LOCUS       sequence-305226-        7295 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Control ("C") lacking sg-1.
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: pJ2044_N2only
FEATURES             Location/Qualifiers
     source          1..7295
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     promoter        complement(164..198)
                     /label=BBa_J23102
                     /note="Anderson constitutive promoter 
                     (https://parts.igem.org/Promoters/Catalog/Anderson). 
                     Measured strength relative to BBa_J223100: 0.86"
     CDS             complement(918..1571)
                     /codon_start=1
                     /product="monomeric Kusabira-Orange 2 fluorescent protein"
                     /label=mKO2
                     /translation="MVSVIKPEMKMRYYMDGSVNGHEFTIEGEGTGRPYEGHQEMTLRV
                     TMAEGGPMPFAFDLVSHVFCYGHRVFTKYPEEIPDYFKQAFPEGLSWERSLEFEDGGSA
                     SVSAHISLRGNTFYHKSKFTGVNFPADGPIMQNQSVDWEPSTEKITASDGVLKGDVTMY
                     LKLEGGGNHKCQMKTTYKAAKEILEMPGDHYIGHRLVRKTEGNITEQVEDAVAHS"
     RBS             1579..1601
                     /note="efficient ribosome binding site from bacteriophage 
                     T7 gene 10 (Olins and Rangwala, 1989)"
     protein_bind    complement(1606..1625)
                     /label=Csy4 Site
                     /bound_moiety="Pseudomonas aeruginosa Csy4 (Cas6f)"
                     /note="cleavage site for the Csy4 endoribonuclease (Cermak 
                     et al. 2017)"
     promoter        complement(1734..2018)
                     /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     complement(1792..1811)
                     /label=pBAD Forward
                     /note="For vectors with E. coli araBAD promoter, forward 
                     primer"
     CDS             2045..2923
                     /codon_start=1
                     /gene="araC"
                     /product="L-arabinose regulatory protein"
                     /label=araC
                     /translation="MAEAQNDPLLPGYSFNAHLVAGLTPIEANGYLDFFIDRPLGMKGY
                     ILNLTIRGQGVVKNQGREFVCRPGDILLFPPGEIHHYGRHPEAREWYHQWVYFRPRAYW
                     HEWLNWPSIFANTGFFRPDEAHQPHFSDLFGQIINAGQGEGRYSELLAINLLEQLLLRR
                     MEAINESLHPPMDNRVREACQYISDHLADSNFDIASVAQHVCLSPSRLSHLFRQQLGIS
                     VLSWREDQRISQAKLLLSTTRMPIATVGRNVGFDDQLYFSRVFKKCTGASPSEFRAGCE
                     EKVNDVAVKLS"
     rep_origin      3074..3709
                     /direction=RIGHT
                     /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     3545..3564
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     promoter        3727..3818
                     /gene="bla"
                     /label=AmpR promoter
     CDS             3819..4634
                     /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             3819..4634
                     /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     complement(3892..3911)
                     /label=Kan-R
                     /note="Kanamycin resistance gene, reverse primer"
     terminator      complement(4822..4849)
                     /label=rrnB T2 terminator
                     /note="transcription terminator T2 from the E. coli rrnB 
                     gene"
     RBS             6076..6098
                     /note="efficient ribosome binding site from bacteriophage 
                     T7 gene 10 (Olins and Rangwala, 1989)"
     protein_bind    complement(6103..6122)
                     /label=Csy4 Site
                     /bound_moiety="Pseudomonas aeruginosa Csy4 (Cas6f)"
                     /note="cleavage site for the Csy4 endoribonuclease (Cermak 
                     et al. 2017)"
     CDS             complement(6542..7255)
                     /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"
     RBS             7263..7285
                     /note="efficient ribosome binding site from bacteriophage 
                     T7 gene 10 (Olins and Rangwala, 1989)"
     protein_bind    complement(join(7290..7295,1..14))
                     /label=Csy4 Site
                     /bound_moiety="Pseudomonas aeruginosa Csy4 (Cas6f)"
                     /note="cleavage site for the Csy4 endoribonuclease (Cermak 
                     et al. 2017)"
ORIGIN
        1 atacggcagt gaacccggac cgcaggctga gacagtgctc aatcgtgtag aaatctcttg
       61 gatcccgggc gtcccagcga tctcaagcta gactctagtg tggccggacc gcaggctgag
      121 caatcaatag tttcgtaagt gtagtccccg ggcgtcccag cgagctagca cagtacctag
      181 gactgagcta gctgtcaacc ggaccgcagg ctgaagctta ttttctacag tctcagtttc
      241 agggatttcg ttcactttac cttcttactc aataacaacc tatctactac caggcagcag
      301 ttctcgcaac agtgtatcaa ttaccaactt actgaaatca cagaactggg tgggactcgg
      361 cagagaaaac tcaatcggag actactttac tatctattat tcgttacagg tcagaagaat
      421 tgagaagaga aaagaaaacc gccgatcctg tccaccgcat tactgcaagg tagtggacaa
      481 gaccggcggt cttaagtttt ttggctgaac catggcgggc gtcccagcga ttattaggaa
      541 ttgtaatgat tcaacggatg caaaaaacgt gactcaccct gcatgttggt catacgatat
      601 ttgtgtggtg gaacgtcaaa gtaattcttg aaggtacgtg taagggtttg ctgactttca
      661 aagccgtaac gctccgccag gtacaaaata ggttcattag actctttcaa tttctgggca
      721 atttcagtca tcttgcgaga acgaatatat tgacccaatg aatgacccgt ttccttctta
      781 aacatgcgct gaaggtgcca ctttgagtaa ccactacgtt cactaacttt ttctaaactc
      841 aacggcgact ccaagttgtc ttcaatccaa tccaggatgc tgtggattgt gatggcgtca
      901 gtattacggc gggacatgct gtgtgccaca gcgtcttcga cctgttcagt aatatttcct
      961 tctgtcttac ggaccaaacg atgcccaatg tagtggtctc cgggcatttc aagaatctct
     1021 tttgcggcct tataggtagt cttcatctga catttatggt tccctccccc ttctaatttt
     1081 aagtacatgg ttacatcgcc ctttaagact ccatcggaag ctgtaatctt ctctgtggac
     1141 ggttcccaat caacactttg attctgcatg attgggccgt ctgcggggaa gttcacgccg
     1201 gtgaactttg acttgtgata aaatgtattg ccgcgaagac taatatgagc tgacacgctg
     1261 gccgagccac cgtcttcaaa ctctaaactg cgctcccaag acaaaccttc cgggaaggct
     1321 tgtttgaaat agtctggaat ttcctcgggg tatttagtaa atacacggtg accgtaacaa
     1381 aagacatgac taacaagatc gaacgcaaac ggcataggcc cgccttcagc cattgtgacg
     1441 cgtaaagtca tttcctggtg cccctcgtat gggcgtcccg taccctcgcc ctcaatagta
     1501 aattcgtgac catttactga accatccata tagtaacgca ttttcatctc tggtttgata
     1561 accgaaacca tatgtatatc tccttcttaa agttaaacaa aattactgcc tatacggcag
     1621 tgaacccgga ccgcaggctg taacggagaa gccctatcaa ctgtttattg ctcgagtaag
     1681 gtaagcctga gctcggacgg taaagtttcc agaatctatc ctatgaattc ggtatggaga
     1741 aacagtagag agttgcgata aaaagcgtca ggtaggaacc gctaatctta tggataaaaa
     1801 tgctatggca tagcaaagtg tgacgccgtg caaataatca atgtggactt ttctgccgtg
     1861 attatagaca cttttgttac gcgtttttgt catggctttg gtcccgcttt gttacagaat
     1921 gcttttaata agcggggtta ccggtttggt tagcgagaag agccagtaaa agacgcagtg
     1981 acggcaatgt ctgatgcaat atggacaatt ggtttcttct ctgaatggcg ggagtatgaa
     2041 aagtatggct gaagcgcaaa atgatcccct gctgccggga tactcgttta atgcccatct
     2101 ggtggcgggt ttaacgccga ttgaggccaa cggttatctc gattttttta tcgaccgacc
     2161 gctgggaatg aaaggttata ttctcaatct caccattcgc ggtcaggggg tggtgaaaaa
     2221 tcagggacga gaatttgttt gccgaccggg tgatattttg ctgttcccgc caggagagat
     2281 tcatcactac ggtcgtcatc cggaggctcg cgaatggtat caccagtggg tttactttcg
     2341 tccgcgcgcc tactggcatg aatggcttaa ctggccgtca atatttgcca atacggggtt
     2401 ctttcgcccg gatgaagcgc accagccgca tttcagcgac ctgtttgggc aaatcattaa
     2461 cgccgggcaa ggggaagggc gctattcgga gctgctggcg ataaatctgc ttgagcaatt
     2521 gttactgcgg cgcatggaag cgattaacga gtcgctccat ccaccgatgg ataatcgggt
     2581 acgcgaggct tgtcagtaca tcagcgatca cctggcagac agcaattttg atatcgccag
     2641 cgtcgcacag catgtttgct tgtcgccgtc gcgtctgtca catcttttcc gccagcagtt
     2701 agggattagc gtcttaagct ggcgcgagga ccaacgtatc agccaggcga agctgctttt
     2761 gagcaccacc cggatgccta tcgccaccgt cggtcgcaat gttggttttg acgatcaact
     2821 ctatttctcg cgggtattta aaaaatgcac cggggccagc ccgagcgagt tccgtgccgg
     2881 ttgtgaagaa aaagtgaatg atgtagccgt caagttgtca taattggtaa cgaatcagac
     2941 cctaggagca acccagtcag ctccttccgg tgggcgcggg gcatgactaa catgagaatt
     3001 acaacttata tcgtatgggg ctgacttcag gtgctacatt tgaagagata aattgcactg
     3061 aaatctagag tgatggtgtc gggaatccgt aaaggatctt cttgagatcc ttttacgatc
     3121 gtcgtaatct cctgctctgt aaacgaaaaa accgcctggg gaggcggttt gatcgaaggt
     3181 taagtcagtt ggggaactgc ttaacctggt aactggcttt agtggagcgc agataccaaa
     3241 tactgtcctt tcagtgtagc ctctgttagg ccaccacttc aagactctcg atatctaaat
     3301 ccactaattc tcagttacca atggctgctg ccagtggcgt tttgtcgtgt ctttccgggt
     3361 tggactcaag atgatagtta ccggataagg cgcagcagtc gggctgaacg gggggttctt
     3421 gcacacagcc cagcttggag cgaactgtct acacggaacg ggacgtggtg atttgggtaa
     3481 agcctccacc acaacacgga cgccgcagga cgggaacagg agagcgcaag agggagccat
     3541 cagggggaaa cgcctggtat ctttatagtc ctgtcgggtt tcgccaccac tgatttgagc
     3601 gtcagatttc gtgatgttcg tcaggggggc ggagcctatg gaaaaacggc ttcgctccgg
     3661 ccttattgtc tctctgctaa gtatcctcct ggcatcttct aggacgtttc tgcgctagca
     3721 tgcctatttg tttatttttc taaatacatt caaatatgta tccgctcatg agacaataac
     3781 cctgataaat gcttcaataa tattgaaaaa ggaagagtat gagccatatt caacgggaaa
     3841 cgtcttgctc taggccgcga ttaaattcca acatggatgc tgatttatat gggtataaat
     3901 gggctcgcga taatgtcggg caatcaggtg cgacaatcta tcgattgtat gggaagcccg
     3961 atgcgccaga gttgtttctg aaacatggca aaggtagcgt tgccaatgat gttacagatg
     4021 agatggtcag actaaactgg ctgacggaat ttatgcctct tccgaccatc aagcatttta
     4081 tccgtactcc tgatgatgca tggttactca ccactgcgat ccccgggaaa acagcattcc
     4141 aggtattaga agaatatcct gattcaggtg aaaatattgt tgatgcgctg gcagtgttcc
     4201 tgcgccggtt gcattcgatt cctgtttgta attgtccttt taacagcgac cgcgtatttc
     4261 gtctcgctca ggcgcaatca cgaatgaata acggtttggt tgatgcgagt gattttgatg
     4321 acgagcgtaa tggctggcct gttgaacaag tctggaaaga aatgcataaa cttttgccat
     4381 tctcaccgga ttcagtcgtc actcatggtg atttctcact tgataacctt atttttgacg
     4441 aggggaaatt aataggttgt attgatgttg gacgagtcgg aatcgcagac cgataccagg
     4501 atcttgccat cctatggaac tgcctcggtg agttttctcc ttcattacag aaacggcttt
     4561 ttcaaaaata tggtattgat aatcctgata tgaataaatt gcagtttcat ttgatgctcg
     4621 atgagttttt ctaagaatta attcatgagc ggatacatat ttgaatgtat ttagaaaaat
     4681 aaacaaatag gggttccgcg cacatttccc cgaaaagtgc cacttgcgga gacccggtcg
     4741 tcagcttgtc gtcggttcag ggcagggtcg ttaaatagcc gcttatgtct attgctggtt
     4801 taccggtaga tctgaaacgc aaaaaggcca tccgtcagga tggccttctg cttagtttga
     4861 tgcctggcag tttatggcgg gcgtcctgcc cgccaccctc cgggccgttg cttcacaacg
     4921 ttcaaatccg ctcccggcgg atttgtccta ctcaggagag cgttcaccga caaaggcgcg
     4981 ccgctgagga cctgtgtaac aggcgcctag tagagaaagt atttatcctc aacagtgact
     5041 cgtcgttcgc tacctttggt cgaaaaaaaa agcccgcact gtcaggtgcg ggcttttttc
     5101 tgtgtttccg cggccgccgg gcgtcccagc gattattaga ctctgtgccc cagtttgcta
     5161 gggaggtcgc agtatctggc cacagccacc tcgtgctgct cgacgtaggt ctctttgtcg
     5221 gcctccttga ttctttccag tcttctgtcc acatagtaga cgccgggcat cttgaggttc
     5281 ttagcgggtt tcttggatct gtatgtggtc ttcaagttgc agatcaggtg gcccccgccc
     5341 acgagcttca gggccatgtc ggctctgcct tccaggccgc cgtcagcggg gtacagggtc
     5401 tcggtggagg cctcccagcc gagtgttttc ttctgcatca cagggccgtt ggatgggaag
     5461 ttcacccctc tgatcttgac gttgtagatg aggcagccgt cctggaggct ggtgtcctgg
     5521 gtagcggtca gcacgccccc gtcttcgtat gtggtgactc tctcccatgt gaagccctca
     5581 gggaaggact gcttaaagaa gtcggggatg ccctgggtgt ggttgatgaa ggttttgctg
     5641 ccgtacatga agctggtagc caggatgtcg aaggcgaagg ggagagggcc gccctcgacg
     5701 gccttgattc tcatggtctg ggtgccctcg tagggcttgc cttcgccctc ggatgtgcac
     5761 ttgaagtggt ggttgttcac ggtgccctcc atgtacagct tcatgtgcat gttctcctta
     5821 atcagtgcca tgttattttc ttctccttta ctaaccatgc cctctgcttt agcaatctcc
     5881 agttgagaga aggctttggt ggcgtcaatt tccttacttt ctcctttgct cttggaaggt
     5941 ttgggaacga aaactcctaa gcgcataagc gcgaccggct ggacagtatt gttggaattg
     6001 actgtaagtt ccttagcctt cgattcgatg tcggcatata agatgtccga gatgaatgac
     6061 tgattcatat gtatatctcc ttcttaaagt taaacaaaat tactgcctat acggcagtga
     6121 acccggaccg caggctgatc ttcagtttcc aacgacgagt gtaataggac gtcttcagta
     6181 agtgcgtcga ctgagttttc aacgattacc aatacttctg actggatttc ctgagcggtt
     6241 ctcttcacgc aactacgagc acctactgtg tcttattaca atctatctgt tatcgctacc
     6301 ctgtcccact gaaaagtcta cggaactgct gagtattatt gatttacctg cgagaacgaa
     6361 caacactacc agacctatcc tttatctatt aggaccaaaa cgaaaaaagg cccccctttc
     6421 gggaggcctc ttttctggaa tttggtaccg agcctgcagg cgggcgtccc agcgattatt
     6481 agtcttcaat ccaatccagg atgctgtgga ttgtgatggc gtcagtatta cggcgggaca
     6541 ttttatacag ttcgtccatg ccgtgggtga tacccgcagc cgtcacaaat tccagcagga
     6601 ccatgtggtc acgtttttcg ttcggatctt ttgacagcac ggactgggta ctcaggtagt
     6661 gattgtccgg cagcaggacc gggccatcac caatcggcgt gttttgctga tagtggtcgg
     6721 ccagctgaac gctaccatct tccacattat ggcggatttt gaaattggct ttaatgccgt
     6781 ttttctgttt atccgcggtg atgtacacgt tgtgggaatt gaagttatat tccagtttat
     6841 gacccagaat attgccgtct tctttgaaat cgataccttt cagttcgata cggttaacca
     6901 gggtatcgcc ttcgaatttc acttccgcgc gggttttata cgtaccatcg tctttaaagc
     6961 taatcgtacg ttcctgcaca tagccttccg gcatggccga tttgaaaaag tcgtgctgtt
     7021 tcatgtgatc cgggtaacgc gcaaaacatt gaacgccata ggtcagcgtg gtcaccagcg
     7081 tcggccacgg gaccggcagt ttacccgtgg tgcagataaa tttcagggtc agtttgccgt
     7141 tcgtcgcatc gccttcacct tcgccgcgaa cactgaattt atgaccatta acatcgccgt
     7201 ccagttccac cagaatcgga accacaccgg taaacagttc ttcgccttta cgcatatgta
     7261 tatctccttc ttaaagttaa acaaaattac tgcct
//
