LOCUS       sequence-305196-        7295 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Control ("C") lacking sg-4.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 7295)
  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 7295)
  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: pJ2040_N2only
FEATURES             Location/Qualifiers
     source          1..7295
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     CDS             complement(20..898)
                     /codon_start=1
                     /gene="araC"
                     /product="L-arabinose regulatory protein"
                     /label=araC
                     /translation="MAEAQNDPLLPGYSFNAHLVAGLTPIEANGYLDFFIDRPLGMKGY
                     ILNLTIRGQGVVKNQGREFVCRPGDILLFPPGEIHHYGRHPEAREWYHQWVYFRPRAYW
                     HEWLNWPSIFANTGFFRPDEAHQPHFSDLFGQIINAGQGEGRYSELLAINLLEQLLLRR
                     MEAINESLHPPMDNRVREACQYISDHLADSNFDIASVAQHVCLSPSRLSHLFRQQLGIS
                     VLSWREDQRISQAKLLLSTTRMPIATVGRNVGFDDQLYFSRVFKKCTGASPSEFRAGCE
                     EKVNDVAVKLS"
     promoter        925..1209
                     /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     1132..1151
                     /label=pBAD Forward
                     /note="For vectors with E. coli araBAD promoter, forward 
                     primer"
     protein_bind    1318..1337
                     /label=Csy4 Site
                     /bound_moiety="Pseudomonas aeruginosa Csy4 (Cas6f)"
                     /note="cleavage site for the Csy4 endoribonuclease (Cermak 
                     et al. 2017)"
     RBS             1342..1364
                     /note="efficient ribosome binding site from bacteriophage 
                     T7 gene 10 (Olins and Rangwala, 1989)"
     CDS             1372..2025
                     /codon_start=1
                     /product="monomeric Kusabira-Orange 2 fluorescent protein"
                     /label=mKO2
                     /translation="MVSVIKPEMKMRYYMDGSVNGHEFTIEGEGTGRPYEGHQEMTLRV
                     TMAEGGPMPFAFDLVSHVFCYGHRVFTKYPEEIPDYFKQAFPEGLSWERSLEFEDGGSA
                     SVSAHISLRGNTFYHKSKFTGVNFPADGPIMQNQSVDWEPSTEKITASDGVLKGDVTMY
                     LKLEGGGNHKCQMKTTYKAAKEILEMPGDHYIGHRLVRKTEGNITEQVEDAVAHS"
     promoter        2745..2779
                     /label=BBa_J23102
                     /note="Anderson constitutive promoter 
                     (https://parts.igem.org/Promoters/Catalog/Anderson). 
                     Measured strength relative to BBa_J223100: 0.86"
     protein_bind    2929..2948
                     /label=Csy4 Site
                     /bound_moiety="Pseudomonas aeruginosa Csy4 (Cas6f)"
                     /note="cleavage site for the Csy4 endoribonuclease (Cermak 
                     et al. 2017)"
     RBS             2953..2975
                     /note="efficient ribosome binding site from bacteriophage 
                     T7 gene 10 (Olins and Rangwala, 1989)"
     CDS             2983..3696
                     /codon_start=1
                     /product="GFP variant that folds robustly even when fused 
                     to poorly folded proteins (Pedelacq et al., 2006)"
                     /label=superfolder GFP
                     /translation="MRKGEELFTGVVPILVELDGDVNGHKFSVRGEGEGDATNGKLTLK
                     FICTTGKLPVPWPTLVTTLTYGVQCFARYPDHMKQHDFFKSAMPEGYVQERTISFKDDG
                     TYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNFNSHNVYITADKQKNGIKA
                     NFKIRHNVEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSVLSKDPNEKRDHMVLLE
                     FVTAAGITHGMDELYK"
     protein_bind    4116..4135
                     /label=Csy4 Site
                     /bound_moiety="Pseudomonas aeruginosa Csy4 (Cas6f)"
                     /note="cleavage site for the Csy4 endoribonuclease (Cermak 
                     et al. 2017)"
     RBS             4140..4162
                     /note="efficient ribosome binding site from bacteriophage 
                     T7 gene 10 (Olins and Rangwala, 1989)"
     terminator      5389..5416
                     /label=rrnB T2 terminator
                     /note="transcription terminator T2 from the E. coli rrnB 
                     gene"
     CDS             complement(5604..6419)
                     /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(5604..6419)
                     /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     6327..6346
                     /label=Kan-R
                     /note="Kanamycin resistance gene, reverse primer"
     promoter        complement(6420..6511)
                     /gene="bla"
                     /label=AmpR promoter
     rep_origin      complement(6529..7164)
                     /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(6674..6693)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
ORIGIN
        1 gggtctgatt cgttaccaat tatgacaact tgacggctac atcattcact ttttcttcac
       61 aaccggcacg gaactcgctc gggctggccc cggtgcattt tttaaatacc cgcgagaaat
      121 agagttgatc gtcaaaacca acattgcgac cgacggtggc gataggcatc cgggtggtgc
      181 tcaaaagcag cttcgcctgg ctgatacgtt ggtcctcgcg ccagcttaag acgctaatcc
      241 ctaactgctg gcggaaaaga tgtgacagac gcgacggcga caagcaaaca tgctgtgcga
      301 cgctggcgat atcaaaattg ctgtctgcca ggtgatcgct gatgtactga caagcctcgc
      361 gtacccgatt atccatcggt ggatggagcg actcgttaat cgcttccatg cgccgcagta
      421 acaattgctc aagcagattt atcgccagca gctccgaata gcgcccttcc ccttgcccgg
      481 cgttaatgat ttgcccaaac aggtcgctga aatgcggctg gtgcgcttca tccgggcgaa
      541 agaaccccgt attggcaaat attgacggcc agttaagcca ttcatgccag taggcgcgcg
      601 gacgaaagta aacccactgg tgataccatt cgcgagcctc cggatgacga ccgtagtgat
      661 gaatctctcc tggcgggaac agcaaaatat cacccggtcg gcaaacaaat tctcgtccct
      721 gatttttcac caccccctga ccgcgaatgg tgagattgag aatataacct ttcattccca
      781 gcggtcggtc gataaaaaaa tcgagataac cgttggcctc aatcggcgtt aaacccgcca
      841 ccagatgggc attaaacgag tatcccggca gcaggggatc attttgcgct tcagccatac
      901 ttttcatact cccgccattc agagaagaaa ccaattgtcc atattgcatc agacattgcc
      961 gtcactgcgt cttttactgg ctcttctcgc taaccaaacc ggtaaccccg cttattaaaa
     1021 gcattctgta acaaagcggg accaaagcca tgacaaaaac gcgtaacaaa agtgtctata
     1081 atcacggcag aaaagtccac attgattatt tgcacggcgt cacactttgc tatgccatag
     1141 catttttatc cataagatta gcggttccta cctgacgctt tttatcgcaa ctctctactg
     1201 tttctccata ccgaattcat aggatagatt ctggaaactt taccgtccga gctcaggctt
     1261 accttactcg agcaataaac agttgatagg gcttctccgt tacagcctgc ggtccgggtt
     1321 cactgccgta taggcagtaa ttttgtttaa ctttaagaag gagatataca tatggtttcg
     1381 gttatcaaac cagagatgaa aatgcgttac tatatggatg gttcagtaaa tggtcacgaa
     1441 tttactattg agggcgaggg tacgggacgc ccatacgagg ggcaccagga aatgacttta
     1501 cgcgtcacaa tggctgaagg cgggcctatg ccgtttgcgt tcgatcttgt tagtcatgtc
     1561 ttttgttacg gtcaccgtgt atttactaaa taccccgagg aaattccaga ctatttcaaa
     1621 caagccttcc cggaaggttt gtcttgggag cgcagtttag agtttgaaga cggtggctcg
     1681 gccagcgtgt cagctcatat tagtcttcgc ggcaatacat tttatcacaa gtcaaagttc
     1741 accggcgtga acttccccgc agacggccca atcatgcaga atcaaagtgt tgattgggaa
     1801 ccgtccacag agaagattac agcttccgat ggagtcttaa agggcgatgt aaccatgtac
     1861 ttaaaattag aagggggagg gaaccataaa tgtcagatga agactaccta taaggccgca
     1921 aaagagattc ttgaaatgcc cggagaccac tacattgggc atcgtttggt ccgtaagaca
     1981 gaaggaaata ttactgaaca ggtcgaagac gctgtggcac acagcatgtc ccgccgtaat
     2041 actgacgcca tcacaatcca cagcatcctg gattggattg aagacaactt ggagtcgccg
     2101 ttgagtttag aaaaagttag tgaacgtagt ggttactcaa agtggcacct tcagcgcatg
     2161 tttaagaagg aaacgggtca ttcattgggt caatatattc gttctcgcaa gatgactgaa
     2221 attgcccaga aattgaaaga gtctaatgaa cctattttgt acctggcgga gcgttacggc
     2281 tttgaaagtc agcaaaccct tacacgtacc ttcaagaatt actttgacgt tccaccacac
     2341 aaatatcgta tgaccaacat gcagggtgag tcacgttttt tgcatccgtt gaatcattac
     2401 aattcctaat aatcgctggg acgcccgcca tggttcagcc aaaaaactta agaccgccgg
     2461 tcttgtccac taccttgcag taatgcggtg gacaggatcg gcggttttct tttctcttct
     2521 caattcttct gacctgtaac gaataataga tagtaaagta gtctccgatt gagttttctc
     2581 tgccgagtcc cacccagttc tgtgatttca gtaagttggt aattgataca ctgttgcgag
     2641 aactgctgcc tggtagtaga taggttgtta ttgagtaaga aggtaaagtg aacgaaatcc
     2701 ctgaaactga gactgtagaa aataagcttc agcctgcggt ccggttgaca gctagctcag
     2761 tcctaggtac tgtgctagct cgctgggacg cccggggact acacttacga aactattgat
     2821 tgctcagcct gcggtccggc catacatact ctaagatgtg tctcgctggg acgcccggga
     2881 tccaagagat ttctacacga ttgagcactg tctcagcctg cggtccgggt tcactgccgt
     2941 ataggcagta attttgttta actttaagaa ggagatatac atatgcgtaa aggcgaagaa
     3001 ctgtttaccg gtgtggttcc gattctggtg gaactggacg gcgatgttaa tggtcataaa
     3061 ttcagtgttc gcggcgaagg tgaaggcgat gcgacgaacg gcaaactgac cctgaaattt
     3121 atctgcacca cgggtaaact gccggtcccg tggccgacgc tggtgaccac gctgacctat
     3181 ggcgttcaat gttttgcgcg ttacccggat cacatgaaac agcacgactt tttcaaatcg
     3241 gccatgccgg aaggctatgt gcaggaacgt acgattagct ttaaagacga tggtacgtat
     3301 aaaacccgcg cggaagtgaa attcgaaggc gataccctgg ttaaccgtat cgaactgaaa
     3361 ggtatcgatt tcaaagaaga cggcaatatt ctgggtcata aactggaata taacttcaat
     3421 tcccacaacg tgtacatcac cgcggataaa cagaaaaacg gcattaaagc caatttcaaa
     3481 atccgccata atgtggaaga tggtagcgtt cagctggccg accactatca gcaaaacacg
     3541 ccgattggtg atggcccggt cctgctgccg gacaatcact acctgagtac ccagtccgtg
     3601 ctgtcaaaag atccgaacga aaaacgtgac cacatggtcc tgctggaatt tgtgacggct
     3661 gcgggtatca cccacggcat ggacgaactg tataaaatgt cccgccgtaa tactgacgcc
     3721 atcacaatcc acagcatcct ggattggatt gaagactaat aatcgctggg acgcccgcct
     3781 gcaggctcgg taccaaattc cagaaaagag gcctcccgaa aggggggcct tttttcgttt
     3841 tggtcctaat agataaagga taggtctggt agtgttgttc gttctcgcag gtaaatcaat
     3901 aatactcagc agttccgtag acttttcagt gggacagggt agcgataaca gatagattgt
     3961 aataagacac agtaggtgct cgtagttgcg tgaagagaac cgctcaggaa atccagtcag
     4021 aagtattggt aatcgttgaa aactcagtcg acgcacttac tgaagacgtc ctattacact
     4081 cgtcgttgga aactgaagat cagcctgcgg tccgggttca ctgccgtata ggcagtaatt
     4141 ttgtttaact ttaagaagga gatatacata tgaatcagtc attcatctcg gacatcttat
     4201 atgccgacat cgaatcgaag gctaaggaac ttacagtcaa ttccaacaat actgtccagc
     4261 cggtcgcgct tatgcgctta ggagttttcg ttcccaaacc ttccaagagc aaaggagaaa
     4321 gtaaggaaat tgacgccacc aaagccttct ctcaactgga gattgctaaa gcagagggca
     4381 tggttagtaa aggagaagaa aataacatgg cactgattaa ggagaacatg cacatgaagc
     4441 tgtacatgga gggcaccgtg aacaaccacc acttcaagtg cacatccgag ggcgaaggca
     4501 agccctacga gggcacccag accatgagaa tcaaggccgt cgagggcggc cctctcccct
     4561 tcgccttcga catcctggct accagcttca tgtacggcag caaaaccttc atcaaccaca
     4621 cccagggcat ccccgacttc tttaagcagt ccttccctga gggcttcaca tgggagagag
     4681 tcaccacata cgaagacggg ggcgtgctga ccgctaccca ggacaccagc ctccaggacg
     4741 gctgcctcat ctacaacgtc aagatcagag gggtgaactt cccatccaac ggccctgtga
     4801 tgcagaagaa aacactcggc tgggaggcct ccaccgagac cctgtacccc gctgacggcg
     4861 gcctggaagg cagagccgac atggccctga agctcgtggg cgggggccac ctgatctgca
     4921 acttgaagac cacatacaga tccaagaaac ccgctaagaa cctcaagatg cccggcgtct
     4981 actatgtgga cagaagactg gaaagaatca aggaggccga caaagagacc tacgtcgagc
     5041 agcacgaggt ggctgtggcc agatactgcg acctccctag caaactgggg cacagagtct
     5101 aataatcgct gggacgcccg gcggccgcgg aaacacagaa aaaagcccgc acctgacagt
     5161 gcgggctttt tttttcgacc aaaggtagcg aacgacgagt cactgttgag gataaatact
     5221 ttctctacta ggcgcctgtt acacaggtcc tcagcggcgc gcctttgtcg gtgaacgctc
     5281 tcctgagtag gacaaatccg ccgggagcgg atttgaacgt tgtgaagcaa cggcccggag
     5341 ggtggcgggc aggacgcccg ccataaactg ccaggcatca aactaagcag aaggccatcc
     5401 tgacggatgg cctttttgcg tttcagatct accggtaaac cagcaataga cataagcggc
     5461 tatttaacga ccctgccctg aaccgacgac aagctgacga ccgggtctcc gcaagtggca
     5521 cttttcgggg aaatgtgcgc ggaaccccta tttgtttatt tttctaaata cattcaaata
     5581 tgtatccgct catgaattaa ttcttagaaa aactcatcga gcatcaaatg aaactgcaat
     5641 ttattcatat caggattatc aataccatat ttttgaaaaa gccgtttctg taatgaagga
     5701 gaaaactcac cgaggcagtt ccataggatg gcaagatcct ggtatcggtc tgcgattccg
     5761 actcgtccaa catcaataca acctattaat ttcccctcgt caaaaataag gttatcaagt
     5821 gagaaatcac catgagtgac gactgaatcc ggtgagaatg gcaaaagttt atgcatttct
     5881 ttccagactt gttcaacagg ccagccatta cgctcgtcat caaaatcact cgcatcaacc
     5941 aaaccgttat tcattcgtga ttgcgcctga gcgagacgaa atacgcggtc gctgttaaaa
     6001 ggacaattac aaacaggaat cgaatgcaac cggcgcagga acactgccag cgcatcaaca
     6061 atattttcac ctgaatcagg atattcttct aatacctgga atgctgtttt cccggggatc
     6121 gcagtggtga gtaaccatgc atcatcagga gtacggataa aatgcttgat ggtcggaaga
     6181 ggcataaatt ccgtcagcca gtttagtctg accatctcat ctgtaacatc attggcaacg
     6241 ctacctttgc catgtttcag aaacaactct ggcgcatcgg gcttcccata caatcgatag
     6301 attgtcgcac ctgattgccc gacattatcg cgagcccatt tatacccata taaatcagca
     6361 tccatgttgg aatttaatcg cggcctagag caagacgttt cccgttgaat atggctcata
     6421 ctcttccttt ttcaatatta ttgaagcatt tatcagggtt attgtctcat gagcggatac
     6481 atatttgaat gtatttagaa aaataaacaa ataggcatgc tagcgcagaa acgtcctaga
     6541 agatgccagg aggatactta gcagagagac aataaggccg gagcgaagcc gtttttccat
     6601 aggctccgcc cccctgacga acatcacgaa atctgacgct caaatcagtg gtggcgaaac
     6661 ccgacaggac tataaagata ccaggcgttt ccccctgatg gctccctctt gcgctctcct
     6721 gttcccgtcc tgcggcgtcc gtgttgtggt ggaggcttta cccaaatcac cacgtcccgt
     6781 tccgtgtaga cagttcgctc caagctgggc tgtgtgcaag aaccccccgt tcagcccgac
     6841 tgctgcgcct tatccggtaa ctatcatctt gagtccaacc cggaaagaca cgacaaaacg
     6901 ccactggcag cagccattgg taactgagaa ttagtggatt tagatatcga gagtcttgaa
     6961 gtggtggcct aacagaggct acactgaaag gacagtattt ggtatctgcg ctccactaaa
     7021 gccagttacc aggttaagca gttccccaac tgacttaacc ttcgatcaaa ccgcctcccc
     7081 aggcggtttt ttcgtttaca gagcaggaga ttacgacgat cgtaaaagga tctcaagaag
     7141 atcctttacg gattcccgac accatcactc tagatttcag tgcaatttat ctcttcaaat
     7201 gtagcacctg aagtcagccc catacgatat aagttgtaat tctcatgtta gtcatgcccc
     7261 gcgcccaccg gaaggagctg actgggttgc tccta
//
