LOCUS       sequence-408094-        2964 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  litR repressor plasmid for LPattB2 integration.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 2964)
  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 2964)
  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: pAJT254-litR
FEATURES             Location/Qualifiers
     source          1..2964
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     protein_bind    862..881
                     /label=lac operator (symmetric)
                     /bound_moiety="lac repressor encoded by lacI"
                     /note="The lac repressor binds to the lac operator to 
                     inhibit transcription in E. coli. This inhibition can be 
                     relieved by adding lactose or 
                     isopropyl-beta-D-thiogalactopyranoside (IPTG). The 
                     symmetric lac operator was optimized for tight binding of 
                     lac repressor."
     promoter        complement(join(887..893,894..909,910..915))
                     /label=tac promoter
                     /note="strong E. coli promoter; hybrid between the trp and 
                     lac UV5 promoters"
     protein_bind    complement(1005..1052)
                     /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)."
     promoter        1104..1195
                     /gene="bla"
                     /label=AmpR promoter
     CDS             1196..2011
                     /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             1196..2011
                     /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     complement(1269..1288)
                     /label=Kan-R
                     /note="Kanamycin resistance gene, reverse primer"
     terminator      2022..2116
                     /label=lambda t0 terminator
                     /note="transcription terminator from phage lambda"
     oriT            2267..2375
                     /note="incP origin of transfer"
     rep_origin      2419..2807
                     /label=R6K gamma ori
                     /note="gamma replication origin from E. coli plasmid R6K; 
                     requires the R6K initiator protein pi for replication"
ORIGIN
        1 gaatctcgcg tagacacata catgagcagt aggaccaaaa cgaaaaaaga cgctcgaaag
       61 cgtctctttt ctggaatttg gtaccgagca atccatgtaa aaaaagggcc ctgaaattca
      121 ggaccctttc tggcatcagc ttatttgtag atgcacagca tattcagaaa gcaatttgcg
      181 gtttcttcca tttcttcgct gctgctatta cgattggcct gaataaacac gctatagcaa
      241 ataccatgca gcattttggt cagattttcc ggtgtatgat cattgcacac ttcattgcgt
      301 tcaataccct cttcaaacat ggttttgatc acctgattgg tattgctatg ggtgctcaga
      361 aacagaggcc aaacttcatc acgggtgctg gttgaccatt caaaccaaac tttaatccat
      421 ttgttgccgg tctgaacgct atcaataatg ttcagcagca gggtattcag attgctacga
      481 acatccagat ccaggctaat gctgttattg atgaactggt gaaactcgtt ttccactttg
      541 ttcagaacat catcaaccag atcttcacgg gtcggaaaat agttaaacac ggttgcaacg
      601 ctaacctgtg caatttctgc aatatctgca tgaccaccac gaccaatacc acgctggcta
      661 aaaacttcaa tggcaatatc cagcagctgt tctttacgtt tttccggact cagacgggta
      721 cgcggacgtt tctgaatggt atccatcgag ggttctcctg tatgaaaaag tccataggac
      781 ttaaacaaaa ttatttgtag aggtcataag tttcgtcgca tttcagcgac tcatcagtgg
      841 gcttagccca gacttatgac taattgtgag cgctcacaat tccacacatt atacgagccg
      901 atgattaatt gtcaacacag ccaacgatcg ttctcccagt acaatctgct ctgatgccct
      961 cgagtgcggc cgcactagtg tcgacggagc tcgaattcgg atccgaagtt cctatacttt
     1021 ctagagaata ggaacttcgg aataggaact tctattttta taggttaatg tcatgataat
     1081 aatggtttct tagaccatgc ctatttgttt atttttctaa atacattcaa atatgtatcc
     1141 gctcatgaga caataaccct gataaatgct tcaataatat tgaaaaagga agagtatgag
     1201 ccatattcaa cgggaaacgt cttgctctag gccgcgatta aattccaaca tggatgctga
     1261 tttatatggg tataaatggg ctcgcgataa tgtcgggcaa tcaggtgcga caatctatcg
     1321 attgtatggg aagcccgatg cgccagagtt gtttctgaaa catggcaaag gtagcgttgc
     1381 caatgatgtt acagatgaga tggtcagact aaactggctg acggaattta tgcctcttcc
     1441 gaccatcaag cattttatcc gtactcctga tgatgcatgg ttactcacca ctgcgatccc
     1501 cgggaaaaca gcattccagg tattagaaga atatcctgat tcaggtgaaa atattgttga
     1561 tgcgctggca gtgttcctgc gccggttgca ttcgattcct gtttgtaatt gtccttttaa
     1621 cagcgaccgc gtatttcgtc tcgctcaggc gcaatcacga atgaataacg gtttggttga
     1681 tgcgagtgat tttgatgacg agcgtaatgg ctggcctgtt gaacaagtct ggaaagaaat
     1741 gcataaactt ttgccattct caccggattc agtcgtcact catggtgatt tctcacttga
     1801 taaccttatt tttgacgagg ggaaattaat aggttgtatt gatgttggac gagtcggaat
     1861 cgcagaccga taccaggatc ttgccatcct atggaactgc ctcggtgagt tttctccttc
     1921 attacagaaa cggctttttc aaaaatatgg tattgataat cctgatatga ataaattgca
     1981 gtttcatttg atgctcgatg agtttttcta aagctcgctt ggactcctgt tgatagatcc
     2041 agtaatgacc tcagaactcc atctggattt gttcagaacg ctcggttgcc gccgggcgtt
     2101 ttttattggt gagaatccaa gcttaaggta tactttccgc tgcataaccc tgcttcgggg
     2161 tcattatagc gattttttcg gtatatccat cctttttcgc acgatataca ggattttgcc
     2221 aaagggttcg tgtagacttt ccttggtgta tccaacggcg tcagccgggc aggataggtg
     2281 aagtaggccc acccgcgagc gggtgttcct tcttcactgt cccttattcg cacctggcgg
     2341 tgctcaacgg gaatcctgct ctgcgaggct ggccgataag ctacaagaag atccggccac
     2401 gatgcgtccg gcgtagagga tctgaagatc agcagttcaa cctgttgata gtacgtacta
     2461 agctctcatg tttcacgtac taagctctca tgtttaacgt actaagctct catgtttaac
     2521 gaactaaacc ctcatggcta acgtactaag ctctcatggc taacgtacta agctctcatg
     2581 tttcacgtac taagctctca tgtttgaaca ataaaattaa tataaatcag caacttaaat
     2641 agcctctaag gttttaagtt ttataagaaa aaaaagaata tataaggctt ttaaagcttt
     2701 taaggtttaa cggttgtgga caacaagcca gggatgtaac gcactgagaa gcccttagag
     2761 cctctcaaag caattttgag tgacacagga acacttaacg gctgacatgg gaattagcca
     2821 tgggcccgtg cgaatcattt ttacggttcc tggccttttg ctggcctttt gctcacatgt
     2881 tctttcctgc gttatcccct gattctgtgg ataaccgtat taccgcctag ccaagagcag
     2941 tgccttgcag aagttctcgg gcga
//
