LOCUS       sequence-408077-        3072 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  RPU integration plasmid (LPattB2).
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 3072)
  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 3072)
  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: pAJT248
FEATURES             Location/Qualifiers
     source          1..3072
                     /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"
     oriT            702..810
                     /note="incP origin of transfer"
     terminator      complement(970..1004)
                     /label=lambda t0 terminator
                     /note="minimal transcription terminator from phage lambda 
                     (Scholtissek and Grosse, 1987)"
     CDS             complement(1065..1880)
                     /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(1065..1880)
                     /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     1788..1807
                     /label=Kan-R
                     /note="Kanamycin resistance gene, reverse primer"
     promoter        complement(1881..1972)
                     /gene="bla"
                     /label=AmpR promoter
     protein_bind    2024..2071
                     /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        2186..2220
                     /label=BBa_J23101
                     /note="Anderson constitutive promoter 
                     (https://parts.igem.org/Promoters/Catalog/Anderson). 
                     Measured strength relative to BBa_J223100: 0.7"
     misc_RNA        2227..2276
                     /label=sTRSV HHRz
                     /note="hammerhead ribozyme from the tobacco ringspot virus 
                     satellite RNA (Khvorova et al., 2003)"
     RBS             2308..2319
                     /note="strong bacterial ribosome binding site (Elowitz and 
                     Leibler, 2000)"
     CDS             join(2326..2328,2329..2331,2332..3045)
                     /codon_start=1
                     /product="enhanced YFP"
                     /label=EYFP
                     /note="mammalian codon-optimized"
                     /translation="MVSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTL
                     KFICTTGKLPVPWPTLVTTFGYGLQCFARYPDHMKLHDFFKSAMPEGYVQERTIFFKDD
                     GNYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIK
                     VNFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSYQSALSKDPNEKRDHMVLL
                     EFVTAAGITLGMDELYK"
     primer_bind     complement(2371..2392)
                     /label=EGFP-N
                     /note="EGFP, reverse primer"
     primer_bind     complement(2632..2651)
                     /label=EXFP-R
                     /note="For distinguishing EGFP variants, reverse primer"
     primer_bind     2979..3000
                     /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 ttgtagctta tcggccagcc tcgcagagca
      721 ggattcccgt tgagcaccgc caggtgcgaa taagggacag tgaagaagga acacccgctc
      781 gcgggtgggc ctacttcacc tatcctgccc ggctgacgcc gttggataca ccaaggaaag
      841 tctacacgaa ccctttggca aaatcctgta tatcgtgcga aaaaggatgg atataccgaa
      901 aaaatcgcta taatgacccc gaagcagggt tatgcagcgg aaagtatacc ttaagcttgg
      961 attctcacaa taaaaaacgc ccggcggcaa ccgagcgttc tgaacaaatc cagatggagt
     1021 tctgaggtca ttactggatc tatcaacagg agtccaagcg agctttagaa aaactcatcg
     1081 agcatcaaat gaaactgcaa tttattcata tcaggattat caataccata tttttgaaaa
     1141 agccgtttct gtaatgaagg agaaaactca ccgaggcagt tccataggat ggcaagatcc
     1201 tggtatcggt ctgcgattcc gactcgtcca acatcaatac aacctattaa tttcccctcg
     1261 tcaaaaataa ggttatcaag tgagaaatca ccatgagtga cgactgaatc cggtgagaat
     1321 ggcaaaagtt tatgcatttc tttccagact tgttcaacag gccagccatt acgctcgtca
     1381 tcaaaatcac tcgcatcaac caaaccgtta ttcattcgtg attgcgcctg agcgagacga
     1441 aatacgcggt cgctgttaaa aggacaatta caaacaggaa tcgaatgcaa ccggcgcagg
     1501 aacactgcca gcgcatcaac aatattttca cctgaatcag gatattcttc taatacctgg
     1561 aatgctgttt tcccggggat cgcagtggtg agtaaccatg catcatcagg agtacggata
     1621 aaatgcttga tggtcggaag aggcataaat tccgtcagcc agtttagtct gaccatctca
     1681 tctgtaacat cattggcaac gctacctttg ccatgtttca gaaacaactc tggcgcatcg
     1741 ggcttcccat acaatcgata gattgtcgca cctgattgcc cgacattatc gcgagcccat
     1801 ttatacccat ataaatcagc atccatgttg gaatttaatc gcggcctaga gcaagacgtt
     1861 tcccgttgaa tatggctcat actcttcctt tttcaatatt attgaagcat ttatcagggt
     1921 tattgtctca tgagcggata catatttgaa tgtatttaga aaaataaaca aataggcatg
     1981 gtctaagaaa ccattattat catgacatta acctataaaa atagaagttc ctattccgaa
     2041 gttcctattc tctagaaagt ataggaactt cggatccgaa ttcgagctcc gtcgacacta
     2101 gtgcggccgc actcgagggc atcagagcag attgtactgg aagaccatac tcgacgtacg
     2161 gtggaagctt gataagtccc taacttttac agctagctca gtcctaggta ttatgctagc
     2221 ctgaagctgt caccggatgt gctttccggt ctgatgagtc cgtgaggacg aaacagcctc
     2281 tacaaataat tttgtttaat actagagaaa gaggagaaat actagatggt gagcaagggc
     2341 gaggagctgt tcaccggggt ggtgcccatc ctggtcgagc tggacggcga cgtaaacggc
     2401 cacaagttca gcgtgtccgg cgagggcgag ggcgatgcca cctacggcaa gctgaccctg
     2461 aagttcatct gcaccacagg caagctgccc gtgccctggc ccaccctcgt gaccaccttc
     2521 ggctacggcc tgcaatgctt cgcccgctac cccgaccaca tgaagctgca cgacttcttc
     2581 aagtccgcca tgcccgaagg ctacgtccag gagcgcacca tcttcttcaa ggacgacggc
     2641 aactacaaga cccgcgccga ggtgaagttc gagggcgaca ccctggtgaa ccgcatcgag
     2701 ctgaagggca tcgacttcaa ggaggacggc aacatcctgg ggcacaagct ggagtacaac
     2761 tacaacagcc acaacgtcta tatcatggcc gacaagcaga agaacggcat caaggtgaac
     2821 ttcaagatcc gccacaacat cgaggacggc agcgtgcagc tcgccgacca ctaccagcag
     2881 aacaccccaa tcggcgacgg ccccgtgctg ctgcccgaca accactacct tagctaccag
     2941 tccgccctga gcaaagaccc caacgagaag cgcgatcaca tggtcctgct ggagttcgtg
     3001 accgccgccg ggatcactct cggcatggac gagctgtaca agtaagctca tgtatgtgtc
     3061 tacgcgagat tc
//
