LOCUS       sequence-273319-        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        58..92
                     /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    236..255
                     /label=Csy4 Site
                     /bound_moiety="Pseudomonas aeruginosa Csy4 (Cas6f)"
                     /note="cleavage site for the Csy4 endoribonuclease (Cermak 
                     et al. 2017)"
     protein_bind    362..381
                     /label=Csy4 Site
                     /bound_moiety="Pseudomonas aeruginosa Csy4 (Cas6f)"
                     /note="cleavage site for the Csy4 endoribonuclease (Cermak 
                     et al. 2017)"
     protein_bind    448..467
                     /label=Csy4 Site
                     /bound_moiety="Pseudomonas aeruginosa Csy4 (Cas6f)"
                     /note="cleavage site for the Csy4 endoribonuclease (Cermak 
                     et al. 2017)"
     RBS             472..494
                     /note="efficient ribosome binding site from bacteriophage 
                     T7 gene 10 (Olins and Rangwala, 1989)"
     CDS             502..1209
                     /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(647..665)
                     /label=mCherry-R
                     /note="mCherry, reverse primer"
     primer_bind     915..934
                     /label=mCherry-F
                     /note="mCherry, forward primer"
     promoter        1929..1963
                     /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    2107..2126
                     /label=Csy4 Site
                     /bound_moiety="Pseudomonas aeruginosa Csy4 (Cas6f)"
                     /note="cleavage site for the Csy4 endoribonuclease (Cermak 
                     et al. 2017)"
     protein_bind    2233..2252
                     /label=Csy4 Site
                     /bound_moiety="Pseudomonas aeruginosa Csy4 (Cas6f)"
                     /note="cleavage site for the Csy4 endoribonuclease (Cermak 
                     et al. 2017)"
     protein_bind    2319..2338
                     /label=Csy4 Site
                     /bound_moiety="Pseudomonas aeruginosa Csy4 (Cas6f)"
                     /note="cleavage site for the Csy4 endoribonuclease (Cermak 
                     et al. 2017)"
     RBS             2343..2365
                     /note="efficient ribosome binding site from bacteriophage 
                     T7 gene 10 (Olins and Rangwala, 1989)"
     CDS             2373..3086
                     /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        3458..3492
                     /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    3636..3655
                     /label=Csy4 Site
                     /bound_moiety="Pseudomonas aeruginosa Csy4 (Cas6f)"
                     /note="cleavage site for the Csy4 endoribonuclease (Cermak 
                     et al. 2017)"
     protein_bind    3762..3781
                     /label=Csy4 Site
                     /bound_moiety="Pseudomonas aeruginosa Csy4 (Cas6f)"
                     /note="cleavage site for the Csy4 endoribonuclease (Cermak 
                     et al. 2017)"
     protein_bind    3848..3867
                     /label=Csy4 Site
                     /bound_moiety="Pseudomonas aeruginosa Csy4 (Cas6f)"
                     /note="cleavage site for the Csy4 endoribonuclease (Cermak 
                     et al. 2017)"
     RBS             3872..3894
                     /note="efficient ribosome binding site from bacteriophage 
                     T7 gene 10 (Olins and Rangwala, 1989)"
     primer_bind     4765..4785
                     /label=GFP-F
                     /note="GFP, forward primer. Does NOT anneal to EGFP"
     terminator      5115..5142
                     /label=rrnB T2 terminator
                     /note="transcription terminator T2 from the E. coli rrnB 
                     gene"
     CDS             complement(5330..6145)
                     /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(5330..6145)
                     /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     6053..6072
                     /label=Kan-R
                     /note="Kanamycin resistance gene, reverse primer"
     promoter        complement(6146..6237)
                     /gene="bla"
                     /label=AmpR promoter
     rep_origin      complement(6255..6890)
                     /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(6400..6419)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
ORIGIN
        1 gaattcatag gatagattct ggaaacttta ccgtccgagc tccagcctgc ggtccggttg
       61 acggctagct cagtcctagg tacagtgcta gctcgctggg acgcccgaga gccgaatcgc
      121 acttatttac agtagttcag cctgcggtcc ggccatacat actctaagat gtgtctcgct
      181 gggacgcccg gggactacac ttacgaaact attgattgct cagcctgcgg tccgggttca
      241 ctgccgtata ggcagatcag tgtgtactaa gtactgtttc agagctatgc tggaaacagc
      301 atagcaagtt gaaataaggc tagtccgtta tcaacttgaa aaagtggcac cgagtcggtg
      361 cgttcactgc cgtataggca gtcgctggga cgcccgctcg agcaataaac agttgatagg
      421 gcttctccgt tacagcctgc ggtccgggtt cactgccgta taggcagtaa ttttgtttaa
      481 ctttaagaag gagatataca tatggtgagc aagggcgagg aggataacat ggccatcatc
      541 aaggagttca tgcgcttcaa ggtgcacatg gagggctccg tgaacggcca cgagttcgag
      601 atcgagggcg agggcgaggg ccgcccctac gagggcaccc agaccgccaa gctgaaggtg
      661 accaagggtg gccccctgcc cttcgcctgg gacatcctgt cccctcagtt catgtacggc
      721 tccaaggcct acgtgaagca ccccgccgac atccccgact acttgaagct gtccttcccc
      781 gagggcttca agtgggagcg cgtgatgaac ttcgaggacg gcggcgtggt gaccgtgacc
      841 caggactcct ctctgcagga cggcgagttc atctacaagg tgaagctgcg cggcaccaac
      901 ttcccctccg acggccccgt aatgcagaag aagactatgg gctgggaggc ctcctccgag
      961 cggatgtacc ccgaggacgg cgcgctgaag ggcgagatca agcagaggct gaagctgaag
     1021 gacggcggcc actacgacgc tgaagtcaag accacctaca aggccaagaa gcccgtgcag
     1081 ctgcccggcg cgtacaacgt caacatcaag ttggacatca cctcccacaa cgaggactac
     1141 accatcgtgg aacagtacga acgcgccgag ggccgccact ccaccggcgg catggacgag
     1201 ctgtacaaga tgtcccgccg taatactgac gccatcacaa tccacagcat cctggattgg
     1261 attgaagaca acttggagtc gccgttgagt ttagaaaaag ttagtgaacg tagtggttac
     1321 tcaaagtggc accttcagcg catgtttaag aaggaaacgg gtcattcatt gggtcaatat
     1381 attcgttctc gcaagatgac tgaaattgcc cagaaattga aagagtctaa tgaacctatt
     1441 ttgtacctgg cggagcgtta cggctttgaa agtcagcaaa cccttacacg taccttcaag
     1501 aattactttg acgttccacc acacaaatat cgtatgacca acatgcaggg tgagtcacgt
     1561 tttttgcatc cgttgaatca ttacaattcc taataatcgc tgggacgccc gccatggttc
     1621 agccaaaaaa cttaagaccg ccggtcttgt ccactacctt gcagtaatgc ggtggacagg
     1681 atcggcggtt ttcttttctc ttctcaattc ttctgacctg taacgaataa tagatagtaa
     1741 agtagtctcc gattgagttt tctctgccga gtcccaccca gttctgtgat ttcagtaagt
     1801 tggtaattga tacactgttg cgagaactgc tgcctggtag tagataggtt gttattgagt
     1861 aagaaggtaa agtgaacgaa atccctgaaa ctgagactgt agaaaataag cttcagcctg
     1921 cggtccggtt gacggctagc tcagtcctag gtacagtgct agctcgctgg gacgcccgag
     1981 atagccgtta cacaggtgac acttatttca gcctgcggtc cggccacact agagtctagc
     2041 ttgagatcgc tgggacgccc gtgaacagtt gctctgattg aaaccacgat tcagcctgcg
     2101 gtccgggttc actgccgtat aggcagtgac tgagctagtg tactctgttt cagagctatg
     2161 ctggaaacag catagcaagt tgaaataagg ctagtccgtt atcaacttga aaaagtggca
     2221 ccgagtcggt gcgttcactg ccgtataggc agtcgctggg acgcccggga tccaagagat
     2281 ttctacacga ttgagcactg tctcagcctg cggtccgggt tcactgccgt ataggcagta
     2341 attttgttta actttaagaa ggagatatac atatgtctaa aggtgaagaa ttattcactg
     2401 gtgttgtccc aattttggtt gaattagatg gtgatgttaa tggtcacaaa ttttctgtct
     2461 ccggtgaagg tgaaggtgat gctacttacg gtaaattgac cttaaaattt atttgtacta
     2521 ctggtaaatt gccagttccg tggccaacct tagtcactac tttaggttat ggtttgatgt
     2581 gttttgctag atacccagat catatgaaac aacatgactt tttcaagtct gccatgccag
     2641 aaggttatgt tcaagaaaga actatttttt tcaaagatga cggtaactac aagaccagag
     2701 ctgaagtcaa gtttgaaggt gataccttag ttaatagaat cgaattaaaa ggtattgatt
     2761 ttaaagaaga tggtaacatt ttaggtcaca aattggaata caactataac tctcacaatg
     2821 tttacatcat ggctgacaaa caaaagaatg gtatcaaagt taacttcaaa attagacaca
     2881 acattgaaga tggttctgtt caattagctg accattatca acaaaatact ccaattggtg
     2941 atggtccagt cttgttacca gacaaccatt acttatccta tcaatctaaa ttatccaaag
     3001 atccaaacga aaagagagac cacatggtct tgttagaatt tgttactgct gctggtatta
     3061 cccacggtat ggatgaattg tacaaaatgt cccgccgtaa tactgacgcc atcacaatcc
     3121 acagcatcct ggattggatt gaagactaat aatcgctggg acgcccgcct gcaggctcgg
     3181 taccaaattc cagaaaagag gcctcccgaa aggggggcct tttttcgttt tggtcctaat
     3241 agataaagga taggtctggt agtgttgttc gttctcgcag gtaaatcaat aatactcagc
     3301 agttccgtag acttttcagt gggacagggt agcgataaca gatagattgt aataagacac
     3361 agtaggtgct cgtagttgcg tgaagagaac cgctcaggaa atccagtcag aagtattggt
     3421 aatcgttgaa aactcagtcg accagcctgc ggtccggttg acggctagct cagtcctagg
     3481 tacagtgcta gctcgctggg acgcccgagt gacgactgcg aagtaacctc tatttatcag
     3541 cctgcggtcc ggccaagtac ttagtacaca ctgattcgct gggacgcccg gtagtgctta
     3601 tcagacccaa tactgttgaa cagcctgcgg tccgggttca ctgccgtata ggcagtctca
     3661 agctagactc tagtggtttc agagctatgc tggaaacagc atagcaagtt gaaataaggc
     3721 tagtccgtta tcaacttgaa aaagtggcac cgagtcggtg cgttcactgc cgtataggca
     3781 gtcgctggga cgcccggacg tcctattaca ctcgtcgttg gaaactgaag atcagcctgc
     3841 ggtccgggtt cactgccgta taggcagtaa ttttgtttaa ctttaagaag gagatataca
     3901 tatgaatcag tcattcatct cggacatctt atatgccgac atcgaatcga aggctaagga
     3961 acttacagtc aattccaaca atactgtcca gccggtcgcg cttatgcgct taggagtttt
     4021 cgttcccaaa ccttccaaga gcaaaggaga aagtaaggaa attgacgcca ccaaagcctt
     4081 ctctcaactg gagattgcta aagcagaggg catgagtaaa ggagaagaac ttttcactgg
     4141 agttgtccca attcttgttg aattagatgg tgatgttaat gggcacaaat tttctgtcag
     4201 tggagagggt gaaggtgatg caacatacgg aaaacttacc cttaaattta tttgcactac
     4261 tggaaaacta cctgttccgt ggccaacact tgtcactact ttgacttggg gtgttcaatg
     4321 ctttgctaga tacccagatc atatgaaaca gcatgacttt ttcaagagtg ccatgcccga
     4381 aggttatgta caggaaagaa ctatattttt caaagatgac gggaactaca agacacgtgc
     4441 tgaagtcaag tttgaaggtg atacccttgt taatagaatc gagttaaaag gtattgattt
     4501 taaagaagat ggaaacattc ttggacacaa attggaatac aacgctattt cagataatgt
     4561 atacatcact gcagacaaac aaaagaatgg aatcaaagct aatttcaaaa ttagacacaa
     4621 cattgaagat ggaagcgttc aactagcaga ccattatcaa caaaatactc caattggcga
     4681 tggccctgtc cttttaccag acaaccatta cctgtccaca caatctaaac tttcgaaaga
     4741 tcccaacgaa aagagagacc acatggtcct tcttgagttt gtaacagctg ctgggattac
     4801 actaggcatg gatgaactat acaaataata atcgctggga cgcccggcgg ccgcggaaac
     4861 acagaaaaaa gcccgcacct gacagtgcgg gctttttttt tcgaccaaag gtagcgaacg
     4921 acgagtcact gttgaggata aatactttct ctactaggcg cctgttacac aggtcctcag
     4981 cggcgcgcct ttgtcggtga acgctctcct gagtaggaca aatccgccgg gagcggattt
     5041 gaacgttgtg aagcaacggc ccggagggtg gcgggcagga cgcccgccat aaactgccag
     5101 gcatcaaact aagcagaagg ccatcctgac ggatggcctt tttgcgtttc agatctaccg
     5161 gtaaaccagc aatagacata agcggctatt taacgaccct gccctgaacc gacgacaagc
     5221 tgacgaccgg gtctccgcaa gtggcacttt tcggggaaat gtgcgcggaa cccctatttg
     5281 tttatttttc taaatacatt caaatatgta tccgctcatg aattaattct tagaaaaact
     5341 catcgagcat caaatgaaac tgcaatttat tcatatcagg attatcaata ccatattttt
     5401 gaaaaagccg tttctgtaat gaaggagaaa actcaccgag gcagttccat aggatggcaa
     5461 gatcctggta tcggtctgcg attccgactc gtccaacatc aatacaacct attaatttcc
     5521 cctcgtcaaa aataaggtta tcaagtgaga aatcaccatg agtgacgact gaatccggtg
     5581 agaatggcaa aagtttatgc atttctttcc agacttgttc aacaggccag ccattacgct
     5641 cgtcatcaaa atcactcgca tcaaccaaac cgttattcat tcgtgattgc gcctgagcga
     5701 gacgaaatac gcggtcgctg ttaaaaggac aattacaaac aggaatcgaa tgcaaccggc
     5761 gcaggaacac tgccagcgca tcaacaatat tttcacctga atcaggatat tcttctaata
     5821 cctggaatgc tgttttcccg gggatcgcag tggtgagtaa ccatgcatca tcaggagtac
     5881 ggataaaatg cttgatggtc ggaagaggca taaattccgt cagccagttt agtctgacca
     5941 tctcatctgt aacatcattg gcaacgctac ctttgccatg tttcagaaac aactctggcg
     6001 catcgggctt cccatacaat cgatagattg tcgcacctga ttgcccgaca ttatcgcgag
     6061 cccatttata cccatataaa tcagcatcca tgttggaatt taatcgcggc ctagagcaag
     6121 acgtttcccg ttgaatatgg ctcatactct tcctttttca atattattga agcatttatc
     6181 agggttattg tctcatgagc ggatacatat ttgaatgtat ttagaaaaat aaacaaatag
     6241 gcatgctagc gcagaaacgt cctagaagat gccaggagga tacttagcag agagacaata
     6301 aggccggagc gaagccgttt ttccataggc tccgcccccc tgacgaacat cacgaaatct
     6361 gacgctcaaa tcagtggtgg cgaaacccga caggactata aagataccag gcgtttcccc
     6421 ctgatggctc cctcttgcgc tctcctgttc ccgtcctgcg gcgtccgtgt tgtggtggag
     6481 gctttaccca aatcaccacg tcccgttccg tgtagacagt tcgctccaag ctgggctgtg
     6541 tgcaagaacc ccccgttcag cccgactgct gcgccttatc cggtaactat catcttgagt
     6601 ccaacccgga aagacacgac aaaacgccac tggcagcagc cattggtaac tgagaattag
     6661 tggatttaga tatcgagagt cttgaagtgg tggcctaaca gaggctacac tgaaaggaca
     6721 gtatttggta tctgcgctcc actaaagcca gttaccaggt taagcagttc cccaactgac
     6781 ttaaccttcg atcaaaccgc ctccccaggc ggttttttcg tttacagagc aggagattac
     6841 gacgatcgta aaaggatctc aagaagatcc tttacggatt cccgacacca tcactctaga
//
