LOCUS       sequence-408080-        3599 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Type IIS cloning plasmid for LPattB5 integrations w/ OriT.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 3599)
  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 3599)
  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: pAJT194
FEATURES             Location/Qualifiers
     source          1..3599
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     rep_origin      259..647
                     /label=R6K gamma ori
                     /note="gamma replication origin from E. coli plasmid R6K; 
                     requires the R6K initiator protein pi for replication"
     oriT            691..799
                     /note="incP origin of transfer"
     terminator      complement(960..994)
                     /label=lambda t0 terminator
                     /note="minimal transcription terminator from phage lambda 
                     (Scholtissek and Grosse, 1987)"
     CDS             1219..2409
                     /codon_start=1
                     /gene="tet"
                     /product="tetracycline efflux protein"
                     /label=TcR
                     /note="confers resistance to tetracycline"
                     /translation="MKSNNALIVILGTVTLDAVGIGLVMPVLPGLLRDIVHSDSIASHY
                     GVLLALYALMQFLCAPVLGALSDRFGRRPVLLASLLGATIDYAIMATTPVLWILYAGRI
                     VAGITGATGAVAGAYIADITDGEDRARHFGLMSACFGVGMVAGPVAGGLLGAISLHAPF
                     LAAAVLNGLNLLLGCFLMQESHKGERRPMPLRAFNPVSSFRWARGMTIVAALMTVFFIM
                     QLVGQVPAALWVIFGEDRFRWSATMSGLSLAVFGILHALAQAFVTGPATKRFGEKQAII
                     AGMAADALGYVLLAFATRGWMAFPIMILLASGGIGMPALQAMLSRQVDDDHQGQLQGSL
                     AALTSLTSITGPLIVTAIYAASASTWNGLAWIVGAALYLVCLPALRRGAWSRATST"
     primer_bind     complement(1571..1590)
                     /label=pBRrevBam
                     /note="pBR322 vectors, tet region, downstream of BamHI, 
                     reverse primer"
     protein_bind    2502..2549
                     /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        2611..2645
                     /label=BBa_J23101
                     /note="Anderson constitutive promoter 
                     (https://parts.igem.org/Promoters/Catalog/Anderson). 
                     Measured strength relative to BBa_J223100: 0.7"
     misc_RNA        2653..2702
                     /label=sTRSV HHRz
                     /note="hammerhead ribozyme from the tobacco ringspot virus 
                     satellite RNA (Khvorova et al., 2003)"
     CDS             join(2745..2747,2748..3461)
                     /codon_start=1
                     /product="GFP variant that folds robustly even when fused 
                     to poorly folded proteins (Pedelacq et al., 2006)"
                     /label=superfolder GFP
                     /note="mammalian codon-optimized"
                     /translation="MSKGEELFTGVVPILVELDGDVNGHKFSVRGEGEGDATNGKLTLK
                     FICTTGKLPVPWPTLVTTLTYGVQCFSRYPDHMKRHDFFKSAMPEGYVQERTISFKDDG
                     TYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNFNSHNVYITADKQKNGIKA
                     NFKIRHNVEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSVLSKDPNEKRDHMVLLE
                     FVTAAGITHGMDELYK"
     CDS             2745..3458
                     /codon_start=1
                     /product="GFP variant that folds robustly even when fused 
                     to poorly folded proteins (Nager et al., 2011)"
                     /label=superfolder GFP
                     /translation="MSKGEELFTGVVPILVELDGDVNGHKFSVRGEGEGDATNGKLTLK
                     FICTTGKLPVPWPTLVTTLTYGVQCFSRYPDHMKRHDFFKSAMPEGYVQERTISFKDDG
                     TYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNFNSHNVYITADKQKNGIKA
                     NFKIRHNVEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSVLSKDPNEKRDHMVLLE
                     FVTAAGITHGMDELYK"
     primer_bind     complement(2775..2803)
                     /label=GFP-R
                     /note="GFP, reverse primer. Does NOT anneal to EGFP"
     primer_bind     3398..3418
                     /label=GFP-F
                     /note="GFP, forward primer. Does NOT anneal to EGFP"
     terminator      3478..3549
                     /label=rrnB T1 terminator
                     /note="transcription terminator T1 from the E. coli rrnB 
                     gene"
ORIGIN
        1 ttgacaactt gcgcaccctg atctgggaaa cacagaaaaa agcccgcacc tgacagtgcg
       61 ggcttttttt ttcgaccaaa ggctcggtac caaattccag aaaagacacc cgaaagggtg
      121 ttttttcgtt ttggtccagg cggtaatacg gttatccaca gaatcagggg ataacgcagg
      181 aaagaacatg tgagcaaaag gccagcaaaa ggccaggaac cgtaaaaatg attcgcacgg
      241 gcccatggct aattcccatg tcagccgtta agtgttcctg tgtcactcaa aattgctttg
      301 agaggctcta agggcttctc agtgcgttac atccctggct tgttgtccac aaccgttaaa
      361 ccttaaaagc tttaaaagcc ttatatattc ttttttttct tataaaactt aaaaccttag
      421 aggctattta agttgctgat ttatattaat tttattgttc aaacatgaga gcttagtacg
      481 tgtaacatga gagcttagta cgttagccat gagagcttag tacgttagcc atgagggttt
      541 agttcgttaa acatgagagc ttagtacgtt aaacatgaga gcttagtacg tgaaacatga
      601 gagcttagta cgtactatca acaggttgaa ctgctgatct tcagatcctc tacgccggac
      661 gcatcgtggc cggatcttct tgtagcttat cggccagcct cgcagagcag gattcccgtt
      721 gagcaccgcc aggtgcgaat aagggacagt gaagaaggaa cacccgctcg cgggtgggcc
      781 tacttcacct atcctgcccg gctgacgccg ttggatacac caaggaaagt ctacacgaac
      841 cctttggcaa aatcctgtat atcgtgcgaa aaaggatgga tataccgaaa aaatcgctat
      901 aatgaccccg aagcagggtt atgcagcgga aagtatacct taagcttgga ttctcaccaa
      961 taaaaaacgc ccggcggcaa ccgagcgttc tgaacaaatc caacatttaa taaaaaaagg
     1021 gcggtcgcaa gatcgccctt ttttacgtat gacacctcgt ctcaaccaaa gcaatcaacc
     1081 agtgaaaaat ggcgcccatc ggcgccattt ttttatggtt ccttatcatc tggcgaatcg
     1141 gattgacagc ttatcatcga taaactgtaa tgcggtagtt tatcacagtt aaattgctaa
     1201 cgcagtcagg caccgtgtat gaaatctaac aatgcgctca tcgtcatcct cggcaccgtc
     1261 accctggatg ctgtaggcat aggcttggtt atgccggtac tgccgggcct cttgcgggat
     1321 atcgtccatt ccgacagcat cgccagtcac tatggcgtgc tgctagcgct atatgcgttg
     1381 atgcaatttc tatgcgcacc cgttctcgga gcactgtccg accgctttgg ccgccgccca
     1441 gtcctgctcg cttcgctact tggagccact atcgactacg cgatcatggc gaccacaccc
     1501 gtcctgtgga ttctctacgc cggacgcatc gtggccggca tcaccggcgc cacaggtgcg
     1561 gttgctggcg cctatatcgc cgacatcacc gatggggaag atcgggctcg ccacttcggg
     1621 ctcatgagcg cttgtttcgg cgtgggtatg gtggcaggcc ccgtggccgg gggactgttg
     1681 ggcgccatct ccctgcacgc accattcctt gcggcggcgg tgctcaacgg cctcaaccta
     1741 ctactgggct gcttcctaat gcaggagtcg cataagggag agcgccgtcc gatgcccttg
     1801 agagccttca acccagtcag ctccttccgg tgggcgcggg gcatgaccat tgtggccgca
     1861 cttatgactg tcttctttat catgcaactc gtaggacagg tgccggcagc gctctgggtc
     1921 attttcggcg aggaccgctt tcgctggagc gcgacgatga gcggcctgtc gcttgcggta
     1981 ttcggaatct tgcacgccct cgctcaagcc ttcgtcactg gtcccgccac caaacgtttc
     2041 ggcgagaagc aggccattat cgccggcatg gcggccgacg cgctgggcta cgtcttgctg
     2101 gcgttcgcga cgcgaggctg gatggccttc cccattatga ttcttctcgc ttccggcggc
     2161 atcgggatgc ccgcgttgca ggccatgctg tccaggcagg tagatgacga ccatcaggga
     2221 cagcttcaag gatcgctcgc ggctcttacc agcctaactt cgatcactgg accgctgatc
     2281 gtcacggcga tttatgccgc ctcggcgagc acatggaacg ggttggcatg gattgtaggc
     2341 gccgccctat accttgtctg cctccccgcg ttgcgtcgcg gtgcatggag ccgggccacc
     2401 tcgacctgag acaattgttt aattaattgc ggaccctaga ggtccccttt tttattttaa
     2461 aaattttttc acaaaacggt ttacaagcat aaaatctctg agaagttcct attccgaagt
     2521 tcctattctc tagaaagtat aggaacttcg gatccgaatt cgagctccgt cgacactagt
     2581 gcggccgcac tcgaggtccc gagaccatca tttacagcta gctcagtcct aggtattatg
     2641 ctagccagtt agctgtcacc ggatgtgctt tccggtctga tgagtccgtg aggacgaaac
     2701 agcctctaca aataattttg tttaatcaca caggaaagta ctagatgagc aaaggagaag
     2761 aacttttcac tggagttgtc ccaattcttg ttgaattaga tggtgatgtt aatgggcaca
     2821 aattttctgt ccgtggagag ggtgaaggtg atgctacaaa cggaaaactc acccttaaat
     2881 ttatttgcac tactggaaaa ctacctgttc cgtggccaac acttgtcact actctgacct
     2941 atggtgttca atgcttttcc cgttatccgg atcacatgaa acggcatgac tttttcaaga
     3001 gtgccatgcc cgaaggttat gtacaggaac gcactatatc tttcaaagat gacgggacct
     3061 acaagacgcg tgctgaagtc aagtttgaag gtgataccct tgttaatcgt atcgagttaa
     3121 agggtattga ttttaaagaa gatggaaaca ttcttggaca caaactcgag tacaacttta
     3181 actcacacaa tgtatacatc acggcagaca aacaaaagaa tggaatcaaa gctaacttca
     3241 aaattcgcca caacgttgaa gatggttccg ttcaactagc agaccattat caacaaaata
     3301 ctccaattgg cgatggccct gtccttttac cagacaacca ttacctgtcg acacaatctg
     3361 tcctttcgaa agatcccaac gaaaagcgtg accacatggt ccttcttgag tttgtaactg
     3421 ctgctgggat tacacatggc atggatgagc tctacaaata atactagagc caggcatcaa
     3481 ataaaacgaa aggctcagtc gaaagactgg gcctttcgtt ttatctgttg tttgtcggtg
     3541 aacgctctca tgtggtctca cgctccctaa tacgcaagtc gataactctc ctgggagcg
//
