LOCUS       sequence-408079-        3008 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  RPU plasmid w/ high RBS (p15a backbone).
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 3008)
  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 3008)
  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: pAJT475
FEATURES             Location/Qualifiers
     source          1..3008
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     promoter        102..136
                     /label=BBa_J23101
                     /note="Anderson constitutive promoter 
                     (https://parts.igem.org/Promoters/Catalog/Anderson). 
                     Measured strength relative to BBa_J223100: 0.7"
     misc_RNA        143..192
                     /label=sTRSV HHRz
                     /note="hammerhead ribozyme from the tobacco ringspot virus 
                     satellite RNA (Khvorova et al., 2003)"
     RBS             224..235
                     /note="strong bacterial ribosome binding site (Elowitz and 
                     Leibler, 2000)"
     CDS             join(242..244,245..247,248..961)
                     /codon_start=1
                     /product="enhanced YFP"
                     /label=EYFP
                     /note="mammalian codon-optimized"
                     /translation="MVSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTL
                     KFICTTGKLPVPWPTLVTTFGYGLQCFARYPDHMKLHDFFKSAMPEGYVQERTIFFKDD
                     GNYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIK
                     VNFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSYQSALSKDPNEKRDHMVLL
                     EFVTAAGITLGMDELYK"
     primer_bind     complement(287..308)
                     /label=EGFP-N
                     /note="EGFP, reverse primer"
     primer_bind     complement(548..567)
                     /label=EXFP-R
                     /note="For distinguishing EGFP variants, reverse primer"
     primer_bind     895..916
                     /label=EGFP-C
                     /note="EGFP, forward primer"
     primer_bind     complement(1241..1260)
                     /label=pENTR-R
                     /note="pENTR vectors, reverse primer"
     CDS             1252..2067
                     /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             1252..2067
                     /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(1325..1344)
                     /label=Kan-R
                     /note="Kanamycin resistance gene, reverse primer"
     rep_origin      2156..2701
                     /direction=RIGHT
                     /label=p15A ori
                     /note="Plasmids containing the medium-copy-number p15A 
                     origin of replication can be propagated in E. coli cells 
                     that contain a second plasmid with the ColE1 origin.
                     "
     primer_bind     2602..2621
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
ORIGIN
        1 ggatccgaat tcgagctccg tcgacactag tgcggccgca ctcgagggca tcagagcaga
       61 ttgggagcga cgtacggtgg aagcttgata agtccctaac ttttacagct agctcagtcc
      121 taggtattat gctagcctga agctgtcacc ggatgtgctt tccggtctga tgagtccgtg
      181 aggacgaaac agcctctaca aataattttg tttaatacta gagaaagagg agaaatacta
      241 gatggtgagc aagggcgagg agctgttcac cggggtggtg cccatcctgg tcgagctgga
      301 cggcgacgta aacggccaca agttcagcgt gtccggcgag ggcgagggcg atgccaccta
      361 cggcaagctg accctgaagt tcatctgcac cacaggcaag ctgcccgtgc cctggcccac
      421 cctcgtgacc accttcggct acggcctgca atgcttcgcc cgctaccccg accacatgaa
      481 gctgcacgac ttcttcaagt ccgccatgcc cgaaggctac gtccaggagc gcaccatctt
      541 cttcaaggac gacggcaact acaagacccg cgccgaggtg aagttcgagg gcgacaccct
      601 ggtgaaccgc atcgagctga agggcatcga cttcaaggag gacggcaaca tcctggggca
      661 caagctggag tacaactaca acagccacaa cgtctatatc atggccgaca agcagaagaa
      721 cggcatcaag gtgaacttca agatccgcca caacatcgag gacggcagcg tgcagctcgc
      781 cgaccactac cagcagaaca ccccaatcgg cgacggcccc gtgctgctgc ccgacaacca
      841 ctaccttagc taccagtccg ccctgagcaa agaccccaac gagaagcgcg atcacatggt
      901 cctgctggag ttcgtgaccg ccgccgggat cactctcggc atggacgagc tgtacaagta
      961 agcttgtgtt ataaacctgt gtgagagtta agtttacatg cctaacctta acttttacgc
     1021 aggttcagct tggaaacaca gaaaaaagcc cgcacctgac agtgcgggct ttttttttcg
     1081 accaaaggct cggtaccaaa ttccagaaaa gacacccgaa agggtgtttt ttcgttttgg
     1141 tccctcagta cttgtgtctc aaaatctctg atgtacattg cacaagataa aaatatatca
     1201 tcatgaacaa taaaactgtc tgcttacata aacagtaata caaggggtgt tatgagccat
     1261 attcaacggg aaacgtcttg ctccaggccg cgattaaatt ccaacatgga tgctgattta
     1321 tatgggtata aatgggctcg cgataatgtc gggcaatcag gtgcgacaat ctatcgattg
     1381 tatgggaagc ccgatgcgcc agagttgttt ctgaaacatg gcaaaggtag cgttgccaat
     1441 gatgttacag atgagatggt cagactaaac tggctgacgg aatttatgcc tcttccgacc
     1501 atcaagcatt ttatccgtac tcctgatgat gcatggttac tcaccactgc gatccccggg
     1561 aaaacagcat tccaggtatt agaagaatat cctgattcag gtgaaaatat tgttgatgcg
     1621 ctggcagtgt tcctgcgccg gttgcattcg attcctgttt gtaattgtcc ttttaacagc
     1681 gatcgcgtat ttcgtctcgc tcaggcgcaa tcacgaatga ataacggttt ggttgatgcg
     1741 agtgattttg atgacgagcg taatggctgg cctgttgaac aagtctggaa agaaatgcat
     1801 aagcttttgc cattctcacc ggattcagtc gtcactcatg gtgatttctc acttgataac
     1861 cttatttttg acgaggggaa attaataggt tgtattgatg ttggacgagt cggaatcgca
     1921 gaccgatacc aggatcttgc catcctatgg aactgcctcg gtgagttttc tccttcatta
     1981 cagaaacggc tttttcaaaa atatggtatt gataatcctg atatgaataa attgcagttt
     2041 catttgatgc tcgatgagtt tttctaaggt aactgtcaga ccaagtttac tcatatatac
     2101 tttagattga tttttcgttc cactgagcgt cagacccctt aataagatga tcttcttgag
     2161 atcgttttgg tctgcgcgta atctcttgct ctgaaaacga aaaaaccgcc ttgcagggcg
     2221 gtttttcgaa ggttctctga gctaccaact ctttgaaccg aggtaactgg cttggaggag
     2281 cgcagtcacc aaaacttgtc ctttcagttt agccttaacc ggcgcatgac ttcaagacta
     2341 actcctctaa atcaattacc agtggctgct gccagtggtg cttttgcatg tctttccggg
     2401 ttggactcaa gacgatagtt accggataag gcgcagcggt cggactgaac ggggggttcg
     2461 tgcatacagt ccagcttgga gcgaactgcc tacccggaac tgagtgtcag gcgtggaatg
     2521 agacaaacgc ggccataaca gcggaatgac accggtaaac cgaaaggcag gaacaggaga
     2581 gcgcacgagg gagccgccag ggggaaacgc ctggtatctt tatagtcctg tcgggtttcg
     2641 ccaccactga tttgagcgtc agatttcgtg atgcttgtca ggggggcgga gcctatggaa
     2701 aaacggcttt gccgcggccc tctcacttcc ctgttaagta tcttcctggc atcttccagg
     2761 aaatctccgc cccgttcgta agccatttcc gctcgccgca gtcgaacgac cgagcgtagc
     2821 gagtcagtga gcgaggaagc ggaatatatc ctgtatcaca tattctgctg acgcaccggt
     2881 gcagcctttt ttctcctgcc acatgaagca cttcactgac accctcatca gtgccaacat
     2941 agtaagccag tatacactcc gctagcgctg aggtctgcct cgtgcagcga gtcagtgagc
     3001 gaggaagc
//
