LOCUS       Exported                3867 bp ds-DNA     circular SYN 12-MAY-2021
DEFINITION  CymRAM + PCymRC-YFP Reporter. Cuma sensor for use in non-Marionette 
            strain..
ACCESSION   .
VERSION     .
KEYWORDS    pAJM.657
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 3867)
  AUTHORS   Meyer AJ, Segall-Shapiro TH, Glassey E, Zhang J, Voigt CA
  TITLE     Escherichia coli ""Marionette"" strains with 12 highly optimized 
            small-molecule sensors.
  JOURNAL   Nat Chem Biol. 2018 Nov 26. pii: 10.1038/s41589-018-0168-3. doi: 
            10.1038/s41589-018-0168-3.
  PUBMED    30478458
REFERENCE   2  (bases 1 to 3867)
  AUTHORS   .
  TITLE     Direct Submission
  JOURNAL   Exported May 12, 2021 from SnapGene Server 1.1.58
            http://www.snapgene.com
FEATURES             Location/Qualifiers
     source          1..3867
                     /organism="synthetic DNA construct"
                     /mol_type="other DNA"
     misc_RNA        1..50
                     /label=sTRSV HHRz
                     /note="hammerhead ribozyme from the tobacco ringspot virus 
                     satellite RNA (Khvorova et al., 2003)"
     CDS             100..819
                     /codon_start=1
                     /product="enhanced YFP"
                     /label=EYFP
                     /note="mammalian codon-optimized"
                     /translation="MVSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTL
                     KFICTTGKLPVPWPTLVTTFGYGLQCFARYPDHMKLHDFFKSAMPEGYVQERTIFFKDD
                     GNYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIK
                     VNFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSYQSALSKDPNEKRDHMVLL
                     EFVTAAGITLGMDELYK"
     primer_bind     complement(145..166)
                     /label=EGFP-N
                     /note="EGFP, reverse primer"
     primer_bind     complement(406..425)
                     /label=EXFP-R
                     /note="For distinguishing EGFP variants, reverse primer"
     primer_bind     753..774
                     /label=EGFP-C
                     /note="EGFP, forward primer"
     primer_bind     complement(1770..1789)
                     /label=pBRrevBam
                     /note="pBR322 vectors, tet region, downstream of BamHI, 
                     reverse primer"
     rep_origin      complement(2154..2699)
                     /direction=LEFT
                     /label=p15A ori
                     /note="Plasmids containing the medium-copy-number p15A 
                     origin of replication can be propagated in E. coli cells 
                     that contain a second plasmid with the ColE1 origin.
                     "
     primer_bind     complement(2234..2253)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(2788..3603)
                     /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     3511..3530
                     /label=Kan-R
                     /note="Kanamycin resistance gene, reverse primer"
     primer_bind     3595..3614
                     /label=pENTR-R
                     /note="pENTR vectors, reverse primer"
     protein_bind    3776..3803
                     /label=CuO
                     /bound_moiety="CymR"
                     /note="CymR-binding P2 operator sequence from the p-cmt 
                     operon of Pseudomonas putida (Mullick et al., 2006)"
     protein_bind    3834..3861
                     /label=CuO
                     /bound_moiety="CymR"
                     /note="CymR-binding P2 operator sequence from the p-cmt 
                     operon of Pseudomonas putida (Mullick et al., 2006)"
ORIGIN
        1 ctgtcaccgg atgtgctttc cggtctgatg agtccgtgag gacgaaacag cctctacaaa
       61 taattttgtt taatactaga gaaagagggg aaatactaga tggtgagcaa gggcgaggag
      121 ctgttcaccg gggtggtgcc catcctggtc gagctggacg gcgacgtaaa cggccacaag
      181 ttcagcgtgt ccggcgaggg cgagggcgat gccacctacg gcaagctgac cctgaagttc
      241 atctgcacca caggcaagct gcccgtgccc tggcccaccc tcgtgaccac cttcggctac
      301 ggcctgcaat gcttcgcccg ctaccccgac cacatgaagc tgcacgactt cttcaagtcc
      361 gccatgcccg aaggctacgt ccaggagcgc accatcttct tcaaggacga cggcaactac
      421 aagacccgcg ccgaggtgaa gttcgagggc gacaccctgg tgaaccgcat cgagctgaag
      481 ggcatcgact tcaaggagga cggcaacatc ctggggcaca agctggagta caactacaac
      541 agccacaacg tctatatcat ggccgacaag cagaagaacg gcatcaaggt gaacttcaag
      601 atccgccaca acatcgagga cggcagcgtg cagctcgccg accactacca gcagaacacc
      661 ccaatcggcg acggccccgt gctgctgccc gacaaccact accttagcta ccagtccgcc
      721 ctgagcaaag accccaacga gaagcgcgat cacatggtcc tgctggagtt cgtgaccgcc
      781 gccgggatca ctctcggcat ggacgagctg tacaagtaac tcggtaccaa attccagaaa
      841 agaggcctcc cgaaaggggg gccttttttc gttttggtcc aatggcggcg cgccatcgaa
      901 tggtgcaaaa cctttcgcgg tatggcatga tagcgcccgg aagagagtca attcagggtg
      961 gtgaatatga gcccgaaacg tcgtacccag gcagaacgtg caatggaaac ccagggtaaa
     1021 ctgattgcag cagcactggg tgttctgcgt gaaaaaggtt atgcaggttt tcgtattgca
     1081 gatgttccgg gtgcagccgg tgttagccgt ggtgcacaga gccatcattt tccgaccaaa
     1141 ctggaactgc tgctggcaac ctttgaatgg ctgtatgagc agattaccga acgtagccgt
     1201 gcacgtctgg caaaactgaa accggaagat gatgttattc agcagatgct ggatgatgca
     1261 gcagaatttt ttctggatga tgattttagc atcggcctgg atctgattgt tgcagcagat
     1321 cgtgatccgg cactgcgtga aggtattcag cgtaccgttg aacgtaatcg ttttgttgtt
     1381 gaagatatgt ggctgggtgt gctggtgagc cgtggtctga gccgtgatga tgccgaagat
     1441 attctgtggc tgatttttaa cagcgttcgt ggtctggtag ttcgtagcct gtggcagaaa
     1501 gataaagaac gttttgaacg tgtgcgtaat agcaccctgg aaattgcacg tgaacgttat
     1561 gcaaaattca aacgttgata aggatcctaa ttggtaacga atcagacaat tgacggctcg
     1621 agggagtagc atagggtttg cagaatccct gcttcgtcca tttgacaggc acattatgca
     1681 tcgatgataa gctgtcaaac atgagcagat cctctacgcc ggacgcatcg tggccggcat
     1741 caccggcgcc acaggtgcgg ttgctggcgc ctatatcgcc gacatcaccg atggggaaga
     1801 tcgggctcgc cacttcgggc tcatgagcaa atattttatc tgaggtgctt cctcgctcac
     1861 tgactcgctg cacgaggcag acctcagcgc tagcggagtg tatactggct tactatgttg
     1921 gcactgatga gggtgtcagt gaagtgcttc atgtggcagg agaaaaaagg ctgcaccggt
     1981 gcgtcagcag aatatgtgat acaggatata ttccgcttcc tcgctcactg actcgctacg
     2041 ctcggtcgtt cgactgcggc gagcggaaat ggcttacgaa cggggcggag atttcctgga
     2101 agatgccagg aagatactta acagggaagt gagagggccg cggcaaagcc gtttttccat
     2161 aggctccgcc cccctgacaa gcatcacgaa atctgacgct caaatcagtg gtggcgaaac
     2221 ccgacaggac tataaagata ccaggcgttt ccccctggcg gctccctcgt gcgctctcct
     2281 gttcctgcct ttcggtttac cggtgtcatt ccgctgttat ggccgcgttt gtctcattcc
     2341 acgcctgaca ctcagttccg ggtaggcagt tcgctccaag ctggactgta tgcacgaacc
     2401 ccccgttcag tccgaccgct gcgccttatc cggtaactat cgtcttgagt ccaacccgga
     2461 aagacatgca aaagcaccac tggcagcagc cactggtaat tgatttagag gagttagtct
     2521 tgaagtcatg cgccggttaa ggctaaactg aaaggacaag ttttggtgac tgcgctcctc
     2581 caagccagtt acctcggttc aaagagttgg tagctcagag aaccttcgaa aaaccgccct
     2641 gcaaggcggt tttttcgttt tcagagcaag agattacgcg cagaccaaaa cgatctcaag
     2701 aagatcatct tattaagggg tctgacgctc agtggaacga aaaatcaatc taaagtatat
     2761 atgagtaaac ttggtctgac agttacctta gaaaaactca tcgagcatca aatgaaactg
     2821 caatttattc atatcaggat tatcaatacc atatttttga aaaagccgtt tctgtaatga
     2881 aggagaaaac tcaccgaggc agttccatag gatggcaaga tcctggtatc ggtctgcgat
     2941 tccgactcgt ccaacatcaa tacaacctat taatttcccc tcgtcaaaaa taaggttatc
     3001 aagtgagaaa tcaccatgag tgacgactga atccggtgag aatggcaaaa gcttatgcat
     3061 ttctttccag acttgttcaa caggccagcc attacgctcg tcatcaaaat cactcgcatc
     3121 aaccaaaccg ttattcattc gtgattgcgc ctgagcgaga cgaaatacgc gatcgctgtt
     3181 aaaaggacaa ttacaaacag gaatcgaatg caaccggcgc aggaacactg ccagcgcatc
     3241 aacaatattt tcacctgaat caggatattc ttctaatacc tggaatgctg ttttcccggg
     3301 gatcgcagtg gtgagtaacc atgcatcatc aggagtacgg ataaaatgct tgatggtcgg
     3361 aagaggcata aattccgtca gccagtttag tctgaccatc tcatctgtaa catcattggc
     3421 aacgctacct ttgccatgtt tcagaaacaa ctctggcgca tcgggcttcc catacaatcg
     3481 atagattgtc gcacctgatt gcccgacatt atcgcgagcc catttatacc catataaatc
     3541 agcatccatg ttggaattta atcgcggcct ggagcaagac gtttcccgtt gaatatggct
     3601 cataacaccc cttgtattac tgtttatgta agcagacagt tttattgttc atgatgatat
     3661 atttttatct tgtgcaatgt acatcagaga ttttgagaca caaccaatta ttgaaggcct
     3721 ccctaacggg gggccttttt ttgtttctgg tctcccgctt aacgatcgtt ggctgaacaa
     3781 acagacaatc tggtctgttt gtattatgga aaatttttct gtataataga ttcaacaaac
     3841 agacaatctg gtctgtttgt attatag
//
