LOCUS       sequence-147691-        3387 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Supplemental Figure 3. Integration Site 4..
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 3387)
  AUTHORS   Perez-Pinera P, Han N, Cleto S, Cao J, Purcell O, Shah KA, Lee K, 
            Ram R, Lu TK
  TITLE     Synthetic biology and microbioreactor platforms for programmable 
            production of biologics at the point-of-care.
  JOURNAL   Nat Commun. 2016 Jul 29;7:12211. doi: 10.1038/ncomms12211.
  PUBMED    27470089
REFERENCE   2  (bases 1 to 3387)
  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"; journalName: "Nat 
            Commun."; date: "2016-07-29"; volume: "7:12211. doi"; pages: " 
            10.1038/ncomms12211"
COMMENT     Sequence Label: PP77
FEATURES             Location/Qualifiers
     source          1..3387
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     terminator      414..661
                     /gene="S. cerevisiae CYC1"
                     /label=CYC1 terminator
                     /note="transcription terminator for CYC1"
     primer_bind     complement(430..448)
                     /label=CYC1
                     /note="CYC1 transcription termination signal, reverse 
                     primer"
     rep_origin      complement(736..1324)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(816..835)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     protein_bind    complement(1415..1448)
                     /label=FRT (minimal)
                     /bound_moiety="FLP recombinase from the Saccharomyces 
                     cerevisiae 2u plasmid"
                     /note="supports FLP-mediated excision but not integration 
                     (Turan and Bode, 2011)"
     protein_bind    2126..2173
                     /label=Bxb1 attP
                     /bound_moiety="Mycobacteriophage Bxb1 integrase"
                     /note="Minimal attP site for the Mycopbacteriophage Bxb1 
                     integrase (Voutev and Mann, 2017)"
     promoter        2489..2902
                     /gene="S. cerevisiae TEF1"
                     /label=TEF1 promoter
                     /note="promoter for EF-1-alpha"
     promoter        2909..2956
                     /label=EM7 promoter
                     /note="synthetic bacterial promoter "
     CDS             join(2981..3387,1..409)
                     /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             join(2981..3387,1..409)
                     /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(3054..3073)
                     /label=Kan-R
                     /note="Kanamycin resistance gene, reverse primer"
ORIGIN
        1 ttgtaattgt ccttttaaca gcgatcgcgt atttcgtctc gctcaggcgc aatcacgaat
       61 gaataacggt ttggttgatg cgagtgattt tgatgacgag cgtaatggct ggcctgttga
      121 acaagtctgg aaagaaatgc ataaactttt gccattctca ccggattcag tcgtcactca
      181 tggtgatttc tcacttgata accttatttt tgacgagggg aaattaatag gttgtattga
      241 tgttggacga gtcggaatcg cagaccgata ccaggatctt gccatcctat ggaactgcct
      301 cggtgagttt tctccttcat tacagaaacg gctttttcaa aaatatggta ttgataatcc
      361 tgatatgaat aaattgcagt ttcatttgat gctcgatgag tttttctaac acatcatgta
      421 attagttatg tcacgcttac attcacgccc tccccccaca tccgctctaa ccgaaaagga
      481 aggagttaga caacctgaag tctaggtccc tatttatttt tttatagtta tgttagtatt
      541 aagaacgtta tttatatttc aaatttttct tttttttctg tacagacgcg tgtacgcatg
      601 taacattata ctgaaaacct tgcttgagaa ggttttggga cgctcgaagg ctttaatttg
      661 caagctggag accaacatgt gagcaaaagg ccagcaaaag gccaggaacc gtaaaaaggc
      721 cgcgttgctg gcgtttttcc ataggctccg cccccctgac gagcatcaca aaaatcgacg
      781 ctcaagtcag aggtggcgaa acccgacagg actataaaga taccaggcgt ttccccctgg
      841 aagctccctc gtgcgctctc ctgttccgac cctgccgctt accggatacc tgtccgcctt
      901 tctcccttcg ggaagcgtgg cgctttctca tagctcacgc tgtaggtatc tcagttcggt
      961 gtaggtcgtt cgctccaagc tgggctgtgt gcacgaaccc cccgttcagc ccgaccgctg
     1021 cgccttatcc ggtaactatc gtcttgagtc caacccggta agacacgact tatcgccact
     1081 ggcagcagcc actggtaaca ggattagcag agcgaggtat gtaggcggtg ctacagagtt
     1141 cttgaagtgg tggcctaact acggctacac tagaagaaca gtatttggta tctgcgctct
     1201 gctgaagcca gttaccttcg gaaaaagagt tggtagctct tgatccggca aacaaaccac
     1261 cgctggtagc ggtggttttt ttgtttgcaa gcagcagatt acgcgcagaa aaaaaggatc
     1321 tcaagaagat cctttgatct tttctacggg gtctgacgct cagtggaacg aaaactcacg
     1381 ttaagggatt ttggtcatgc atgagatcag atctgaagtt cctatacttt ctagagaata
     1441 ggaacttcaa gcttcacttg cgctggagac agtgcgatag ggagctctat ggttcgagct
     1501 aaccaagttg tggcactaca acagggcaat ttcagactag tctgctgatt tataatagac
     1561 cctatgaaac agttataacg ccgccaagaa ttactagatt cccagtccta aacattacgt
     1621 catagtcaaa cgtatccaat tgcgtacatt gaatataacc gaacttgagc aaaaactgat
     1681 gaataacacc tcaagaagtc accccctccg cgaacgatta ctaaacttac gatatatggt
     1741 gtgtttgtca tatgcaattg tcacgtgaat tgtgaaatag ggcccaatat tcaagcagct
     1801 tgaaccacga cgttctagag ttccagccct tcttgaaatt gaagacagcg accgaaaatt
     1861 gatctaccta ggaaggatac accaggtgag tagcctctga gacgtacata tttttgtgcg
     1921 tctgagtctt tgcgttcatg agattattgg aaaccaccag aatcgaatat aaaaggcgaa
     1981 cacctttccc aattttggtt tctcctgacc caaagacttt aaatttaatt tatttgtccc
     2041 tatttcaatc aattgaacaa ctatttcggc tggacggcga cgtaaacggc cacaagttta
     2101 tggccgtgat gacctgtgtc ttcgtggttt gtctggtcaa ccaccgcggt ctcagtggtg
     2161 tacggtacaa acccaaagca gcacgacacg gcaactacaa gacccgcgcc gagggcatgt
     2221 tccccaaagc gataccactt gaagcagtgg tactgcttgt gggtacactc tgcgggtgtg
     2281 aagttcgagg gcgacaccct ggtgaaccgc atcgagctga agggcatctt ccaactcgct
     2341 taattgcgag tttttatttc gtttatttca attaaggtaa ctaaaaaact ccttttacac
     2401 atgaagcagc acgacttctt caagtccgcc atgcccgaaa aacgcctctt cagagtacag
     2461 aagattaagt gagaccttcg tttgtgcgga tcccccacac accatagctt caaaatgttt
     2521 ctactccttt tttactcttc cagattttct cggactccgc gcatcgccgt accacttcaa
     2581 aacacccaag cacagcatac taaatttccc ctctttcttc ctctagggtg tcgttaatta
     2641 cccgtactaa aggtttggaa aagaaaaaag agaccgcctc gtttcttttt cttcgtcgaa
     2701 aaaggcaata aaaattttta tcacgtttct ttttcttgaa aatttttttt tttgattttt
     2761 ttctctttcg atgacctccc attgatattt aagttaataa acggtcttca atttctcaag
     2821 tttcagtttc atttttcttg ttctattaca acttttttta cttcttgctc attagaaaga
     2881 aagcatagca atctaatcta agggcggtgt tgacaattaa tcatcggcat agtatatcgg
     2941 catagtataa tacgacaagg tgaggaacta aaccatggta atgagccata ttcaacggga
     3001 aacgtcttgc tctaggccgc gattaaattc caacatggat gctgatttat atgggtataa
     3061 atgggctcgc gataatgtcg ggcaatcagg tgcgacaatc tatcgattgt atgggaagcc
     3121 cgatgcgcca gagttgtttc tgaaacatgg caaaggtagc gttgccaatg atgttacaga
     3181 tgagatggtc agactaaact ggctgacgga atttatgcct cttccgacca tcaagcattt
     3241 tatccgtact cctgatgatg catggttact caccactgcg atccccggga aaacagcatt
     3301 ccaggtatta gaagaatatc ctgattcagg tgaaaatatt gttgatgcgc tggcagtgtt
     3361 cctgcgccgg ttgcattcga ttcctgt
//
