LOCUS       Exported                5853 bp ds-DNA     circular SYN 12-MAY-2021
DEFINITION  1-color stripe ("GREEN").
ACCESSION   .
VERSION     .
KEYWORDS    pJ2048.2_Gs
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 5853)
  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 5853)
  AUTHORS   .
  TITLE     Direct Submission
  JOURNAL   Exported May 12, 2021 from SnapGene Server 1.1.58
            http://www.snapgene.com
FEATURES             Location/Qualifiers
     source          1..5853
                     /organism="synthetic DNA construct"
                     /mol_type="other DNA"
     protein_bind    16..30
                     /label=Csy4 site
                     /bound_moiety="Pseudomonas aeruginosa Csy4 (Cas6f)"
                     /note="cleavage site for the Csy4 endoribonuclease 
                     (Haurwitz et al., 2010)"
     RBS             35..57
                     /note="efficient ribosome binding site from bacteriophage 
                     T7 gene 10 (Olins and Rangwala, 1989)"
     CDS             65..778
                     /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    1333..1347
                     /label=Csy4 site
                     /bound_moiety="Pseudomonas aeruginosa Csy4 (Cas6f)"
                     /note="cleavage site for the Csy4 endoribonuclease 
                     (Haurwitz et al., 2010)"
     protein_bind    1459..1473
                     /label=Csy4 site
                     /bound_moiety="Pseudomonas aeruginosa Csy4 (Cas6f)"
                     /note="cleavage site for the Csy4 endoribonuclease 
                     (Haurwitz et al., 2010)"
     terminator      1793..1820
                     /label=rrnB T2 terminator
                     /note="transcription terminator T2 from the E. coli rrnB 
                     gene"
     CDS             complement(1945..2805)
                     /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     2568..2587
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(2806..2910)
                     /gene="bla"
                     /label=AmpR promoter
     rep_origin      complement(2928..3563)
                     /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(3073..3092)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(3714..4592)
                     /codon_start=1
                     /gene="araC"
                     /product="L-arabinose regulatory protein"
                     /label=araC
                     /translation="MAEAQNDPLLPGYSFNAHLVAGLTPIEANGYLDFFIDRPLGMKGY
                     ILNLTIRGQGVVKNQGREFVCRPGDILLFPPGEIHHYGRHPEAREWYHQWVYFRPRAYW
                     HEWLNWPSIFANTGFFRPDEAHQPHFSDLFGQIINAGQGEGRYSELLAINLLEQLLLRR
                     MEAINESLHPPMDNRVREACQYISDHLADSNFDIASVAQHVCLSPSRLSHLFRQQLGIS
                     VLSWREDQRISQAKLLLSTTRMPIATVGRNVGFDDQLYFSRVFKKCTGASPSEFRAGCE
                     EKVNDVAVKLS"
     promoter        4619..4903
                     /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     4826..4845
                     /label=pBAD Forward
                     /note="For vectors with E. coli araBAD promoter, forward 
                     primer"
     protein_bind    4969..4983
                     /label=Csy4 site
                     /bound_moiety="Pseudomonas aeruginosa Csy4 (Cas6f)"
                     /note="cleavage site for the Csy4 endoribonuclease 
                     (Haurwitz et al., 2010)"
     protein_bind    5095..5109
                     /label=Csy4 site
                     /bound_moiety="Pseudomonas aeruginosa Csy4 (Cas6f)"
                     /note="cleavage site for the Csy4 endoribonuclease 
                     (Haurwitz et al., 2010)"
     protein_bind    5175..5189
                     /label=Csy4 site
                     /bound_moiety="Pseudomonas aeruginosa Csy4 (Cas6f)"
                     /note="cleavage site for the Csy4 endoribonuclease 
                     (Haurwitz et al., 2010)"
     protein_bind    5297..5311
                     /label=Csy4 site
                     /bound_moiety="Pseudomonas aeruginosa Csy4 (Cas6f)"
                     /note="cleavage site for the Csy4 endoribonuclease 
                     (Haurwitz et al., 2010)"
ORIGIN
        1 tgcggtccgg gttcactgcc gtataggcag taattttgtt taactttaag aaggagatat
       61 acatatgcgt aaaggcgaag aactgtttac cggtgtggtt ccgattctgg tggaactgga
      121 cggcgatgtt aatggtcata aattcagtgt tcgcggcgaa ggtgaaggcg atgcgacgaa
      181 cggcaaactg accctgaaat ttatctgcac cacgggtaaa ctgccggtcc cgtggccgac
      241 gctggtgacc acgctgacct atggcgttca atgttttgcg cgttacccgg atcacatgaa
      301 acagcacgac tttttcaaat cggccatgcc ggaaggctat gtgcaggaac gtacgattag
      361 ctttaaagac gatggtacgt ataaaacccg cgcggaagtg aaattcgaag gcgataccct
      421 ggttaaccgt atcgaactga aaggtatcga tttcaaagaa gacggcaata ttctgggtca
      481 taaactggaa tataacttca attcccacaa cgtgtacatc accgcggata aacagaaaaa
      541 cggcattaaa gccaatttca aaatccgcca taatgtggaa gatggtagcg ttcagctggc
      601 cgaccactat cagcaaaaca cgccgattgg tgatggcccg gtcctgctgc cggacaatca
      661 ctacctgagt acccagtccg tgctgtcaaa agatccgaac gaaaaacgtg accacatggt
      721 cctgctggaa tttgtgacgg ctgcgggtat cacccacggc atggacgaac tgtataaaat
      781 gtcccgccgt aatactgacg ccatcacaat ccacagcatc ctggattgga ttgaagacta
      841 ataatcgctg ggacgcccgc ctgcaggctc ggtaccaaat tccagaaaag aggcctcccg
      901 aaaggggggc cttttttcgt tttggtccta atagataaag gataggtctg gtagtgttgt
      961 tcgttctcgc aggtaaatca ataatactca gcagttccgt agacttttca gtgggacagg
     1021 gtagcgataa cagatagatt gtaataagac acagtaggtg ctcgtagttg cgtgaagaga
     1081 accgctcagg aaatccagtc agaagtattg gtaatcgttg aaaactcagt cgaccagcct
     1141 gcggtccggt ttacggctag ctcagtccta ggtattatgc tagctcgctg ggacgcccga
     1201 gtgacgactg cgaagtaacc tctatttatc agcctgcggt ccggccaaga gtacactagc
     1261 tcagtcatcg ctgggacgcc cggtagtgct tatcagaccc aatactgttg aacagcctgc
     1321 ggtccgggtt cactgccgta taggcaggac acatcttaga gtatgtagtt tcagagctat
     1381 gctggaaaca gcatagcaag ttgaaataag gctagtccgt tatcaacttg aaaaagtggc
     1441 accgagtcgg tgcgttcact gccgtatagg cagtcgctgg gacgcccgga cgtcctatta
     1501 cactcgtcgt tggaaactga agatgcggcc gcggaaacac agaaaaaagc ccgcacctga
     1561 cagtgcgggc tttttttttc gaccaaaggt agcgaacgac gagtcactgt tgaggataaa
     1621 tactttctct actaggcgcc tgttacacag gtcctcagcg gcgcgccttt gtcggtgaac
     1681 gctctcctga gtaggacaaa tccgccggga gcggatttga acgttgtgaa gcaacggccc
     1741 ggagggtggc gggcaggacg cccgccataa actgccaggc atcaaactaa gcagaaggcc
     1801 atcctgacgg atggcctttt tgcgtttcag atctgggatt ttggtcatga gattatcaaa
     1861 aaggatcttc acctagatcc ttttaaatta aaaatgaagt tttaaatcaa tctaaagtat
     1921 atatgagtaa acttggtctg acagttacca atgcttaatc agtgaggcac ctatctcagc
     1981 gatctgtcta tttcgttcat ccatagttgc ctgactcccc gtcgtgtaga taactacgat
     2041 acgggagggc ttaccatctg gccccagtgc tgcaatgata ccgcgagacc cacgctcacc
     2101 ggctccagat ttatcagcaa taaaccagcc agccggaagg gccgagcgca gaagtggtcc
     2161 tgcaacttta tccgcctcca tccagtctat taattgttgc cgggaagcta gagtaagtag
     2221 ttcgccagtt aatagtttgc gcaacgttgt tgccattgct gcaggcatcg tggtgtcacg
     2281 ctcgtcgttt ggtatggctt cattcagctc cggttcccaa cgatcaaggc gagttacatg
     2341 atcccccatg ttgtgcaaaa aagcggttag ctccttcggt cctccgatcg ttgtcagaag
     2401 taagttggcc gcagtgttat cactcatggt tatggcagca ctgcataatt ctcttactgt
     2461 catgccatcc gtaagatgct tttctgtgac tggtgagtac tcaaccaagt cattctgaga
     2521 atagtgtatg cggcgaccga gttgctcttg cccggcgtca acacgggata ataccgcgcc
     2581 acatagcaga actttaaaag tgctcatcat tggaaaacgt tcttcggggc gaaaactctc
     2641 aaggatctta ccgctgttga gatccagttc gatgtaaccc actcgtgcac ccaactgatc
     2701 ttcagcatct tttactttca ccagcgtttc tgggtgagca aaaacaggaa ggcaaaatgc
     2761 cgcaaaaaag ggaataaggg cgacacggaa atgttgaata ctcatactct tcctttttca
     2821 atattattga agcatttatc agggttattg tctcatgagc ggatacatat ttgaatgtat
     2881 ttagaaaaat aaacaaatag gggttccgcg cagcatgcta gcggcagaaa cgtcctagaa
     2941 gatgccagga ggatacttag cagagagaca ataaggccgg agcgaagccg tttttccata
     3001 ggctccgccc ccctgacgaa catcacgaaa tctgacgctc aaatcagtgg tggcgaaacc
     3061 cgacaggact ataaagatac caggcgtttc cccctgatgg ctccctcttg cgctctcctg
     3121 ttcccgtcct gcggcgtccg tgttgtggtg gaggctttac ccaaatcacc acgtcccgtt
     3181 ccgtgtagac agttcgctcc aagctgggct gtgtgcaaga accccccgtt cagcccgact
     3241 gctgcgcctt atccggtaac tatcatcttg agtccaaccc ggaaagacac gacaaaacgc
     3301 cactggcagc agccattggt aactgagaat tagtggattt agatatcgag agtcttgaag
     3361 tggtggccta acagaggcta cactgaaagg acagtatttg gtatctgcgc tccactaaag
     3421 ccagttacca ggttaagcag ttccccaact gacttaacct tcgatcaaac cgcctcccca
     3481 ggcggttttt tcgtttacag agcaggagat tacgacgatc gtaaaaggat ctcaagaaga
     3541 tcctttacgg attcccgaca ccatcactct agatttcagt gcaatttatc tcttcaaatg
     3601 tagcacctga agtcagcccc atacgatata agttgtaatt ctcatgttag tcatgccccg
     3661 cgcccaccgg aaggagctga ctgggttgct cctagggtct gattcgttac caattatgac
     3721 aacttgacgg ctacatcatt cactttttct tcacaaccgg cacggaactc gctcgggctg
     3781 gccccggtgc attttttaaa tacccgcgag aaatagagtt gatcgtcaaa accaacattg
     3841 cgaccgacgg tggcgatagg catccgggtg gtgctcaaaa gcagcttcgc ctggctgata
     3901 cgttggtcct cgcgccagct taagacgcta atccctaact gctggcggaa aagatgtgac
     3961 agacgcgacg gcgacaagca aacatgctgt gcgacgctgg cgatatcaaa attgctgtct
     4021 gccaggtgat cgctgatgta ctgacaagcc tcgcgtaccc gattatccat cggtggatgg
     4081 agcgactcgt taatcgcttc catgcgccgc agtaacaatt gctcaagcag atttatcgcc
     4141 agcagctccg aatagcgccc ttccccttgc ccggcgttaa tgatttgccc aaacaggtcg
     4201 ctgaaatgcg gctggtgcgc ttcatccggg cgaaagaacc ccgtattggc aaatattgac
     4261 ggccagttaa gccattcatg ccagtaggcg cgcggacgaa agtaaaccca ctggtgatac
     4321 cattcgcgag cctccggatg acgaccgtag tgatgaatct ctcctggcgg gaacagcaaa
     4381 atatcacccg gtcggcaaac aaattctcgt ccctgatttt tcaccacccc ctgaccgcga
     4441 atggtgagat tgagaatata acctttcatt cccagcggtc ggtcgataaa aaaatcgaga
     4501 taaccgttgg cctcaatcgg cgttaaaccc gccaccagat gggcattaaa cgagtatccc
     4561 ggcagcaggg gatcattttg cgcttcagcc atacttttca tactcccgcc attcagagaa
     4621 gaaaccaatt gtccatattg catcagacat tgccgtcact gcgtctttta ctggctcttc
     4681 tcgctaacca aaccggtaac cccgcttatt aaaagcattc tgtaacaaag cgggaccaaa
     4741 gccatgacaa aaacgcgtaa caaaagtgtc tataatcacg gcagaaaagt ccacattgat
     4801 tatttgcacg gcgtcacact ttgctatgcc atagcatttt tatccataag attagcggtt
     4861 cctacctgac gctttttatc gcaactctct actgtttctc cataccgaat tcataggata
     4921 gattctggaa actttaccgt ccgagctcca gcctgcggtc cgggttcact gccgtatagg
     4981 cagtgactga gctagtgtac tctgtttcag agctatgctg gaaacagcat agcaagttga
     5041 aataaggcta gtccgttatc aacttgaaaa agtggcaccg agtcggtgcg ttcactgccg
     5101 tataggcagt cgctgggacg cccggggact acacttacga aactattgat tgctcagcct
     5161 gcggtccggg ttcactgccg tataggcagc atcttagagt atgtagtttc agagctatgc
     5221 tggaaacagc atagcaagtt gaaataaggc tagtccgtta tcaacttgaa aaagtggcac
     5281 cgagtcggtg cgttcactgc cgtataggca gtcgctggga cgcccgctcg agcaataaac
     5341 agttgatagg gcttctccgt taccatggtt cagccaaaaa acttaagacc gccggtcttg
     5401 tccactacct tgcagtaatg cggtggacag gatcggcggt tttcttttct cttctcaatt
     5461 cttctgacct gtaacgaata atagatagta aagtagtctc cgattgagtt ttctctgccg
     5521 agtcccaccc agttctgtga tttcagtaag ttggtaattg atacactgtt gcgagaactg
     5581 ctgcctggta gtagataggt tgttattgag taagaaggta aagtgaacga aatccctgaa
     5641 actgagactg tagaaaataa gcttcagcct gcggtccggt tgacggctag ctcagtccta
     5701 ggtacagtgc tagctcgctg ggacgcccga gatagccgtt acacaggtga cacttatttc
     5761 agcctgcggt ccggccatac atactctaag atgtgtctcg ctgggacgcc cgggatccaa
     5821 gagatttcta cacgattgag cactgtctca gcc
//
