LOCUS       Exported                5858 bp ds-DNA     circular SYN 12-MAY-2021
DEFINITION  1-color stripe.
ACCESSION   .
VERSION     .
KEYWORDS    pJ2042.2_GFPonly
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 5858)
  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 5858)
  AUTHORS   .
  TITLE     Direct Submission
  JOURNAL   Exported May 12, 2021 from SnapGene Server 1.1.58
            http://www.snapgene.com
FEATURES             Location/Qualifiers
     source          1..5858
                     /organism="synthetic DNA construct"
                     /mol_type="other DNA"
     promoter        25..309
                     /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     232..251
                     /label=pBAD Forward
                     /note="For vectors with E. coli araBAD promoter, forward 
                     primer"
     protein_bind    375..389
                     /label=Csy4 site
                     /bound_moiety="Pseudomonas aeruginosa Csy4 (Cas6f)"
                     /note="cleavage site for the Csy4 endoribonuclease 
                     (Haurwitz et al., 2010)"
     protein_bind    497..511
                     /label=Csy4 site
                     /bound_moiety="Pseudomonas aeruginosa Csy4 (Cas6f)"
                     /note="cleavage site for the Csy4 endoribonuclease 
                     (Haurwitz et al., 2010)"
     protein_bind    577..591
                     /label=Csy4 site
                     /bound_moiety="Pseudomonas aeruginosa Csy4 (Cas6f)"
                     /note="cleavage site for the Csy4 endoribonuclease 
                     (Haurwitz et al., 2010)"
     protein_bind    703..717
                     /label=Csy4 site
                     /bound_moiety="Pseudomonas aeruginosa Csy4 (Cas6f)"
                     /note="cleavage site for the Csy4 endoribonuclease 
                     (Haurwitz et al., 2010)"
     protein_bind    1275..1289
                     /label=Csy4 site
                     /bound_moiety="Pseudomonas aeruginosa Csy4 (Cas6f)"
                     /note="cleavage site for the Csy4 endoribonuclease 
                     (Haurwitz et al., 2010)"
     RBS             1294..1316
                     /note="efficient ribosome binding site from bacteriophage 
                     T7 gene 10 (Olins and Rangwala, 1989)"
     CDS             1324..2037
                     /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    2592..2606
                     /label=Csy4 site
                     /bound_moiety="Pseudomonas aeruginosa Csy4 (Cas6f)"
                     /note="cleavage site for the Csy4 endoribonuclease 
                     (Haurwitz et al., 2010)"
     protein_bind    2718..2732
                     /label=Csy4 site
                     /bound_moiety="Pseudomonas aeruginosa Csy4 (Cas6f)"
                     /note="cleavage site for the Csy4 endoribonuclease 
                     (Haurwitz et al., 2010)"
     terminator      3052..3079
                     /label=rrnB T2 terminator
                     /note="transcription terminator T2 from the E. coli rrnB 
                     gene"
     CDS             complement(3267..4082)
                     /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     3990..4009
                     /label=Kan-R
                     /note="Kanamycin resistance gene, reverse primer"
     promoter        complement(4083..4174)
                     /gene="bla"
                     /label=AmpR promoter
     rep_origin      complement(4192..4827)
                     /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(4337..4356)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(4978..5856)
                     /codon_start=1
                     /gene="araC"
                     /product="L-arabinose regulatory protein"
                     /label=araC
                     /translation="MAEAQNDPLLPGYSFNAHLVAGLTPIEANGYLDFFIDRPLGMKGY
                     ILNLTIRGQGVVKNQGREFVCRPGDILLFPPGEIHHYGRHPEAREWYHQWVYFRPRAYW
                     HEWLNWPSIFANTGFFRPDEAHQPHFSDLFGQIINAGQGEGRYSELLAINLLEQLLLRR
                     MEAINESLHPPMDNRVREACQYISDHLADSNFDIASVAQHVCLSPSRLSHLFRQQLGIS
                     VLSWREDQRISQAKLLLSTTRMPIATVGRNVGFDDQLYFSRVFKKCTGASPSEFRAGCE
                     EKVNDVAVKLS"
ORIGIN
        1 ttttcatact cccgccattc agagaagaaa ccaattgtcc atattgcatc agacattgcc
       61 gtcactgcgt cttttactgg ctcttctcgc taaccaaacc ggtaaccccg cttattaaaa
      121 gcattctgta acaaagcggg accaaagcca tgacaaaaac gcgtaacaaa agtgtctata
      181 atcacggcag aaaagtccac attgattatt tgcacggcgt cacactttgc tatgccatag
      241 catttttatc cataagatta gcggttccta cctgacgctt tttatcgcaa ctctctactg
      301 tttctccata ccgaattcat aggatagatt ctggaaactt taccgtccga gctccagcct
      361 gcggtccggg ttcactgccg tataggcaga agctagactc tagtggtttc agagctatgc
      421 tggaaacagc atagcaagtt gaaataaggc tagtccgtta tcaacttgaa aaagtggcac
      481 cgagtcggtg cgttcactgc cgtataggca gtcgctggga cgcccgggga ctacacttac
      541 gaaactattg attgctcagc ctgcggtccg ggttcactgc cgtataggca gatcagtgtg
      601 tactaagtac tgtttcagag ctatgctgga aacagcatag caagttgaaa taaggctagt
      661 ccgttatcaa cttgaaaaag tggcaccgag tcggtgcgtt cactgccgta taggcagtcg
      721 ctgggacgcc cgctcgagca ataaacagtt gatagggctt ctccgttacc atggttcagc
      781 caaaaaactt aagaccgccg gtcttgtcca ctaccttgca gtaatgcggt ggacaggatc
      841 ggcggttttc ttttctcttc tcaattcttc tgacctgtaa cgaataatag atagtaaagt
      901 agtctccgat tgagttttct ctgccgagtc ccacccagtt ctgtgatttc agtaagttgg
      961 taattgatac actgttgcga gaactgctgc ctggtagtag ataggttgtt attgagtaag
     1021 aaggtaaagt gaacgaaatc cctgaaactg agactgtaga aaataagctt cagcctgcgg
     1081 tccggttgac ggctagctca gtcctaggta cagtgctagc tcgctgggac gcccgagata
     1141 gccgttacac aggtgacact tatttcagcc tgcggtccgg ccacactaga gtctagcttg
     1201 agatcgctgg gacgcccggg atccaagaga tttctacacg attgagcact gtctcagcct
     1261 gcggtccggg ttcactgccg tataggcagt aattttgttt aactttaaga aggagatata
     1321 catatgcgta aaggcgaaga actgtttacc ggtgtggttc cgattctggt ggaactggac
     1381 ggcgatgtta atggtcataa attcagtgtt cgcggcgaag gtgaaggcga tgcgacgaac
     1441 ggcaaactga ccctgaaatt tatctgcacc acgggtaaac tgccggtccc gtggccgacg
     1501 ctggtgacca cgctgaccta tggcgttcaa tgttttgcgc gttacccgga tcacatgaaa
     1561 cagcacgact ttttcaaatc ggccatgccg gaaggctatg tgcaggaacg tacgattagc
     1621 tttaaagacg atggtacgta taaaacccgc gcggaagtga aattcgaagg cgataccctg
     1681 gttaaccgta tcgaactgaa aggtatcgat ttcaaagaag acggcaatat tctgggtcat
     1741 aaactggaat ataacttcaa ttcccacaac gtgtacatca ccgcggataa acagaaaaac
     1801 ggcattaaag ccaatttcaa aatccgccat aatgtggaag atggtagcgt tcagctggcc
     1861 gaccactatc agcaaaacac gccgattggt gatggcccgg tcctgctgcc ggacaatcac
     1921 tacctgagta cccagtccgt gctgtcaaaa gatccgaacg aaaaacgtga ccacatggtc
     1981 ctgctggaat ttgtgacggc tgcgggtatc acccacggca tggacgaact gtataaaatg
     2041 tcccgccgta atactgacgc catcacaatc cacagcatcc tggattggat tgaagactaa
     2101 taatcgctgg gacgcccgcc tgcaggctcg gtaccaaatt ccagaaaaga ggcctcccga
     2161 aaggggggcc ttttttcgtt ttggtcctaa tagataaagg ataggtctgg tagtgttgtt
     2221 cgttctcgca ggtaaatcaa taatactcag cagttccgta gacttttcag tgggacaggg
     2281 tagcgataac agatagattg taataagaca cagtaggtgc tcgtagttgc gtgaagagaa
     2341 ccgctcagga aatccagtca gaagtattgg taatcgttga aaactcagtc gaccagcctg
     2401 cggtccggtt tacggctagc tcagtcctag gtattatgct agctcgctgg gacgcccgag
     2461 tgacgactgc gaagtaacct ctatttatca gcctgcggtc cggccaagta cttagtacac
     2521 actgattcgc tgggacgccc ggtagtgctt atcagaccca atactgttga acagcctgcg
     2581 gtccgggttc actgccgtat aggcagtctc aagctagact ctagtggttt cagagctatg
     2641 ctggaaacag catagcaagt tgaaataagg ctagtccgtt atcaacttga aaaagtggca
     2701 ccgagtcggt gcgttcactg ccgtataggc agtcgctggg acgcccggac gtcctattac
     2761 actcgtcgtt ggaaactgaa gatgcggccg cggaaacaca gaaaaaagcc cgcacctgac
     2821 agtgcgggct ttttttttcg accaaaggta gcgaacgacg agtcactgtt gaggataaat
     2881 actttctcta ctaggcgcct gttacacagg tcctcagcgg cgcgcctttg tcggtgaacg
     2941 ctctcctgag taggacaaat ccgccgggag cggatttgaa cgttgtgaag caacggcccg
     3001 gagggtggcg ggcaggacgc ccgccataaa ctgccaggca tcaaactaag cagaaggcca
     3061 tcctgacgga tggccttttt gcgtttcaga tctaccggta aaccagcaat agacataagc
     3121 ggctatttaa cgaccctgcc ctgaaccgac gacaagctga cgaccgggtc tccgcaagtg
     3181 gcacttttcg gggaaatgtg cgcggaaccc ctatttgttt atttttctaa atacattcaa
     3241 atatgtatcc gctcatgaat taattcttag aaaaactcat cgagcatcaa atgaaactgc
     3301 aatttattca tatcaggatt atcaatacca tatttttgaa aaagccgttt ctgtaatgaa
     3361 ggagaaaact caccgaggca gttccatagg atggcaagat cctggtatcg gtctgcgatt
     3421 ccgactcgtc caacatcaat acaacctatt aatttcccct cgtcaaaaat aaggttatca
     3481 agtgagaaat caccatgagt gacgactgaa tccggtgaga atggcaaaag tttatgcatt
     3541 tctttccaga cttgttcaac aggccagcca ttacgctcgt catcaaaatc actcgcatca
     3601 accaaaccgt tattcattcg tgattgcgcc tgagcgagac gaaatacgcg gtcgctgtta
     3661 aaaggacaat tacaaacagg aatcgaatgc aaccggcgca ggaacactgc cagcgcatca
     3721 acaatatttt cacctgaatc aggatattct tctaatacct ggaatgctgt tttcccgggg
     3781 atcgcagtgg tgagtaacca tgcatcatca ggagtacgga taaaatgctt gatggtcgga
     3841 agaggcataa attccgtcag ccagtttagt ctgaccatct catctgtaac atcattggca
     3901 acgctacctt tgccatgttt cagaaacaac tctggcgcat cgggcttccc atacaatcga
     3961 tagattgtcg cacctgattg cccgacatta tcgcgagccc atttataccc atataaatca
     4021 gcatccatgt tggaatttaa tcgcggccta gagcaagacg tttcccgttg aatatggctc
     4081 atactcttcc tttttcaata ttattgaagc atttatcagg gttattgtct catgagcgga
     4141 tacatatttg aatgtattta gaaaaataaa caaataggca tgctagcgca gaaacgtcct
     4201 agaagatgcc aggaggatac ttagcagaga gacaataagg ccggagcgaa gccgtttttc
     4261 cataggctcc gcccccctga cgaacatcac gaaatctgac gctcaaatca gtggtggcga
     4321 aacccgacag gactataaag ataccaggcg tttccccctg atggctccct cttgcgctct
     4381 cctgttcccg tcctgcggcg tccgtgttgt ggtggaggct ttacccaaat caccacgtcc
     4441 cgttccgtgt agacagttcg ctccaagctg ggctgtgtgc aagaaccccc cgttcagccc
     4501 gactgctgcg ccttatccgg taactatcat cttgagtcca acccggaaag acacgacaaa
     4561 acgccactgg cagcagccat tggtaactga gaattagtgg atttagatat cgagagtctt
     4621 gaagtggtgg cctaacagag gctacactga aaggacagta tttggtatct gcgctccact
     4681 aaagccagtt accaggttaa gcagttcccc aactgactta accttcgatc aaaccgcctc
     4741 cccaggcggt tttttcgttt acagagcagg agattacgac gatcgtaaaa ggatctcaag
     4801 aagatccttt acggattccc gacaccatca ctctagattt cagtgcaatt tatctcttca
     4861 aatgtagcac ctgaagtcag ccccatacga tataagttgt aattctcatg ttagtcatgc
     4921 cccgcgccca ccggaaggag ctgactgggt tgctcctagg gtctgattcg ttaccaatta
     4981 tgacaacttg acggctacat cattcacttt ttcttcacaa ccggcacgga actcgctcgg
     5041 gctggccccg gtgcattttt taaatacccg cgagaaatag agttgatcgt caaaaccaac
     5101 attgcgaccg acggtggcga taggcatccg ggtggtgctc aaaagcagct tcgcctggct
     5161 gatacgttgg tcctcgcgcc agcttaagac gctaatccct aactgctggc ggaaaagatg
     5221 tgacagacgc gacggcgaca agcaaacatg ctgtgcgacg ctggcgatat caaaattgct
     5281 gtctgccagg tgatcgctga tgtactgaca agcctcgcgt acccgattat ccatcggtgg
     5341 atggagcgac tcgttaatcg cttccatgcg ccgcagtaac aattgctcaa gcagatttat
     5401 cgccagcagc tccgaatagc gcccttcccc ttgcccggcg ttaatgattt gcccaaacag
     5461 gtcgctgaaa tgcggctggt gcgcttcatc cgggcgaaag aaccccgtat tggcaaatat
     5521 tgacggccag ttaagccatt catgccagta ggcgcgcgga cgaaagtaaa cccactggtg
     5581 ataccattcg cgagcctccg gatgacgacc gtagtgatga atctctcctg gcgggaacag
     5641 caaaatatca cccggtcggc aaacaaattc tcgtccctga tttttcacca ccccctgacc
     5701 gcgaatggtg agattgagaa tataaccttt cattcccagc ggtcggtcga taaaaaaatc
     5761 gagataaccg ttggcctcaa tcggcgttaa acccgccacc agatgggcat taaacgagta
     5821 tcccggcagc aggggatcat tttgcgcttc agccatac
//
