LOCUS       sequence-305195-        5864 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  1-color stripe ("BLUE").
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1978 to 2012)
  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 5864)
  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: pJ2042.2_Bs
FEATURES             Location/Qualifiers
     source          1..5864
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     CDS             complement(12..890)
                     /codon_start=1
                     /gene="araC"
                     /product="L-arabinose regulatory protein"
                     /label=araC
                     /translation="MAEAQNDPLLPGYSFNAHLVAGLTPIEANGYLDFFIDRPLGMKGY
                     ILNLTIRGQGVVKNQGREFVCRPGDILLFPPGEIHHYGRHPEAREWYHQWVYFRPRAYW
                     HEWLNWPSIFANTGFFRPDEAHQPHFSDLFGQIINAGQGEGRYSELLAINLLEQLLLRR
                     MEAINESLHPPMDNRVREACQYISDHLADSNFDIASVAQHVCLSPSRLSHLFRQQLGIS
                     VLSWREDQRISQAKLLLSTTRMPIATVGRNVGFDDQLYFSRVFKKCTGASPSEFRAGCE
                     EKVNDVAVKLS"
     promoter        917..1201
                     /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     1124..1143
                     /label=pBAD Forward
                     /note="For vectors with E. coli araBAD promoter, forward 
                     primer"
     protein_bind    1262..1281
                     /label=Csy4 Site
                     /bound_moiety="Pseudomonas aeruginosa Csy4 (Cas6f)"
                     /note="cleavage site for the Csy4 endoribonuclease (Cermak 
                     et al. 2017)"
     protein_bind    1384..1403
                     /label=Csy4 Site
                     /bound_moiety="Pseudomonas aeruginosa Csy4 (Cas6f)"
                     /note="cleavage site for the Csy4 endoribonuclease (Cermak 
                     et al. 2017)"
     protein_bind    1464..1483
                     /label=Csy4 Site
                     /bound_moiety="Pseudomonas aeruginosa Csy4 (Cas6f)"
                     /note="cleavage site for the Csy4 endoribonuclease (Cermak 
                     et al. 2017)"
     protein_bind    1590..1609
                     /label=Csy4 Site
                     /bound_moiety="Pseudomonas aeruginosa Csy4 (Cas6f)"
                     /note="cleavage site for the Csy4 endoribonuclease (Cermak 
                     et al. 2017)"
     promoter        1978..2012
                     /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    2162..2181
                     /label=Csy4 Site
                     /bound_moiety="Pseudomonas aeruginosa Csy4 (Cas6f)"
                     /note="cleavage site for the Csy4 endoribonuclease (Cermak 
                     et al. 2017)"
     RBS             2186..2208
                     /note="efficient ribosome binding site from bacteriophage 
                     T7 gene 10 (Olins and Rangwala, 1989)"
     promoter        3307..3341
                     /label=BBa_J23105
                     /note="Anderson constitutive promoter 
                     (https://parts.igem.org/Promoters/Catalog/Anderson). 
                     Measured strength relative to BBa_J223100: 0.24"
     promoter        3307..3341
                     /label=BBa_J23107
                     /note="Anderson constitutive promoter 
                     (https://parts.igem.org/Promoters/Catalog/Anderson). 
                     Measured strength relative to BBa_J223100: 0.36"
     promoter        3307..3341
                     /label=BBa_J23101
                     /note="Anderson constitutive promoter 
                     (https://parts.igem.org/Promoters/Catalog/Anderson). 
                     Measured strength relative to BBa_J223100: 0.7"
     protein_bind    3485..3504
                     /label=Csy4 Site
                     /bound_moiety="Pseudomonas aeruginosa Csy4 (Cas6f)"
                     /note="cleavage site for the Csy4 endoribonuclease (Cermak 
                     et al. 2017)"
     protein_bind    3611..3630
                     /label=Csy4 Site
                     /bound_moiety="Pseudomonas aeruginosa Csy4 (Cas6f)"
                     /note="cleavage site for the Csy4 endoribonuclease (Cermak 
                     et al. 2017)"
     terminator      3950..3977
                     /label=rrnB T2 terminator
                     /note="transcription terminator T2 from the E. coli rrnB 
                     gene"
     CDS             complement(4165..4980)
                     /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(4165..4980)
                     /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     4888..4907
                     /label=Kan-R
                     /note="Kanamycin resistance gene, reverse primer"
     promoter        complement(4981..5072)
                     /gene="bla"
                     /label=AmpR promoter
     rep_origin      complement(5090..5725)
                     /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(5235..5254)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
ORIGIN
        1 ttcgttacca attatgacaa cttgacggct acatcattca ctttttcttc acaaccggca
       61 cggaactcgc tcgggctggc cccggtgcat tttttaaata cccgcgagaa atagagttga
      121 tcgtcaaaac caacattgcg accgacggtg gcgataggca tccgggtggt gctcaaaagc
      181 agcttcgcct ggctgatacg ttggtcctcg cgccagctta agacgctaat ccctaactgc
      241 tggcggaaaa gatgtgacag acgcgacggc gacaagcaaa catgctgtgc gacgctggcg
      301 atatcaaaat tgctgtctgc caggtgatcg ctgatgtact gacaagcctc gcgtacccga
      361 ttatccatcg gtggatggag cgactcgtta atcgcttcca tgcgccgcag taacaattgc
      421 tcaagcagat ttatcgccag cagctccgaa tagcgccctt ccccttgccc ggcgttaatg
      481 atttgcccaa acaggtcgct gaaatgcggc tggtgcgctt catccgggcg aaagaacccc
      541 gtattggcaa atattgacgg ccagttaagc cattcatgcc agtaggcgcg cggacgaaag
      601 taaacccact ggtgatacca ttcgcgagcc tccggatgac gaccgtagtg atgaatctct
      661 cctggcggga acagcaaaat atcacccggt cggcaaacaa attctcgtcc ctgatttttc
      721 accaccccct gaccgcgaat ggtgagattg agaatataac ctttcattcc cagcggtcgg
      781 tcgataaaaa aatcgagata accgttggcc tcaatcggcg ttaaacccgc caccagatgg
      841 gcattaaacg agtatcccgg cagcagggga tcattttgcg cttcagccat acttttcata
      901 ctcccgccat tcagagaaga aaccaattgt ccatattgca tcagacattg ccgtcactgc
      961 gtcttttact ggctcttctc gctaaccaaa ccggtaaccc cgcttattaa aagcattctg
     1021 taacaaagcg ggaccaaagc catgacaaaa acgcgtaaca aaagtgtcta taatcacggc
     1081 agaaaagtcc acattgatta tttgcacggc gtcacacttt gctatgccat agcattttta
     1141 tccataagat tagcggttcc tacctgacgc tttttatcgc aactctctac tgtttctcca
     1201 taccgaattc ataggataga ttctggaaac tttaccgtcc gagctccagc ctgcggtccg
     1261 ggttcactgc cgtataggca gaagctagac tctagtggtt tcagagctat gctggaaaca
     1321 gcatagcaag ttgaaataag gctagtccgt tatcaacttg aaaaagtggc accgagtcgg
     1381 tgcgttcact gccgtatagg cagtcgctgg gacgcccggg gactacactt acgaaactat
     1441 tgattgctca gcctgcggtc cgggttcact gccgtatagg cagatcagtg tgtactaagt
     1501 actgtttcag agctatgctg gaaacagcat agcaagttga aataaggcta gtccgttatc
     1561 aacttgaaaa agtggcaccg agtcggtgcg ttcactgccg tataggcagt cgctgggacg
     1621 cccgctcgag caataaacag ttgatagggc ttctccgtta ccatggttca gccaaaaaac
     1681 ttaagaccgc cggtcttgtc cactaccttg cagtaatgcg gtggacagga tcggcggttt
     1741 tcttttctct tctcaattct tctgacctgt aacgaataat agatagtaaa gtagtctccg
     1801 attgagtttt ctctgccgag tcccacccag ttctgtgatt tcagtaagtt ggtaattgat
     1861 acactgttgc gagaactgct gcctggtagt agataggttg ttattgagta agaaggtaaa
     1921 gtgaacgaaa tccctgaaac tgagactgta gaaaataagc ttcagcctgc ggtccggttg
     1981 acggctagct cagtcctagg tacagtgcta gctcgctggg acgcccgaga tagccgttac
     2041 acaggtgaca cttatttcag cctgcggtcc ggccacacta gagtctagct tgagatcgct
     2101 gggacgcccg ggatccaaga gatttctaca cgattgagca ctgtctcagc ctgcggtccg
     2161 ggttcactgc cgtataggca gtaattttgt ttaactttaa gaaggagata tacatatggt
     2221 tagtaaagga gaagaaaata acatggcact gattaaggag aacatgcaca tgaagctgta
     2281 catggagggc accgtgaaca accaccactt caagtgcaca tccgagggcg aaggcaagcc
     2341 ctacgagggc acccagacca tgagaatcaa ggccgtcgag ggcggccctc tccccttcgc
     2401 cttcgacatc ctggctacca gcttcatgta cggcagcaaa accttcatca accacaccca
     2461 gggcatcccc gacttcttta agcagtcctt ccctgagggc ttcacatggg agagagtcac
     2521 cacatacgaa gacgggggcg tgctgaccgc tacccaggac accagcctcc aggacggctg
     2581 cctcatctac aacgtcaaga tcagaggggt gaacttccca tccaacggcc ctgtgatgca
     2641 gaagaaaaca ctcggctggg aggcctccac cgagaccctg taccccgctg acggcggcct
     2701 ggaaggcaga gccgacatgg ccctgaagct cgtgggcggg ggccacctga tctgcaactt
     2761 gaagaccaca tacagatcca agaaacccgc taagaacctc aagatgcccg gcgtctacta
     2821 tgtggacaga agactggaaa gaatcaagga ggccgacaaa gagacctacg tcgagcagca
     2881 cgaggtggct gtggccagat actgcgacct ccctagcaaa ctggggcaca gagtcatgtc
     2941 ccgccgtaat actgacgcca tcacaatcca cagcatcctg gattggattg aagactaata
     3001 atcgctggga cgcccgcctg caggctcggt accaaattcc agaaaagagg cctcccgaaa
     3061 ggggggcctt ttttcgtttt ggtcctaata gataaaggat aggtctggta gtgttgttcg
     3121 ttctcgcagg taaatcaata atactcagca gttccgtaga cttttcagtg ggacagggta
     3181 gcgataacag atagattgta ataagacaca gtaggtgctc gtagttgcgt gaagagaacc
     3241 gctcaggaaa tccagtcaga agtattggta atcgttgaaa actcagtcga ccagcctgcg
     3301 gtccggttta cggctagctc agtcctaggt attatgctag ctcgctggga cgcccgagtg
     3361 acgactgcga agtaacctct atttatcagc ctgcggtccg gccaagtact tagtacacac
     3421 tgattcgctg ggacgcccgg tagtgcttat cagacccaat actgttgaac agcctgcggt
     3481 ccgggttcac tgccgtatag gcagtctcaa gctagactct agtggtttca gagctatgct
     3541 ggaaacagca tagcaagttg aaataaggct agtccgttat caacttgaaa aagtggcacc
     3601 gagtcggtgc gttcactgcc gtataggcag tcgctgggac gcccggacgt cctattacac
     3661 tcgtcgttgg aaactgaaga tgcggccgcg gaaacacaga aaaaagcccg cacctgacag
     3721 tgcgggcttt ttttttcgac caaaggtagc gaacgacgag tcactgttga ggataaatac
     3781 tttctctact aggcgcctgt tacacaggtc ctcagcggcg cgcctttgtc ggtgaacgct
     3841 ctcctgagta ggacaaatcc gccgggagcg gatttgaacg ttgtgaagca acggcccgga
     3901 gggtggcggg caggacgccc gccataaact gccaggcatc aaactaagca gaaggccatc
     3961 ctgacggatg gcctttttgc gtttcagatc taccggtaaa ccagcaatag acataagcgg
     4021 ctatttaacg accctgccct gaaccgacga caagctgacg accgggtctc cgcaagtggc
     4081 acttttcggg gaaatgtgcg cggaacccct atttgtttat ttttctaaat acattcaaat
     4141 atgtatccgc tcatgaatta attcttagaa aaactcatcg agcatcaaat gaaactgcaa
     4201 tttattcata tcaggattat caataccata tttttgaaaa agccgtttct gtaatgaagg
     4261 agaaaactca ccgaggcagt tccataggat ggcaagatcc tggtatcggt ctgcgattcc
     4321 gactcgtcca acatcaatac aacctattaa tttcccctcg tcaaaaataa ggttatcaag
     4381 tgagaaatca ccatgagtga cgactgaatc cggtgagaat ggcaaaagtt tatgcatttc
     4441 tttccagact tgttcaacag gccagccatt acgctcgtca tcaaaatcac tcgcatcaac
     4501 caaaccgtta ttcattcgtg attgcgcctg agcgagacga aatacgcggt cgctgttaaa
     4561 aggacaatta caaacaggaa tcgaatgcaa ccggcgcagg aacactgcca gcgcatcaac
     4621 aatattttca cctgaatcag gatattcttc taatacctgg aatgctgttt tcccggggat
     4681 cgcagtggtg agtaaccatg catcatcagg agtacggata aaatgcttga tggtcggaag
     4741 aggcataaat tccgtcagcc agtttagtct gaccatctca tctgtaacat cattggcaac
     4801 gctacctttg ccatgtttca gaaacaactc tggcgcatcg ggcttcccat acaatcgata
     4861 gattgtcgca cctgattgcc cgacattatc gcgagcccat ttatacccat ataaatcagc
     4921 atccatgttg gaatttaatc gcggcctaga gcaagacgtt tcccgttgaa tatggctcat
     4981 actcttcctt tttcaatatt attgaagcat ttatcagggt tattgtctca tgagcggata
     5041 catatttgaa tgtatttaga aaaataaaca aataggcatg ctagcgcaga aacgtcctag
     5101 aagatgccag gaggatactt agcagagaga caataaggcc ggagcgaagc cgtttttcca
     5161 taggctccgc ccccctgacg aacatcacga aatctgacgc tcaaatcagt ggtggcgaaa
     5221 cccgacagga ctataaagat accaggcgtt tccccctgat ggctccctct tgcgctctcc
     5281 tgttcccgtc ctgcggcgtc cgtgttgtgg tggaggcttt acccaaatca ccacgtcccg
     5341 ttccgtgtag acagttcgct ccaagctggg ctgtgtgcaa gaaccccccg ttcagcccga
     5401 ctgctgcgcc ttatccggta actatcatct tgagtccaac ccggaaagac acgacaaaac
     5461 gccactggca gcagccattg gtaactgaga attagtggat ttagatatcg agagtcttga
     5521 agtggtggcc taacagaggc tacactgaaa ggacagtatt tggtatctgc gctccactaa
     5581 agccagttac caggttaagc agttccccaa ctgacttaac cttcgatcaa accgcctccc
     5641 caggcggttt tttcgtttac agagcaggag attacgacga tcgtaaaagg atctcaagaa
     5701 gatcctttac ggattcccga caccatcact ctagatttca gtgcaattta tctcttcaaa
     5761 tgtagcacct gaagtcagcc ccatacgata taagttgtaa ttctcatgtt agtcatgccc
     5821 cgcgcccacc ggaaggagct gactgggttg ctcctagggt ctga
//
