LOCUS       Exported                6900 bp ds-DNA     circular SYN 12-MAY-2021
DEFINITION  CRISPRlator.
ACCESSION   .
VERSION     .
KEYWORDS    pJ2072.2_CRISPRlator
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 6900)
  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 6900)
  AUTHORS   .
  TITLE     Direct Submission
  JOURNAL   Exported May 12, 2021 from SnapGene Server 1.1.58
            http://www.snapgene.com
FEATURES             Location/Qualifiers
     source          1..6900
                     /organism="synthetic DNA construct"
                     /mol_type="other DNA"
     protein_bind    88..102
                     /label=Csy4 site
                     /bound_moiety="Pseudomonas aeruginosa Csy4 (Cas6f)"
                     /note="cleavage site for the Csy4 endoribonuclease 
                     (Haurwitz et al., 2010)"
     protein_bind    214..228
                     /label=Csy4 site
                     /bound_moiety="Pseudomonas aeruginosa Csy4 (Cas6f)"
                     /note="cleavage site for the Csy4 endoribonuclease 
                     (Haurwitz et al., 2010)"
     protein_bind    300..314
                     /label=Csy4 site
                     /bound_moiety="Pseudomonas aeruginosa Csy4 (Cas6f)"
                     /note="cleavage site for the Csy4 endoribonuclease 
                     (Haurwitz et al., 2010)"
     RBS             319..341
                     /note="efficient ribosome binding site from bacteriophage 
                     T7 gene 10 (Olins and Rangwala, 1989)"
     CDS             349..1056
                     /codon_start=1
                     /product="monomeric derivative of DsRed fluorescent protein
                     (Shaner et al., 2004)"
                     /label=mCherry
                     /note="mammalian codon-optimized"
                     /translation="MVSKGEEDNMAIIKEFMRFKVHMEGSVNGHEFEIEGEGEGRPYEG
                     TQTAKLKVTKGGPLPFAWDILSPQFMYGSKAYVKHPADIPDYLKLSFPEGFKWERVMNF
                     EDGGVVTVTQDSSLQDGEFIYKVKLRGTNFPSDGPVMQKKTMGWEASSERMYPEDGALK
                     GEIKQRLKLKDGGHYDAEVKTTYKAKKPVQLPGAYNVNIKLDITSHNEDYTIVEQYERA
                     EGRHSTGGMDELYK"
     primer_bind     complement(494..512)
                     /label=mCherry-R
                     /note="mCherry, reverse primer"
     primer_bind     762..781
                     /label=mCherry-F
                     /note="mCherry, forward primer"
     protein_bind    1959..1973
                     /label=Csy4 site
                     /bound_moiety="Pseudomonas aeruginosa Csy4 (Cas6f)"
                     /note="cleavage site for the Csy4 endoribonuclease 
                     (Haurwitz et al., 2010)"
     protein_bind    2085..2099
                     /label=Csy4 site
                     /bound_moiety="Pseudomonas aeruginosa Csy4 (Cas6f)"
                     /note="cleavage site for the Csy4 endoribonuclease 
                     (Haurwitz et al., 2010)"
     protein_bind    2171..2185
                     /label=Csy4 site
                     /bound_moiety="Pseudomonas aeruginosa Csy4 (Cas6f)"
                     /note="cleavage site for the Csy4 endoribonuclease 
                     (Haurwitz et al., 2010)"
     RBS             2190..2212
                     /note="efficient ribosome binding site from bacteriophage 
                     T7 gene 10 (Olins and Rangwala, 1989)"
     protein_bind    3488..3502
                     /label=Csy4 site
                     /bound_moiety="Pseudomonas aeruginosa Csy4 (Cas6f)"
                     /note="cleavage site for the Csy4 endoribonuclease 
                     (Haurwitz et al., 2010)"
     protein_bind    3614..3628
                     /label=Csy4 site
                     /bound_moiety="Pseudomonas aeruginosa Csy4 (Cas6f)"
                     /note="cleavage site for the Csy4 endoribonuclease 
                     (Haurwitz et al., 2010)"
     protein_bind    3700..3714
                     /label=Csy4 site
                     /bound_moiety="Pseudomonas aeruginosa Csy4 (Cas6f)"
                     /note="cleavage site for the Csy4 endoribonuclease 
                     (Haurwitz et al., 2010)"
     RBS             3719..3741
                     /note="efficient ribosome binding site from bacteriophage 
                     T7 gene 10 (Olins and Rangwala, 1989)"
     primer_bind     complement(3989..4017)
                     /label=GFP-R
                     /note="GFP, reverse primer. Does NOT anneal to EGFP"
     primer_bind     4612..4632
                     /label=GFP-F
                     /note="GFP, forward primer. Does NOT anneal to EGFP"
     terminator      4962..4989
                     /label=rrnB T2 terminator
                     /note="transcription terminator T2 from the E. coli rrnB 
                     gene"
     CDS             complement(5177..5992)
                     /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     5900..5919
                     /label=Kan-R
                     /note="Kanamycin resistance gene, reverse primer"
     promoter        complement(5993..6084)
                     /gene="bla"
                     /label=AmpR promoter
     rep_origin      complement(6102..6737)
                     /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(6247..6266)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
ORIGIN
        1 catacatact ctaagatgtg tctcgctggg acgcccgggg actacactta cgaaactatt
       61 gattgctcag cctgcggtcc gggttcactg ccgtataggc agatcagtgt gtactaagta
      121 ctgtttcaga gctatgctgg aaacagcata gcaagttgaa ataaggctag tccgttatca
      181 acttgaaaaa gtggcaccga gtcggtgcgt tcactgccgt ataggcagtc gctgggacgc
      241 ccgctcgagc aataaacagt tgatagggct tctccgttac agcctgcggt ccgggttcac
      301 tgccgtatag gcagtaattt tgtttaactt taagaaggag atatacatat ggtgagcaag
      361 ggcgaggagg ataacatggc catcatcaag gagttcatgc gcttcaaggt gcacatggag
      421 ggctccgtga acggccacga gttcgagatc gagggcgagg gcgagggccg cccctacgag
      481 ggcacccaga ccgccaagct gaaggtgacc aagggtggcc ccctgccctt cgcctgggac
      541 atcctgtccc ctcagttcat gtacggctcc aaggcctacg tgaagcaccc cgccgacatc
      601 cccgactact tgaagctgtc cttccccgag ggcttcaagt gggagcgcgt gatgaacttc
      661 gaggacggcg gcgtggtgac cgtgacccag gactcctctc tgcaggacgg cgagttcatc
      721 tacaaggtga agctgcgcgg caccaacttc ccctccgacg gccccgtaat gcagaagaag
      781 actatgggct gggaggcctc ctccgagcgg atgtaccccg aggacggcgc gctgaagggc
      841 gagatcaagc agaggctgaa gctgaaggac ggcggccact acgacgctga agtcaagacc
      901 acctacaagg ccaagaagcc cgtgcagctg cccggcgcgt acaacgtcaa catcaagttg
      961 gacatcacct cccacaacga ggactacacc atcgtggaac agtacgaacg cgccgagggc
     1021 cgccactcca ccggcggcat ggacgagctg tacaagatgt cccgccgtaa tactgacgcc
     1081 atcacaatcc acagcatcct ggattggatt gaagacaact tggagtcgcc gttgagttta
     1141 gaaaaagtta gtgaacgtag tggttactca aagtggcacc ttcagcgcat gtttaagaag
     1201 gaaacgggtc attcattggg tcaatatatt cgttctcgca agatgactga aattgcccag
     1261 aaattgaaag agtctaatga acctattttg tacctggcgg agcgttacgg ctttgaaagt
     1321 cagcaaaccc ttacacgtac cttcaagaat tactttgacg ttccaccaca caaatatcgt
     1381 atgaccaaca tgcagggtga gtcacgtttt ttgcatccgt tgaatcatta caattcctaa
     1441 taatcgctgg gacgcccgcc atggttcagc caaaaaactt aagaccgccg gtcttgtcca
     1501 ctaccttgca gtaatgcggt ggacaggatc ggcggttttc ttttctcttc tcaattcttc
     1561 tgacctgtaa cgaataatag atagtaaagt agtctccgat tgagttttct ctgccgagtc
     1621 ccacccagtt ctgtgatttc agtaagttgg taattgatac actgttgcga gaactgctgc
     1681 ctggtagtag ataggttgtt attgagtaag aaggtaaagt gaacgaaatc cctgaaactg
     1741 agactgtaga aaataagctt cagcctgcgg tccggttgac ggctagctca gtcctaggta
     1801 cagtgctagc tcgctgggac gcccgagata gccgttacac aggtgacact tatttcagcc
     1861 tgcggtccgg ccacactaga gtctagcttg agatcgctgg gacgcccgtg aacagttgct
     1921 ctgattgaaa ccacgattca gcctgcggtc cgggttcact gccgtatagg caggacacat
     1981 cttagagtat gtagtttcag agctatgctg gaaacagcat agcaagttga aataaggcta
     2041 gtccgttatc aacttgaaaa agtggcaccg agtcggtgcg ttcactgccg tataggcagt
     2101 cgctgggacg cccgggatcc aagagatttc tacacgattg agcactgtct cagcctgcgg
     2161 tccgggttca ctgccgtata ggcagtaatt ttgtttaact ttaagaagga gatatacata
     2221 tgtctaaagg tgaagaatta ttcactggtg ttgtcccaat tttggttgaa ttagatggtg
     2281 atgttaatgg tcacaaattt tctgtctccg gtgaaggtga aggtgatgct acttacggta
     2341 aattgacctt aaaatttatt tgtactactg gtaaattgcc agttccgtgg ccaaccttag
     2401 tcactacttt aggttatggt ttgatgtgtt ttgctagata cccagatcat atgaaacaac
     2461 atgacttttt caagtctgcc atgccagaag gttatgttca agaaagaact atttttttca
     2521 aagatgacgg taactacaag accagagctg aagtcaagtt tgaaggtgat accttagtta
     2581 atagaatcga attaaaaggt attgatttta aagaagatgg taacatttta ggtcacaaat
     2641 tggaatacaa ctataactct cacaatgttt acatcatggc tgacaaacaa aagaatggta
     2701 tcaaagttaa cttcaaaatt agacacaaca ttgaagatgg ttctgttcaa ttagctgacc
     2761 attatcaaca aaatactcca attggtgatg gtccagtctt gttaccagac aaccattact
     2821 tatcctatca atctaaatta tccaaagatc caaacgaaaa gagagaccac atggtcttgt
     2881 tagaatttgt tactgctgct ggtattaccc acggtatgga tgaattgtac aaaatgtccc
     2941 gccgtaatac tgacgccatc acaatccaca gcatcctgga ttggattgaa gactaataat
     3001 cgctgggacg cccgcctgca ggctcggtac caaattccag aaaagaggcc tcccgaaagg
     3061 ggggcctttt ttcgttttgg tcctaataga taaaggatag gtctggtagt gttgttcgtt
     3121 ctcgcaggta aatcaataat actcagcagt tccgtagact tttcagtggg acagggtagc
     3181 gataacagat agattgtaat aagacacagt aggtgctcgt agttgcgtga agagaaccgc
     3241 tcaggaaatc cagtcagaag tattggtaat cgttgaaaac tcagtcgacc agcctgcggt
     3301 ccggttgacg gctagctcag tcctaggtac agtgctagct cgctgggacg cccgagtgac
     3361 gactgcgaag taacctctat ttatcagcct gcggtccggc caagtactta gtacacactg
     3421 attcgctggg acgcccggta gtgcttatca gacccaatac tgttgaacag cctgcggtcc
     3481 gggttcactg ccgtataggc agtctcaagc tagactctag tggtttcaga gctatgctgg
     3541 aaacagcata gcaagttgaa ataaggctag tccgttatca acttgaaaaa gtggcaccga
     3601 gtcggtgcgt tcactgccgt ataggcagtc gctgggacgc ccggacgtcc tattacactc
     3661 gtcgttggaa actgaagatc agcctgcggt ccgggttcac tgccgtatag gcagtaattt
     3721 tgtttaactt taagaaggag atatacatat gaatcagtca ttcatctcgg acatcttata
     3781 tgccgacatc gaatcgaagg ctaaggaact tacagtcaat tccaacaata ctgtccagcc
     3841 ggtcgcgctt atgcgcttag gagttttcgt tcccaaacct tccaagagca aaggagaaag
     3901 taaggaaatt gacgccacca aagccttctc tcaactggag attgctaaag cagagggcat
     3961 gagtaaagga gaagaacttt tcactggagt tgtcccaatt cttgttgaat tagatggtga
     4021 tgttaatggg cacaaatttt ctgtcagtgg agagggtgaa ggtgatgcaa catacggaaa
     4081 acttaccctt aaatttattt gcactactgg aaaactacct gttccgtggc caacacttgt
     4141 cactactttg acttggggtg ttcaatgctt tgctagatac ccagatcata tgaaacagca
     4201 tgactttttc aagagtgcca tgcccgaagg ttatgtacag gaaagaacta tatttttcaa
     4261 agatgacggg aactacaaga cacgtgctga agtcaagttt gaaggtgata cccttgttaa
     4321 tagaatcgag ttaaaaggta ttgattttaa agaagatgga aacattcttg gacacaaatt
     4381 ggaatacaac gctatttcag ataatgtata catcactgca gacaaacaaa agaatggaat
     4441 caaagctaat ttcaaaatta gacacaacat tgaagatgga agcgttcaac tagcagacca
     4501 ttatcaacaa aatactccaa ttggcgatgg ccctgtcctt ttaccagaca accattacct
     4561 gtccacacaa tctaaacttt cgaaagatcc caacgaaaag agagaccaca tggtccttct
     4621 tgagtttgta acagctgctg ggattacact aggcatggat gaactataca aataataatc
     4681 gctgggacgc ccggcggccg cggaaacaca gaaaaaagcc cgcacctgac agtgcgggct
     4741 ttttttttcg accaaaggta gcgaacgacg agtcactgtt gaggataaat actttctcta
     4801 ctaggcgcct gttacacagg tcctcagcgg cgcgcctttg tcggtgaacg ctctcctgag
     4861 taggacaaat ccgccgggag cggatttgaa cgttgtgaag caacggcccg gagggtggcg
     4921 ggcaggacgc ccgccataaa ctgccaggca tcaaactaag cagaaggcca tcctgacgga
     4981 tggccttttt gcgtttcaga tctaccggta aaccagcaat agacataagc ggctatttaa
     5041 cgaccctgcc ctgaaccgac gacaagctga cgaccgggtc tccgcaagtg gcacttttcg
     5101 gggaaatgtg cgcggaaccc ctatttgttt atttttctaa atacattcaa atatgtatcc
     5161 gctcatgaat taattcttag aaaaactcat cgagcatcaa atgaaactgc aatttattca
     5221 tatcaggatt atcaatacca tatttttgaa aaagccgttt ctgtaatgaa ggagaaaact
     5281 caccgaggca gttccatagg atggcaagat cctggtatcg gtctgcgatt ccgactcgtc
     5341 caacatcaat acaacctatt aatttcccct cgtcaaaaat aaggttatca agtgagaaat
     5401 caccatgagt gacgactgaa tccggtgaga atggcaaaag tttatgcatt tctttccaga
     5461 cttgttcaac aggccagcca ttacgctcgt catcaaaatc actcgcatca accaaaccgt
     5521 tattcattcg tgattgcgcc tgagcgagac gaaatacgcg gtcgctgtta aaaggacaat
     5581 tacaaacagg aatcgaatgc aaccggcgca ggaacactgc cagcgcatca acaatatttt
     5641 cacctgaatc aggatattct tctaatacct ggaatgctgt tttcccgggg atcgcagtgg
     5701 tgagtaacca tgcatcatca ggagtacgga taaaatgctt gatggtcgga agaggcataa
     5761 attccgtcag ccagtttagt ctgaccatct catctgtaac atcattggca acgctacctt
     5821 tgccatgttt cagaaacaac tctggcgcat cgggcttccc atacaatcga tagattgtcg
     5881 cacctgattg cccgacatta tcgcgagccc atttataccc atataaatca gcatccatgt
     5941 tggaatttaa tcgcggccta gagcaagacg tttcccgttg aatatggctc atactcttcc
     6001 tttttcaata ttattgaagc atttatcagg gttattgtct catgagcgga tacatatttg
     6061 aatgtattta gaaaaataaa caaataggca tgctagcgca gaaacgtcct agaagatgcc
     6121 aggaggatac ttagcagaga gacaataagg ccggagcgaa gccgtttttc cataggctcc
     6181 gcccccctga cgaacatcac gaaatctgac gctcaaatca gtggtggcga aacccgacag
     6241 gactataaag ataccaggcg tttccccctg atggctccct cttgcgctct cctgttcccg
     6301 tcctgcggcg tccgtgttgt ggtggaggct ttacccaaat caccacgtcc cgttccgtgt
     6361 agacagttcg ctccaagctg ggctgtgtgc aagaaccccc cgttcagccc gactgctgcg
     6421 ccttatccgg taactatcat cttgagtcca acccggaaag acacgacaaa acgccactgg
     6481 cagcagccat tggtaactga gaattagtgg atttagatat cgagagtctt gaagtggtgg
     6541 cctaacagag gctacactga aaggacagta tttggtatct gcgctccact aaagccagtt
     6601 accaggttaa gcagttcccc aactgactta accttcgatc aaaccgcctc cccaggcggt
     6661 tttttcgttt acagagcagg agattacgac gatcgtaaaa ggatctcaag aagatccttt
     6721 acggattccc gacaccatca ctctagagaa ttcataggat agattctgga aactttaccg
     6781 tccgagctcc agcctgcggt ccggttgacg gctagctcag tcctaggtac agtgctagct
     6841 cgctgggacg cccgagagcc gaatcgcact tatttacagt agttcagcct gcggtccggc
//
