LOCUS       sequence-408095-        2995 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  ameRS repressor plasmid for LPattB2 integration.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 2995)
  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 2995)
  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: pAJT255-ameRS
FEATURES             Location/Qualifiers
     source          1..2995
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     protein_bind    893..912
                     /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(918..924,925..940,941..946))
                     /label=tac promoter
                     /note="strong E. coli promoter; hybrid between the trp and 
                     lac UV5 promoters"
     protein_bind    complement(1036..1083)
                     /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        1135..1226
                     /gene="bla"
                     /label=AmpR promoter
     CDS             1227..2042
                     /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             1227..2042
                     /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(1300..1319)
                     /label=Kan-R
                     /note="Kanamycin resistance gene, reverse primer"
     terminator      2053..2147
                     /label=lambda t0 terminator
                     /note="transcription terminator from phage lambda"
     oriT            2298..2406
                     /note="incP origin of transfer"
     rep_origin      2450..2838
                     /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 catgagcagc gcataaaaaa atggcgccga tgggcgccat
       61 ttttcactgt gtcatacgta aaaaagggcg atcttgcgac cgcccttttt ttattaaatg
      121 tttattttgc cagcggatta cgcagacctg catcaatcag atcaaccagc tgatcacaac
      181 gggttgccag attaccaata tcttcttgtg caatcagaac cggatgaata acgctggtca
      241 gtgcatgcag aacggtttct gccagaaccg gaatatcggt tgcaatatac agacctgctt
      301 caacaccatc acgaataatt tcggccagca gttttgcaat cacgcttttg taataatcgc
      361 cacatttcat atcctgtttg gcggtcatca ggatctgaat aaaaacccgt atgtgtttga
      421 aatgatcctg atgatgctgt tccatcagat tacgtgccag atgacgcaga cgatccagcg
      481 gtgcatggct tttatccagc ggacaaatct gacgttcaaa aacaccaatc tgaccaaaac
      541 caattgcatc aaccagtgcg tttttgctgc taaaatgttt aaacacattt gccggactca
      601 tattcagtgc gcttgcaata tctgcaatgg caactgcatt aaaaccacgt tcactaaaca
      661 gttcttcggc ttttgccaga atatcacgac gggtttcttc ggcactatga cgcggacgac
      721 gacgaaccgg cagatcgctt ttacgatcac ctttacgcac ctgatcaatg gttttgttca
      781 tctattagct cctgtttgaa acatagtcca tagcgtatta aacaaaatta tttgtagagg
      841 gggtttcgtc atcactgact catcagagtt gaaagcacat caactgaccc ctaattgtga
      901 gcgctcacaa ttccacacat tatacgagcc gatgattaat tgtcaacaca gccaacgatc
      961 gttctcccag tacaatctgc tctgatgccc tcgagtgcgg ccgcactagt gtcgacggag
     1021 ctcgaattcg gatccgaagt tcctatactt tctagagaat aggaacttcg gaataggaac
     1081 ttctattttt ataggttaat gtcatgataa taatggtttc ttagaccatg cctatttgtt
     1141 tatttttcta aatacattca aatatgtatc cgctcatgag acaataaccc tgataaatgc
     1201 ttcaataata ttgaaaaagg aagagtatga gccatattca acgggaaacg tcttgctcta
     1261 ggccgcgatt aaattccaac atggatgctg atttatatgg gtataaatgg gctcgcgata
     1321 atgtcgggca atcaggtgcg acaatctatc gattgtatgg gaagcccgat gcgccagagt
     1381 tgtttctgaa acatggcaaa ggtagcgttg ccaatgatgt tacagatgag atggtcagac
     1441 taaactggct gacggaattt atgcctcttc cgaccatcaa gcattttatc cgtactcctg
     1501 atgatgcatg gttactcacc actgcgatcc ccgggaaaac agcattccag gtattagaag
     1561 aatatcctga ttcaggtgaa aatattgttg atgcgctggc agtgttcctg cgccggttgc
     1621 attcgattcc tgtttgtaat tgtcctttta acagcgaccg cgtatttcgt ctcgctcagg
     1681 cgcaatcacg aatgaataac ggtttggttg atgcgagtga ttttgatgac gagcgtaatg
     1741 gctggcctgt tgaacaagtc tggaaagaaa tgcataaact tttgccattc tcaccggatt
     1801 cagtcgtcac tcatggtgat ttctcacttg ataaccttat ttttgacgag gggaaattaa
     1861 taggttgtat tgatgttgga cgagtcggaa tcgcagaccg ataccaggat cttgccatcc
     1921 tatggaactg cctcggtgag ttttctcctt cattacagaa acggcttttt caaaaatatg
     1981 gtattgataa tcctgatatg aataaattgc agtttcattt gatgctcgat gagtttttct
     2041 aaagctcgct tggactcctg ttgatagatc cagtaatgac ctcagaactc catctggatt
     2101 tgttcagaac gctcggttgc cgccgggcgt tttttattgg tgagaatcca agcttaaggt
     2161 atactttccg ctgcataacc ctgcttcggg gtcattatag cgattttttc ggtatatcca
     2221 tcctttttcg cacgatatac aggattttgc caaagggttc gtgtagactt tccttggtgt
     2281 atccaacggc gtcagccggg caggataggt gaagtaggcc cacccgcgag cgggtgttcc
     2341 ttcttcactg tcccttattc gcacctggcg gtgctcaacg ggaatcctgc tctgcgaggc
     2401 tggccgataa gctacaagaa gatccggcca cgatgcgtcc ggcgtagagg atctgaagat
     2461 cagcagttca acctgttgat agtacgtact aagctctcat gtttcacgta ctaagctctc
     2521 atgtttaacg tactaagctc tcatgtttaa cgaactaaac cctcatggct aacgtactaa
     2581 gctctcatgg ctaacgtact aagctctcat gtttcacgta ctaagctctc atgtttgaac
     2641 aataaaatta atataaatca gcaacttaaa tagcctctaa ggttttaagt tttataagaa
     2701 aaaaaagaat atataaggct tttaaagctt ttaaggttta acggttgtgg acaacaagcc
     2761 agggatgtaa cgcactgaga agcccttaga gcctctcaaa gcaattttga gtgacacagg
     2821 aacacttaac ggctgacatg ggaattagcc atgggcccgt gcgaatcatt tttacggttc
     2881 ctggcctttt gctggccttt tgctcacatg ttctttcctg cgttatcccc tgattctgtg
     2941 gataaccgta ttaccgccta gccaagagca gtgccttgca gaagttctcg ggcga
//
