LOCUS       sequence-408081-        3055 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Type IIS cloning plasmid for LPattB7 integrations w/ OriT.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 3055)
  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 3055)
  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: pAJT195
FEATURES             Location/Qualifiers
     source          1..3055
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     rep_origin      265..653
                     /label=R6K gamma ori
                     /note="gamma replication origin from E. coli plasmid R6K; 
                     requires the R6K initiator protein pi for replication"
     oriT            697..805
                     /note="incP origin of transfer"
     terminator      complement(956..1049)
                     /label=lambda t0 terminator
                     /note="transcription terminator from phage lambda"
     CDS             complement(1060..1719)
                     /codon_start=1
                     /gene="cat"
                     /product="chloramphenicol acetyltransferase"
                     /label=CmR
                     /note="confers resistance to chloramphenicol"
                     /translation="MEKKITGYTTVDISQWHRKEHFEAFQSVAQCTYNQTVQLDITAFL
                     NTVKKNKHKFYPAFIHILARLMNAHPEFRMAMKDGELVIWDSVHPCYTVFHEQTETFSS
                     LWSEYHDDFRQFLHIYSQDVACYDENLAYFPKGFIENMFFVSANPWVSFTSFDLNVANM
                     DNFFAPVFTMGKYYTQGDKVLMPLAIQVHHAVCDGFHVGRMLNELQQYCDEWQGGA"
     primer_bind     1634..1653
                     /label=CAT-R
                     /note="Chloramphenicol resistance gene, reverse primer"
     promoter        complement(1720..1822)
                     /label=cat promoter
                     /note="promoter of the E. coli cat gene encoding 
                     chloramphenicol acetyltransferase"
     protein_bind    1939..1986
                     /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        2066..2100
                     /label=BBa_J23101
                     /note="Anderson constitutive promoter 
                     (https://parts.igem.org/Promoters/Catalog/Anderson). 
                     Measured strength relative to BBa_J223100: 0.7"
     misc_RNA        2108..2157
                     /label=sTRSV HHRz
                     /note="hammerhead ribozyme from the tobacco ringspot virus 
                     satellite RNA (Khvorova et al., 2003)"
     CDS             join(2200..2202,2203..2916)
                     /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             2200..2913
                     /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(2230..2258)
                     /label=GFP-R
                     /note="GFP, reverse primer. Does NOT anneal to EGFP"
     primer_bind     2853..2873
                     /label=GFP-F
                     /note="GFP, forward primer. Does NOT anneal to EGFP"
     terminator      2933..3004
                     /label=rrnB T1 terminator
                     /note="transcription terminator T1 from the E. coli rrnB 
                     gene"
ORIGIN
        1 cctaacctta acttttacgc aggttcagct tggaaacaca gaaaaaagcc cgcacctgac
       61 agtgcgggct ttttttttcg accaaaggct cggtaccaaa ttccagaaaa gacacccgaa
      121 agggtgtttt ttcgttttgg tccaggcggt aatacggtta tccacagaat ccggggataa
      181 cgcaggaaag aacatgttag caaaaggcca gcaaaaggcc aggaaccgta aaaatgattc
      241 gcacgggccc atggctaatt cccatgtcag ccgttaagtg ttcctgtgtc actcaaaatt
      301 gctttgagag gctctaaggg cttctcagtg cgttacatcc ctggcttgtt gtccacaacc
      361 gttaaacctt aaaagcttta aaagccttat atattctttt ttttcttata aaacttaaaa
      421 ccttagaggc tatttaagtt gctgatttat attaatttta ttgttcaaac atgagagctt
      481 agtacgtgaa acatgagagc ttagtacgtt agccatgaga gcttagtacg ttagccatga
      541 gggtttagtt cgttaaacat gagagcttag tacgttaaac atgagagctt agtacgtgaa
      601 acatgagagc ttagtacgta ctatcaacag gttgaactgc tgatcttcag atcctctacg
      661 ccggacgcat cgtggccgga tcttcttgta gcttatcggc cagcctcgca gagcaggatt
      721 cccgttgagc accgccaggt gcgaataagg gacagtgaag aaggaacacc cgctcgcggg
      781 tgggcctact tcacctatcc tgcccggctg acgccgttgg atacaccaag gaaagtctac
      841 acgaaccctt tggcaaaatc ctgtatatcg tgcgaaaaag gatggatata ccgaaaaaat
      901 cgctataatg accccgaagc agggttatgc agcggaaagt ataccttaag cttggattct
      961 caccataaaa aacgcccggc ggcaaccgag cgttctgaac aaatccagat ggagttctga
     1021 ggtcattact ggatctatca acaggagtcc aagcgagctt tacgccccgc cctgccactc
     1081 atcgcagtac tgttgtaatt cattaagcat tctgccgaca tggaagccat cacaaacggc
     1141 atgatgaacc tgaatcgcca gcggcatcag caccttgtcg ccttgcgtat aatatttgcc
     1201 catggtgaaa acgggggcga agaagttgtc catattggcc acgtttaaat caaaactggt
     1261 gaaactcacc cagggattgg ctgagacgaa aaacatattc tcaataaacc ctttagggaa
     1321 ataggccagg ttttcatcgt aacacgccac atcttgcgaa tatatgtgta gaaactgccg
     1381 gaaatcgtcg tggtattcac tccagagcga tgaaaacgtt tcagtttgct catggaaaac
     1441 ggtgtaacaa gggtgaacac tatcccatat caccagctca ccgtctttca ttgccatacg
     1501 aaattccgga tgagcattca tcaggcgggc aagaatgtga ataaaggccg gataaaactt
     1561 gtgcttattt ttctttacgg tatttaaaaa ggccgtaata tccagctgaa cggtctggtt
     1621 ataggtacat tgagcaactg actgaaatgc ctcaaaatgt tctttacgat gccattggga
     1681 tatatcaacg gtggtatatc cagtgatttt tttctccatt ttagcttcct tagctcctga
     1741 aaatctcgat aactcaaaaa atacgcccgg tagtgatctt atttcattat ggtgaaagtt
     1801 ggaacctctt acgtgccgat caacgtctca ttttcgccaa tttaaatcgt aattattggg
     1861 gaccctgtat ttagaaaaat aaacaaatag gcatggtcta agaaaccatt attatcatga
     1921 cattaaccta taaaaataga agttcctatt ccgaagttcc tattctctag aaagtatagg
     1981 aacttcggat ccgaattcga gctccgtcga cactagtgcg gccgcactcg agggcatcag
     2041 agcagattgg gaggtgtctt catcatttac agctagctca gtcctaggta ttatgctagc
     2101 cagttagctg tcaccggatg tgctttccgg tctgatgagt ccgtgaggac gaaacagcct
     2161 ctacaaataa ttttgtttaa tcacacagga aagtactaga tgagcaaagg agaagaactt
     2221 ttcactggag ttgtcccaat tcttgttgaa ttagatggtg atgttaatgg gcacaaattt
     2281 tctgtccgtg gagagggtga aggtgatgct acaaacggaa aactcaccct taaatttatt
     2341 tgcactactg gaaaactacc tgttccgtgg ccaacacttg tcactactct gacctatggt
     2401 gttcaatgct tttcccgtta tccggatcac atgaaacggc atgacttttt caagagtgcc
     2461 atgcccgaag gttatgtaca ggaacgcact atatctttca aagatgacgg gacctacaag
     2521 acgcgtgctg aagtcaagtt tgaaggtgat acccttgtta atcgtatcga gttaaagggt
     2581 attgatttta aagaagatgg aaacattctt ggacacaaac tcgagtacaa ctttaactca
     2641 cacaatgtat acatcacggc agacaaacaa aagaatggaa tcaaagctaa cttcaaaatt
     2701 cgccacaacg ttgaagatgg ttccgttcaa ctagcagacc attatcaaca aaatactcca
     2761 attggcgatg gccctgtcct tttaccagac aaccattacc tgtcgacaca atctgtcctt
     2821 tcgaaagatc ccaacgaaaa gcgtgaccac atggtccttc ttgagtttgt aactgctgct
     2881 gggattacac atggcatgga tgagctctac aaataatact agagccaggc atcaaataaa
     2941 acgaaaggct cagtcgaaag actgggcctt tcgttttatc tgttgtttgt cggtgaacgc
     3001 tctcatgtga agacatgctt gtgttataaa cctgtgtgag agttaagttt acatg
//
