LOCUS       sequence-408084-        2809 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  PTet-YFP plasmid for LPattB2 integration.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 2809)
  AUTHORS   Triassi AJ, Fields BD, Monahan CE, Means JM, Park Y, Doosthosseini 
            H, Padmakumar JP, Isabella VM, Voigt CA
  TITLE     Redesign of an Escherichia coli Nissle treatment for phenylketonuria
            using insulated genomic landing pads and genetic circuits to reduce 
            burden.
  JOURNAL   Cell Syst. 2023 Jun 21;14(6):512-524.e12. doi: 
            10.1016/j.cels.2023.05.004.
  PUBMED    37348465
REFERENCE   2  (bases 1 to 2809)
  AUTHORS   .
  TITLE     Direct Submission
  JOURNAL   Exported Dec 15, 2025 from SnapGene Server 8.0.1
            https://www.snapgene.com
COMMENT     SGRef: number: 1; type: "Journal Article"; doi: 
            "10.1016/j.cels.2023.05"; journalName: "Cell Syst"; date: 
            "2023-06-21- 21"; volume: "14"; issue: "6"; pages: "512-524.e12"
COMMENT     Sequence Label: plYJP067-Ptet
FEATURES             Location/Qualifiers
     source          1..2809
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     rep_origin      270..658
                     /label=R6K gamma ori
                     /note="gamma replication origin from E. coli plasmid R6K; 
                     requires the R6K initiator protein pi for replication"
     terminator      complement(701..795)
                     /label=lambda t0 terminator
                     /note="transcription terminator from phage lambda"
     CDS             complement(806..1621)
                     /codon_start=1
                     /product="aminoglycoside phosphotransferase"
                     /label=KanR
                     /note="confers resistance to kanamycin in bacteria or G418 
                     (Geneticin(R)) in eukaryotes"
                     /translation="MSHIQRETSCSRPRLNSNMDADLYGYKWARDNVGQSGATIYRLYG
                     KPDAPELFLKHGKGSVANDVTDEMVRLNWLTEFMPLPTIKHFIRTPDDAWLLTTAIPGK
                     TAFQVLEEYPDSGENIVDALAVFLRRLHSIPVCNCPFNSDRVFRLAQAQSRMNNGLVDA
                     SDFDDERNGWPVEQVWKEMHKLLPFSPDSVVTHGDFSLDNLIFDEGKLIGCIDVGRVGI
                     ADRYQDLAILWNCLGEFSPSLQKRLFQKYGIDNPDMNKLQFHLMLDEFF"
     CDS             complement(806..1621)
                     /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     1529..1548
                     /label=Kan-R
                     /note="Kanamycin resistance gene, reverse primer"
     promoter        complement(1622..1713)
                     /gene="bla"
                     /label=AmpR promoter
     protein_bind    1765..1812
                     /label=FRT
                     /bound_moiety="FLP recombinase from the Saccharomyces 
                     cerevisiae 2u plasmid"
                     /note="FLP-mediated recombination occurs in the 8-bp core 
                     sequence TCTAGAAA (Turan and Bode, 2011)."
     protein_bind    1908..1926
                     /gene="tetO"
                     /label=tet operator
                     /bound_moiety="tetracycline repressor TetR"
                     /note="bacterial operator O2 for the tetR and tetA genes"
     misc_RNA        1964..2013
                     /label=sTRSV HHRz
                     /note="hammerhead ribozyme from the tobacco ringspot virus 
                     satellite RNA (Khvorova et al., 2003)"
     RBS             2045..2056
                     /note="strong bacterial ribosome binding site (Elowitz and 
                     Leibler, 2000)"
     CDS             join(2063..2065,2066..2068,2069..2782)
                     /codon_start=1
                     /product="enhanced YFP"
                     /label=EYFP
                     /note="mammalian codon-optimized"
                     /translation="MVSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTL
                     KFICTTGKLPVPWPTLVTTFGYGLQCFARYPDHMKLHDFFKSAMPEGYVQERTIFFKDD
                     GNYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIK
                     VNFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSYQSALSKDPNEKRDHMVLL
                     EFVTAAGITLGMDELYK"
     primer_bind     complement(2108..2129)
                     /label=EGFP-N
                     /note="EGFP, reverse primer"
     primer_bind     complement(2369..2388)
                     /label=EXFP-R
                     /note="For distinguishing EGFP variants, reverse primer"
     primer_bind     2716..2737
                     /label=EGFP-C
                     /note="EGFP, forward primer"
ORIGIN
        1 tcgcccgaga acttctgcaa ggcactgctc ttggctggaa acacagaaaa aagcccgcac
       61 ctgacagtgc gggctttttt tttcgaccaa aggctcggta ccaaattcca gaaaagacac
      121 ccgaaagggt gttttttcgt tttggtccag gcggtaatac ggttatccac agaatccggg
      181 gataacgcag gaaagaacat gtgagcaaaa ggccagcaaa aggccaggaa ccgtaaaaat
      241 gattcgcacg ggcccatggc taattcccat gtcagccgtt aagtgttcct gtgtcactca
      301 aaattgcttt gagaggctct aagggcttct cagtgcgtta catccctggc ttgttgtcca
      361 caaccgttaa accttaaaag ctttaaaagc cttatatatt cttttttttc ttataaaact
      421 taaaacctta gaggctattt aagttgctga tttatattaa ttttattgtt caaacatgag
      481 agcttagtac gtgaaacatg agagcttagt acgttagcca tgagagctta gtacgttagc
      541 catgagggtt tagttcgtta aacatgagag cttagtacgt taaacatgag agcttagtac
      601 gtgaaacatg agagcttagt acgtactatc aacaggttga actgctgatc ttcagatcct
      661 ctacgccgga cgcatcgtgg ccggatcttc ttgtgcttgg attctcacca ataaaaaacg
      721 cccggcggca accgagcgtt ctgaacaaat ccagatggag ttctgaggtc attactggat
      781 ctatcaacag gagtccaagc gagctttaga aaaactcatc gagcatcaaa tgaaactgca
      841 atttattcat atcaggatta tcaataccat atttttgaaa aagccgtttc tgtaatgaag
      901 gagaaaactc accgaggcag ttccatagga tggcaagatc ctggtatcgg tctgcgattc
      961 cgactcgtcc aacatcaata caacctatta atttcccctc gtcaaaaata aggttatcaa
     1021 gtgagaaatc accatgagtg acgactgaat ccggtgagaa tggcaaaagt ttatgcattt
     1081 ctttccagac ttgttcaaca ggccagccat tacgctcgtc atcaaaatca ctcgcatcaa
     1141 ccaaaccgtt attcattcgt gattgcgcct gagcgagacg aaatacgcgg tcgctgttaa
     1201 aaggacaatt acaaacagga atcgaatgca accggcgcag gaacactgcc agcgcatcaa
     1261 caatattttc acctgaatca ggatattctt ctaatacctg gaatgctgtt ttcccgggga
     1321 tcgcagtggt gagtaaccat gcatcatcag gagtacggat aaaatgcttg atggtcggaa
     1381 gaggcataaa ttccgtcagc cagtttagtc tgaccatctc atctgtaaca tcattggcaa
     1441 cgctaccttt gccatgtttc agaaacaact ctggcgcatc gggcttccca tacaatcgat
     1501 agattgtcgc acctgattgc ccgacattat cgcgagccca tttataccca tataaatcag
     1561 catccatgtt ggaatttaat cgcggcctag agcaagacgt ttcccgttga atatggctca
     1621 tactcttcct ttttcaatat tattgaagca tttatcaggg ttattgtctc atgagcggat
     1681 acatatttga atgtatttag aaaaataaac aaataggcat ggtctaagaa accattatta
     1741 tcatgacatt aacctataaa aatagaagtt cctattccga agttcctatt ctctagaaag
     1801 tataggaact tcggatccga attcgagctc cgtcgacact agtgcggccg cactcgaggg
     1861 catcagagca gattgtactg gaagaccagg agaacgatcg ttggctgtcc ctatcagtga
     1921 tagagattga catccctatc agtgatagat ataatgagca cagctgtcac cggatgtgct
     1981 ttccggtctg atgagtccgt gaggacgaaa cagcctctac aaataatttt gtttaatact
     2041 agagaaagag gagaaatact agatggtgag caagggcgag gagctgttca ccggggtggt
     2101 gcccatcctg gtcgagctgg acggcgacgt aaacggccac aagttcagcg tgtccggcga
     2161 gggcgagggc gatgccacct acggcaagct gaccctgaag ttcatctgca ccacaggcaa
     2221 gctgcccgtg ccctggccca ccctcgtgac caccttcggc tacggcctgc aatgcttcgc
     2281 ccgctacccc gaccacatga agctgcacga cttcttcaag tccgccatgc ccgaaggcta
     2341 cgtccaggag cgcaccatct tcttcaagga cgacggcaac tacaagaccc gcgccgaggt
     2401 gaagttcgag ggcgacaccc tggtgaaccg catcgagctg aagggcatcg acttcaagga
     2461 ggacggcaac atcctggggc acaagctgga gtacaactac aacagccaca acgtctatat
     2521 catggccgac aagcagaaga acggcatcaa ggtgaacttc aagatccgcc acaacatcga
     2581 ggacggcagc gtgcagctcg ccgaccacta ccagcagaac accccaatcg gcgacggccc
     2641 cgtgctgctg cccgacaacc actaccttag ctaccagtcc gccctgagca aagaccccaa
     2701 cgagaagcgc gatcacatgg tcctgctgga gttcgtgacc gccgccggga tcactctcgg
     2761 catggacgag ctgtacaagt aagctcatgt atgtgtctac gcgagattc
//
