LOCUS       Exported                5651 bp ds-DNA     circular SYN 12-MAY-2021
DEFINITION  Double-inverter.
ACCESSION   .
VERSION     .
KEYWORDS    pJ2048_2xNOT
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 5651)
  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 5651)
  AUTHORS   .
  TITLE     Direct Submission
  JOURNAL   Exported May 12, 2021 from SnapGene Server 1.1.58
            http://www.snapgene.com
FEATURES             Location/Qualifiers
     source          1..5651
                     /organism="synthetic DNA construct"
                     /mol_type="other DNA"
     protein_bind    80..94
                     /label=Csy4 site
                     /bound_moiety="Pseudomonas aeruginosa Csy4 (Cas6f)"
                     /note="cleavage site for the Csy4 endoribonuclease 
                     (Haurwitz et al., 2010)"
     RBS             99..121
                     /note="efficient ribosome binding site from bacteriophage 
                     T7 gene 10 (Olins and Rangwala, 1989)"
     CDS             129..842
                     /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    1397..1411
                     /label=Csy4 site
                     /bound_moiety="Pseudomonas aeruginosa Csy4 (Cas6f)"
                     /note="cleavage site for the Csy4 endoribonuclease 
                     (Haurwitz et al., 2010)"
     protein_bind    1523..1537
                     /label=Csy4 site
                     /bound_moiety="Pseudomonas aeruginosa Csy4 (Cas6f)"
                     /note="cleavage site for the Csy4 endoribonuclease 
                     (Haurwitz et al., 2010)"
     terminator      1857..1884
                     /label=rrnB T2 terminator
                     /note="transcription terminator T2 from the E. coli rrnB 
                     gene"
     CDS             complement(2009..2869)
                     /codon_start=1
                     /gene="bla"
                     /product="beta-lactamase"
                     /label=AmpR
                     /note="confers resistance to ampicillin, carbenicillin, and
                     related antibiotics"
                     /translation="MSIQHFRVALIPFFAAFCLPVFAHPETLVKVKDAEDQLGARVGYI
                     ELDLNSGKILESFRPEERFPMMSTFKVLLCGAVLSRVDAGQEQLGRRIHYSQNDLVEYS
                     PVTEKHLTDGMTVRELCSAAITMSDNTAANLLLTTIGGPKELTAFLHNMGDHVTRLDRW
                     EPELNEAIPNDERDTTMPAAMATTLRKLLTGELLTLASRQQLIDWMEADKVAGPLLRSA
                     LPAGWFIADKSGAGERGSRGIIAALGPDGKPSRIVVIYTTGSQATMDERNRQIAEIGAS
                     LIKHW"
     primer_bind     2632..2651
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(2870..2974)
                     /gene="bla"
                     /label=AmpR promoter
     rep_origin      complement(2992..3627)
                     /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(3137..3156)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(3778..4656)
                     /codon_start=1
                     /gene="araC"
                     /product="L-arabinose regulatory protein"
                     /label=araC
                     /translation="MAEAQNDPLLPGYSFNAHLVAGLTPIEANGYLDFFIDRPLGMKGY
                     ILNLTIRGQGVVKNQGREFVCRPGDILLFPPGEIHHYGRHPEAREWYHQWVYFRPRAYW
                     HEWLNWPSIFANTGFFRPDEAHQPHFSDLFGQIINAGQGEGRYSELLAINLLEQLLLRR
                     MEAINESLHPPMDNRVREACQYISDHLADSNFDIASVAQHVCLSPSRLSHLFRQQLGIS
                     VLSWREDQRISQAKLLLSTTRMPIATVGRNVGFDDQLYFSRVFKKCTGASPSEFRAGCE
                     EKVNDVAVKLS"
     promoter        4683..4967
                     /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     4890..4909
                     /label=pBAD Forward
                     /note="For vectors with E. coli araBAD promoter, forward 
                     primer"
     protein_bind    5033..5047
                     /label=Csy4 site
                     /bound_moiety="Pseudomonas aeruginosa Csy4 (Cas6f)"
                     /note="cleavage site for the Csy4 endoribonuclease 
                     (Haurwitz et al., 2010)"
     protein_bind    5159..5173
                     /label=Csy4 site
                     /bound_moiety="Pseudomonas aeruginosa Csy4 (Cas6f)"
                     /note="cleavage site for the Csy4 endoribonuclease 
                     (Haurwitz et al., 2010)"
ORIGIN
        1 gatgtgtctc gctgggacgc ccgggatcca agagatttct acacgattga gcactgtctc
       61 agcctgcggt ccgggttcac tgccgtatag gcagtaattt tgtttaactt taagaaggag
      121 atatacatat gcgtaaaggc gaagaactgt ttaccggtgt ggttccgatt ctggtggaac
      181 tggacggcga tgttaatggt cataaattca gtgttcgcgg cgaaggtgaa ggcgatgcga
      241 cgaacggcaa actgaccctg aaatttatct gcaccacggg taaactgccg gtcccgtggc
      301 cgacgctggt gaccacgctg acctatggcg ttcaatgttt tgcgcgttac ccggatcaca
      361 tgaaacagca cgactttttc aaatcggcca tgccggaagg ctatgtgcag gaacgtacga
      421 ttagctttaa agacgatggt acgtataaaa cccgcgcgga agtgaaattc gaaggcgata
      481 ccctggttaa ccgtatcgaa ctgaaaggta tcgatttcaa agaagacggc aatattctgg
      541 gtcataaact ggaatataac ttcaattccc acaacgtgta catcaccgcg gataaacaga
      601 aaaacggcat taaagccaat ttcaaaatcc gccataatgt ggaagatggt agcgttcagc
      661 tggccgacca ctatcagcaa aacacgccga ttggtgatgg cccggtcctg ctgccggaca
      721 atcactacct gagtacccag tccgtgctgt caaaagatcc gaacgaaaaa cgtgaccaca
      781 tggtcctgct ggaatttgtg acggctgcgg gtatcaccca cggcatggac gaactgtata
      841 aaatgtcccg ccgtaatact gacgccatca caatccacag catcctggat tggattgaag
      901 actaataatc gctgggacgc ccgcctgcag gctcggtacc aaattccaga aaagaggcct
      961 cccgaaaggg gggccttttt tcgttttggt cctaatagat aaaggatagg tctggtagtg
     1021 ttgttcgttc tcgcaggtaa atcaataata ctcagcagtt ccgtagactt ttcagtggga
     1081 cagggtagcg ataacagata gattgtaata agacacagta ggtgctcgta gttgcgtgaa
     1141 gagaaccgct caggaaatcc agtcagaagt attggtaatc gttgaaaact cagtcgacca
     1201 gcctgcggtc cggtttacgg ctagctcagt cctaggtatt atgctagctc gctgggacgc
     1261 ccgagtgacg actgcgaagt aacctctatt tatcagcctg cggtccggcc aagagtacac
     1321 tagctcagtc atcgctggga cgcccggtag tgcttatcag acccaatact gttgaacagc
     1381 ctgcggtccg ggttcactgc cgtataggca ggacacatct tagagtatgt agtttcagag
     1441 ctatgctgga aacagcatag caagttgaaa taaggctagt ccgttatcaa cttgaaaaag
     1501 tggcaccgag tcggtgcgtt cactgccgta taggcagtcg ctgggacgcc cggacgtcct
     1561 attacactcg tcgttggaaa ctgaagatgc ggccgcggaa acacagaaaa aagcccgcac
     1621 ctgacagtgc gggctttttt tttcgaccaa aggtagcgaa cgacgagtca ctgttgagga
     1681 taaatacttt ctctactagg cgcctgttac acaggtcctc agcggcgcgc ctttgtcggt
     1741 gaacgctctc ctgagtagga caaatccgcc gggagcggat ttgaacgttg tgaagcaacg
     1801 gcccggaggg tggcgggcag gacgcccgcc ataaactgcc aggcatcaaa ctaagcagaa
     1861 ggccatcctg acggatggcc tttttgcgtt tcagatctgg gattttggtc atgagattat
     1921 caaaaaggat cttcacctag atccttttaa attaaaaatg aagttttaaa tcaatctaaa
     1981 gtatatatga gtaaacttgg tctgacagtt accaatgctt aatcagtgag gcacctatct
     2041 cagcgatctg tctatttcgt tcatccatag ttgcctgact ccccgtcgtg tagataacta
     2101 cgatacggga gggcttacca tctggcccca gtgctgcaat gataccgcga gacccacgct
     2161 caccggctcc agatttatca gcaataaacc agccagccgg aagggccgag cgcagaagtg
     2221 gtcctgcaac tttatccgcc tccatccagt ctattaattg ttgccgggaa gctagagtaa
     2281 gtagttcgcc agttaatagt ttgcgcaacg ttgttgccat tgctgcaggc atcgtggtgt
     2341 cacgctcgtc gtttggtatg gcttcattca gctccggttc ccaacgatca aggcgagtta
     2401 catgatcccc catgttgtgc aaaaaagcgg ttagctcctt cggtcctccg atcgttgtca
     2461 gaagtaagtt ggccgcagtg ttatcactca tggttatggc agcactgcat aattctctta
     2521 ctgtcatgcc atccgtaaga tgcttttctg tgactggtga gtactcaacc aagtcattct
     2581 gagaatagtg tatgcggcga ccgagttgct cttgcccggc gtcaacacgg gataataccg
     2641 cgccacatag cagaacttta aaagtgctca tcattggaaa acgttcttcg gggcgaaaac
     2701 tctcaaggat cttaccgctg ttgagatcca gttcgatgta acccactcgt gcacccaact
     2761 gatcttcagc atcttttact ttcaccagcg tttctgggtg agcaaaaaca ggaaggcaaa
     2821 atgccgcaaa aaagggaata agggcgacac ggaaatgttg aatactcata ctcttccttt
     2881 ttcaatatta ttgaagcatt tatcagggtt attgtctcat gagcggatac atatttgaat
     2941 gtatttagaa aaataaacaa ataggggttc cgcgcagcat gctagcggca gaaacgtcct
     3001 agaagatgcc aggaggatac ttagcagaga gacaataagg ccggagcgaa gccgtttttc
     3061 cataggctcc gcccccctga cgaacatcac gaaatctgac gctcaaatca gtggtggcga
     3121 aacccgacag gactataaag ataccaggcg tttccccctg atggctccct cttgcgctct
     3181 cctgttcccg tcctgcggcg tccgtgttgt ggtggaggct ttacccaaat caccacgtcc
     3241 cgttccgtgt agacagttcg ctccaagctg ggctgtgtgc aagaaccccc cgttcagccc
     3301 gactgctgcg ccttatccgg taactatcat cttgagtcca acccggaaag acacgacaaa
     3361 acgccactgg cagcagccat tggtaactga gaattagtgg atttagatat cgagagtctt
     3421 gaagtggtgg cctaacagag gctacactga aaggacagta tttggtatct gcgctccact
     3481 aaagccagtt accaggttaa gcagttcccc aactgactta accttcgatc aaaccgcctc
     3541 cccaggcggt tttttcgttt acagagcagg agattacgac gatcgtaaaa ggatctcaag
     3601 aagatccttt acggattccc gacaccatca ctctagattt cagtgcaatt tatctcttca
     3661 aatgtagcac ctgaagtcag ccccatacga tataagttgt aattctcatg ttagtcatgc
     3721 cccgcgccca ccggaaggag ctgactgggt tgctcctagg gtctgattcg ttaccaatta
     3781 tgacaacttg acggctacat cattcacttt ttcttcacaa ccggcacgga actcgctcgg
     3841 gctggccccg gtgcattttt taaatacccg cgagaaatag agttgatcgt caaaaccaac
     3901 attgcgaccg acggtggcga taggcatccg ggtggtgctc aaaagcagct tcgcctggct
     3961 gatacgttgg tcctcgcgcc agcttaagac gctaatccct aactgctggc ggaaaagatg
     4021 tgacagacgc gacggcgaca agcaaacatg ctgtgcgacg ctggcgatat caaaattgct
     4081 gtctgccagg tgatcgctga tgtactgaca agcctcgcgt acccgattat ccatcggtgg
     4141 atggagcgac tcgttaatcg cttccatgcg ccgcagtaac aattgctcaa gcagatttat
     4201 cgccagcagc tccgaatagc gcccttcccc ttgcccggcg ttaatgattt gcccaaacag
     4261 gtcgctgaaa tgcggctggt gcgcttcatc cgggcgaaag aaccccgtat tggcaaatat
     4321 tgacggccag ttaagccatt catgccagta ggcgcgcgga cgaaagtaaa cccactggtg
     4381 ataccattcg cgagcctccg gatgacgacc gtagtgatga atctctcctg gcgggaacag
     4441 caaaatatca cccggtcggc aaacaaattc tcgtccctga tttttcacca ccccctgacc
     4501 gcgaatggtg agattgagaa tataaccttt cattcccagc ggtcggtcga taaaaaaatc
     4561 gagataaccg ttggcctcaa tcggcgttaa acccgccacc agatgggcat taaacgagta
     4621 tcccggcagc aggggatcat tttgcgcttc agccatactt ttcatactcc cgccattcag
     4681 agaagaaacc aattgtccat attgcatcag acattgccgt cactgcgtct tttactggct
     4741 cttctcgcta accaaaccgg taaccccgct tattaaaagc attctgtaac aaagcgggac
     4801 caaagccatg acaaaaacgc gtaacaaaag tgtctataat cacggcagaa aagtccacat
     4861 tgattatttg cacggcgtca cactttgcta tgccatagca tttttatcca taagattagc
     4921 ggttcctacc tgacgctttt tatcgcaact ctctactgtt tctccatacc gaattcatag
     4981 gatagattct ggaaacttta ccgtccgagc tccagcctgc ggtccgggtt cactgccgta
     5041 taggcagtga ctgagctagt gtactctgtt tcagagctat gctggaaaca gcatagcaag
     5101 ttgaaataag gctagtccgt tatcaacttg aaaaagtggc accgagtcgg tgcgttcact
     5161 gccgtatagg cagtcgctgg gacgcccgct cgagcaataa acagttgata gggcttctcc
     5221 gttaccatgg ttcagccaaa aaacttaaga ccgccggtct tgtccactac cttgcagtaa
     5281 tgcggtggac aggatcggcg gttttctttt ctcttctcaa ttcttctgac ctgtaacgaa
     5341 taatagatag taaagtagtc tccgattgag ttttctctgc cgagtcccac ccagttctgt
     5401 gatttcagta agttggtaat tgatacactg ttgcgagaac tgctgcctgg tagtagatag
     5461 gttgttattg agtaagaagg taaagtgaac gaaatccctg aaactgagac tgtagaaaat
     5521 aagcttcagc ctgcggtccg gttgacggct agctcagtcc taggtacagt gctagctcgc
     5581 tgggacgccc gagatagccg ttacacaggt gacacttatt tcagcctgcg gtccggccat
     5641 acatactcta a
//
