LOCUS       sequence-305230-        7295 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Control ("C") lacking sg-3.
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: pJ2039_N2only
FEATURES             Location/Qualifiers
     source          1..7295
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     CDS             complement(8..886)
                     /codon_start=1
                     /gene="araC"
                     /product="L-arabinose regulatory protein"
                     /label=araC
                     /translation="MAEAQNDPLLPGYSFNAHLVAGLTPIEANGYLDFFIDRPLGMKGY
                     ILNLTIRGQGVVKNQGREFVCRPGDILLFPPGEIHHYGRHPEAREWYHQWVYFRPRAYW
                     HEWLNWPSIFANTGFFRPDEAHQPHFSDLFGQIINAGQGEGRYSELLAINLLEQLLLRR
                     MEAINESLHPPMDNRVREACQYISDHLADSNFDIASVAQHVCLSPSRLSHLFRQQLGIS
                     VLSWREDQRISQAKLLLSTTRMPIATVGRNVGFDDQLYFSRVFKKCTGASPSEFRAGCE
                     EKVNDVAVKLS"
     promoter        913..1197
                     /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     1120..1139
                     /label=pBAD Forward
                     /note="For vectors with E. coli araBAD promoter, forward 
                     primer"
     protein_bind    1306..1325
                     /label=Csy4 Site
                     /bound_moiety="Pseudomonas aeruginosa Csy4 (Cas6f)"
                     /note="cleavage site for the Csy4 endoribonuclease (Cermak 
                     et al. 2017)"
     RBS             1330..1352
                     /note="efficient ribosome binding site from bacteriophage 
                     T7 gene 10 (Olins and Rangwala, 1989)"
     CDS             1360..2013
                     /codon_start=1
                     /product="monomeric Kusabira-Orange 2 fluorescent protein"
                     /label=mKO2
                     /translation="MVSVIKPEMKMRYYMDGSVNGHEFTIEGEGTGRPYEGHQEMTLRV
                     TMAEGGPMPFAFDLVSHVFCYGHRVFTKYPEEIPDYFKQAFPEGLSWERSLEFEDGGSA
                     SVSAHISLRGNTFYHKSKFTGVNFPADGPIMQNQSVDWEPSTEKITASDGVLKGDVTMY
                     LKLEGGGNHKCQMKTTYKAAKEILEMPGDHYIGHRLVRKTEGNITEQVEDAVAHS"
     promoter        2733..2767
                     /label=BBa_J23102
                     /note="Anderson constitutive promoter 
                     (https://parts.igem.org/Promoters/Catalog/Anderson). 
                     Measured strength relative to BBa_J223100: 0.86"
     protein_bind    2917..2936
                     /label=Csy4 Site
                     /bound_moiety="Pseudomonas aeruginosa Csy4 (Cas6f)"
                     /note="cleavage site for the Csy4 endoribonuclease (Cermak 
                     et al. 2017)"
     RBS             2941..2963
                     /note="efficient ribosome binding site from bacteriophage 
                     T7 gene 10 (Olins and Rangwala, 1989)"
     CDS             2971..3684
                     /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    4104..4123
                     /label=Csy4 Site
                     /bound_moiety="Pseudomonas aeruginosa Csy4 (Cas6f)"
                     /note="cleavage site for the Csy4 endoribonuclease (Cermak 
                     et al. 2017)"
     RBS             4128..4150
                     /note="efficient ribosome binding site from bacteriophage 
                     T7 gene 10 (Olins and Rangwala, 1989)"
     terminator      5377..5404
                     /label=rrnB T2 terminator
                     /note="transcription terminator T2 from the E. coli rrnB 
                     gene"
     CDS             complement(5592..6407)
                     /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(5592..6407)
                     /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     6315..6334
                     /label=Kan-R
                     /note="Kanamycin resistance gene, reverse primer"
     promoter        complement(6408..6499)
                     /gene="bla"
                     /label=AmpR promoter
     rep_origin      complement(6517..7152)
                     /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(6662..6681)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
ORIGIN
        1 ttaccaatta tgacaacttg acggctacat cattcacttt ttcttcacaa ccggcacgga
       61 actcgctcgg gctggccccg gtgcattttt taaatacccg cgagaaatag agttgatcgt
      121 caaaaccaac attgcgaccg acggtggcga taggcatccg ggtggtgctc aaaagcagct
      181 tcgcctggct gatacgttgg tcctcgcgcc agcttaagac gctaatccct aactgctggc
      241 ggaaaagatg tgacagacgc gacggcgaca agcaaacatg ctgtgcgacg ctggcgatat
      301 caaaattgct gtctgccagg tgatcgctga tgtactgaca agcctcgcgt acccgattat
      361 ccatcggtgg atggagcgac tcgttaatcg cttccatgcg ccgcagtaac aattgctcaa
      421 gcagatttat cgccagcagc tccgaatagc gcccttcccc ttgcccggcg ttaatgattt
      481 gcccaaacag gtcgctgaaa tgcggctggt gcgcttcatc cgggcgaaag aaccccgtat
      541 tggcaaatat tgacggccag ttaagccatt catgccagta ggcgcgcgga cgaaagtaaa
      601 cccactggtg ataccattcg cgagcctccg gatgacgacc gtagtgatga atctctcctg
      661 gcgggaacag caaaatatca cccggtcggc aaacaaattc tcgtccctga tttttcacca
      721 ccccctgacc gcgaatggtg agattgagaa tataaccttt cattcccagc ggtcggtcga
      781 taaaaaaatc gagataaccg ttggcctcaa tcggcgttaa acccgccacc agatgggcat
      841 taaacgagta tcccggcagc aggggatcat tttgcgcttc agccatactt ttcatactcc
      901 cgccattcag agaagaaacc aattgtccat attgcatcag acattgccgt cactgcgtct
      961 tttactggct cttctcgcta accaaaccgg taaccccgct tattaaaagc attctgtaac
     1021 aaagcgggac caaagccatg acaaaaacgc gtaacaaaag tgtctataat cacggcagaa
     1081 aagtccacat tgattatttg cacggcgtca cactttgcta tgccatagca tttttatcca
     1141 taagattagc ggttcctacc tgacgctttt tatcgcaact ctctactgtt tctccatacc
     1201 gaattcatag gatagattct ggaaacttta ccgtccgagc tcaggcttac cttactcgag
     1261 caataaacag ttgatagggc ttctccgtta cagcctgcgg tccgggttca ctgccgtata
     1321 ggcagtaatt ttgtttaact ttaagaagga gatatacata tggtttcggt tatcaaacca
     1381 gagatgaaaa tgcgttacta tatggatggt tcagtaaatg gtcacgaatt tactattgag
     1441 ggcgagggta cgggacgccc atacgagggg caccaggaaa tgactttacg cgtcacaatg
     1501 gctgaaggcg ggcctatgcc gtttgcgttc gatcttgtta gtcatgtctt ttgttacggt
     1561 caccgtgtat ttactaaata ccccgaggaa attccagact atttcaaaca agccttcccg
     1621 gaaggtttgt cttgggagcg cagtttagag tttgaagacg gtggctcggc cagcgtgtca
     1681 gctcatatta gtcttcgcgg caatacattt tatcacaagt caaagttcac cggcgtgaac
     1741 ttccccgcag acggcccaat catgcagaat caaagtgttg attgggaacc gtccacagag
     1801 aagattacag cttccgatgg agtcttaaag ggcgatgtaa ccatgtactt aaaattagaa
     1861 gggggaggga accataaatg tcagatgaag actacctata aggccgcaaa agagattctt
     1921 gaaatgcccg gagaccacta cattgggcat cgtttggtcc gtaagacaga aggaaatatt
     1981 actgaacagg tcgaagacgc tgtggcacac agcatgtccc gccgtaatac tgacgccatc
     2041 acaatccaca gcatcctgga ttggattgaa gacaacttgg agtcgccgtt gagtttagaa
     2101 aaagttagtg aacgtagtgg ttactcaaag tggcaccttc agcgcatgtt taagaaggaa
     2161 acgggtcatt cattgggtca atatattcgt tctcgcaaga tgactgaaat tgcccagaaa
     2221 ttgaaagagt ctaatgaacc tattttgtac ctggcggagc gttacggctt tgaaagtcag
     2281 caaaccctta cacgtacctt caagaattac tttgacgttc caccacacaa atatcgtatg
     2341 accaacatgc agggtgagtc acgttttttg catccgttga atcattacaa ttcctaataa
     2401 tcgctgggac gcccgccatg gttcagccaa aaaacttaag accgccggtc ttgtccacta
     2461 ccttgcagta atgcggtgga caggatcggc ggttttcttt tctcttctca attcttctga
     2521 cctgtaacga ataatagata gtaaagtagt ctccgattga gttttctctg ccgagtccca
     2581 cccagttctg tgatttcagt aagttggtaa ttgatacact gttgcgagaa ctgctgcctg
     2641 gtagtagata ggttgttatt gagtaagaag gtaaagtgaa cgaaatccct gaaactgaga
     2701 ctgtagaaaa taagcttcag cctgcggtcc ggttgacagc tagctcagtc ctaggtactg
     2761 tgctagctcg ctgggacgcc cggggactac acttacgaaa ctattgattg ctcagcctgc
     2821 ggtccggcca agagtacact agctcagtca tcgctgggac gcccgggatc caagagattt
     2881 ctacacgatt gagcactgtc tcagcctgcg gtccgggttc actgccgtat aggcagtaat
     2941 tttgtttaac tttaagaagg agatatacat atgcgtaaag gcgaagaact gtttaccggt
     3001 gtggttccga ttctggtgga actggacggc gatgttaatg gtcataaatt cagtgttcgc
     3061 ggcgaaggtg aaggcgatgc gacgaacggc aaactgaccc tgaaatttat ctgcaccacg
     3121 ggtaaactgc cggtcccgtg gccgacgctg gtgaccacgc tgacctatgg cgttcaatgt
     3181 tttgcgcgtt acccggatca catgaaacag cacgactttt tcaaatcggc catgccggaa
     3241 ggctatgtgc aggaacgtac gattagcttt aaagacgatg gtacgtataa aacccgcgcg
     3301 gaagtgaaat tcgaaggcga taccctggtt aaccgtatcg aactgaaagg tatcgatttc
     3361 aaagaagacg gcaatattct gggtcataaa ctggaatata acttcaattc ccacaacgtg
     3421 tacatcaccg cggataaaca gaaaaacggc attaaagcca atttcaaaat ccgccataat
     3481 gtggaagatg gtagcgttca gctggccgac cactatcagc aaaacacgcc gattggtgat
     3541 ggcccggtcc tgctgccgga caatcactac ctgagtaccc agtccgtgct gtcaaaagat
     3601 ccgaacgaaa aacgtgacca catggtcctg ctggaatttg tgacggctgc gggtatcacc
     3661 cacggcatgg acgaactgta taaaatgtcc cgccgtaata ctgacgccat cacaatccac
     3721 agcatcctgg attggattga agactaataa tcgctgggac gcccgcctgc aggctcggta
     3781 ccaaattcca gaaaagaggc ctcccgaaag gggggccttt tttcgttttg gtcctaatag
     3841 ataaaggata ggtctggtag tgttgttcgt tctcgcaggt aaatcaataa tactcagcag
     3901 ttccgtagac ttttcagtgg gacagggtag cgataacaga tagattgtaa taagacacag
     3961 taggtgctcg tagttgcgtg aagagaaccg ctcaggaaat ccagtcagaa gtattggtaa
     4021 tcgttgaaaa ctcagtcgac gcacttactg aagacgtcct attacactcg tcgttggaaa
     4081 ctgaagatca gcctgcggtc cgggttcact gccgtatagg cagtaatttt gtttaacttt
     4141 aagaaggaga tatacatatg aatcagtcat tcatctcgga catcttatat gccgacatcg
     4201 aatcgaaggc taaggaactt acagtcaatt ccaacaatac tgtccagccg gtcgcgctta
     4261 tgcgcttagg agttttcgtt cccaaacctt ccaagagcaa aggagaaagt aaggaaattg
     4321 acgccaccaa agccttctct caactggaga ttgctaaagc agagggcatg gttagtaaag
     4381 gagaagaaaa taacatggca ctgattaagg agaacatgca catgaagctg tacatggagg
     4441 gcaccgtgaa caaccaccac ttcaagtgca catccgaggg cgaaggcaag ccctacgagg
     4501 gcacccagac catgagaatc aaggccgtcg agggcggccc tctccccttc gccttcgaca
     4561 tcctggctac cagcttcatg tacggcagca aaaccttcat caaccacacc cagggcatcc
     4621 ccgacttctt taagcagtcc ttccctgagg gcttcacatg ggagagagtc accacatacg
     4681 aagacggggg cgtgctgacc gctacccagg acaccagcct ccaggacggc tgcctcatct
     4741 acaacgtcaa gatcagaggg gtgaacttcc catccaacgg ccctgtgatg cagaagaaaa
     4801 cactcggctg ggaggcctcc accgagaccc tgtaccccgc tgacggcggc ctggaaggca
     4861 gagccgacat ggccctgaag ctcgtgggcg ggggccacct gatctgcaac ttgaagacca
     4921 catacagatc caagaaaccc gctaagaacc tcaagatgcc cggcgtctac tatgtggaca
     4981 gaagactgga aagaatcaag gaggccgaca aagagaccta cgtcgagcag cacgaggtgg
     5041 ctgtggccag atactgcgac ctccctagca aactggggca cagagtctaa taatcgctgg
     5101 gacgcccggc ggccgcggaa acacagaaaa aagcccgcac ctgacagtgc gggctttttt
     5161 tttcgaccaa aggtagcgaa cgacgagtca ctgttgagga taaatacttt ctctactagg
     5221 cgcctgttac acaggtcctc agcggcgcgc ctttgtcggt gaacgctctc ctgagtagga
     5281 caaatccgcc gggagcggat ttgaacgttg tgaagcaacg gcccggaggg tggcgggcag
     5341 gacgcccgcc ataaactgcc aggcatcaaa ctaagcagaa ggccatcctg acggatggcc
     5401 tttttgcgtt tcagatctac cggtaaacca gcaatagaca taagcggcta tttaacgacc
     5461 ctgccctgaa ccgacgacaa gctgacgacc gggtctccgc aagtggcact tttcggggaa
     5521 atgtgcgcgg aacccctatt tgtttatttt tctaaataca ttcaaatatg tatccgctca
     5581 tgaattaatt cttagaaaaa ctcatcgagc atcaaatgaa actgcaattt attcatatca
     5641 ggattatcaa taccatattt ttgaaaaagc cgtttctgta atgaaggaga aaactcaccg
     5701 aggcagttcc ataggatggc aagatcctgg tatcggtctg cgattccgac tcgtccaaca
     5761 tcaatacaac ctattaattt cccctcgtca aaaataaggt tatcaagtga gaaatcacca
     5821 tgagtgacga ctgaatccgg tgagaatggc aaaagtttat gcatttcttt ccagacttgt
     5881 tcaacaggcc agccattacg ctcgtcatca aaatcactcg catcaaccaa accgttattc
     5941 attcgtgatt gcgcctgagc gagacgaaat acgcggtcgc tgttaaaagg acaattacaa
     6001 acaggaatcg aatgcaaccg gcgcaggaac actgccagcg catcaacaat attttcacct
     6061 gaatcaggat attcttctaa tacctggaat gctgttttcc cggggatcgc agtggtgagt
     6121 aaccatgcat catcaggagt acggataaaa tgcttgatgg tcggaagagg cataaattcc
     6181 gtcagccagt ttagtctgac catctcatct gtaacatcat tggcaacgct acctttgcca
     6241 tgtttcagaa acaactctgg cgcatcgggc ttcccataca atcgatagat tgtcgcacct
     6301 gattgcccga cattatcgcg agcccattta tacccatata aatcagcatc catgttggaa
     6361 tttaatcgcg gcctagagca agacgtttcc cgttgaatat ggctcatact cttccttttt
     6421 caatattatt gaagcattta tcagggttat tgtctcatga gcggatacat atttgaatgt
     6481 atttagaaaa ataaacaaat aggcatgcta gcgcagaaac gtcctagaag atgccaggag
     6541 gatacttagc agagagacaa taaggccgga gcgaagccgt ttttccatag gctccgcccc
     6601 cctgacgaac atcacgaaat ctgacgctca aatcagtggt ggcgaaaccc gacaggacta
     6661 taaagatacc aggcgtttcc ccctgatggc tccctcttgc gctctcctgt tcccgtcctg
     6721 cggcgtccgt gttgtggtgg aggctttacc caaatcacca cgtcccgttc cgtgtagaca
     6781 gttcgctcca agctgggctg tgtgcaagaa ccccccgttc agcccgactg ctgcgcctta
     6841 tccggtaact atcatcttga gtccaacccg gaaagacacg acaaaacgcc actggcagca
     6901 gccattggta actgagaatt agtggattta gatatcgaga gtcttgaagt ggtggcctaa
     6961 cagaggctac actgaaagga cagtatttgg tatctgcgct ccactaaagc cagttaccag
     7021 gttaagcagt tccccaactg acttaacctt cgatcaaacc gcctccccag gcggtttttt
     7081 cgtttacaga gcaggagatt acgacgatcg taaaaggatc tcaagaagat cctttacgga
     7141 ttcccgacac catcactcta gatttcagtg caatttatct cttcaaatgt agcacctgaa
     7201 gtcagcccca tacgatataa gttgtaattc tcatgttagt catgccccgc gcccaccgga
     7261 aggagctgac tgggttgctc ctagggtctg attcg
//
