggcaacgcta cctttgccat gtttcagaaa caactctggc gcatcgggct tcccatacaa
     6061 tcgatagatt gtcgcacctg attgcccgac attatcgcga gcccatttat acccatataa
     6121 atcagcatcc atgttggaat ttaatcgcgg cctagagcaa gacgtttccc gttgaatatg
     6181 gctcatactc ttcctttttc aatattattg aagcatttat cagggttatt gtctcatgag
     6241 cggatacata tttgaatgta tttagaaaaa taaacaaata ggcatgctag cgcagaaacg
     6301 tcctagaaga tgccaggagg atacttagca gagagacaat aaggccggag cgaagccgtt
     6361 tttccatagg ctccgccccc ctgacgaaca tcacgaaatc tgacgctcaa atcagtggtg
     6421 gcgaaacccg acaggactat aaagatacca ggcgtttccc cctgatggct ccctcttgcg
     6481 ctctcctgtt cccgtcctgc ggcgtccgtg ttgtggtgga ggctttaccc aaatcaccac
     6541 gtcccgttcc gtgtagacag ttcgctccaa gctgggctgt gtgcaagaac cccccgttca
     6601 gcccgactgc tgcgccttat ccggtaacta tcatcttgag tccaacccgg aaagacacga
     6661 caaaacgcca ctggcagcag ccattggtaa ctgagaatta gtggatttag atatcgagag
     6721 tcttgaagtg gtggcctaac agaggctaca ctgaaaggac agtatttggt atctgcgctc
     6781 cactaaagcc agttaccagg ttaagcagtt ccccaactga cttaaccttc gatcaaaccg
     6841 cctccccagg cggttttttc gtttacagag caggagatta cgacgatcgt aaaaggatct
//
