LOCUS       sequence-361532-        3987 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Tripartite split-GFP. E. coli vector. Inducible coexpression of 
            GFP10-FRB and FKBP-GFP11 coiled coils.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 3987)
  AUTHORS   Pedelacq JD, Waldo GS, Cabantous S
  TITLE     High-Throughput Protein-Protein Interaction Assays Using Tripartite 
            Split-GFP Complementation.
  JOURNAL   Methods Mol Biol. 2019;2025:423-437. doi: 
            10.1007/978-1-4939-9624-7_20.
  PUBMED    31267465
REFERENCE   2  (bases 1 to 3987)
  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: "Methods Mol
            Biol."; date: "2019"; volume: "2025:423-437. doi"; pages: " 
            10.1007/978-1-4939-9624-7_20"
COMMENT     Sequence Label: pTET GFP10-FRB/FKBP-GFP11
FEATURES             Location/Qualifiers
     source          1..3987
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     promoter        join(23..41,42..47,48..64,65..70,71..96)
                     /label=PLtetO-1 promoter
                     /note="modified phage lambda PL promoter with tet operator 
                     sites (Lutz and Bujard, 1997)"
     protein_bind    23..41
                     /gene="tetO"
                     /label=tet operator
                     /bound_moiety="tetracycline repressor TetR"
                     /note="bacterial operator O2 for the tetR and tetA genes"
     protein_bind    48..66
                     /gene="tetO"
                     /label=tet operator
                     /bound_moiety="tetracycline repressor TetR"
                     /note="bacterial operator O2 for the tetR and tetA genes"
     RBS             106..117
                     /note="strong bacterial ribosome binding site (Elowitz and 
                     Leibler, 2000)"
     CDS             257..535
                     /codon_start=1
                     /product="FKBP-rapamycin binding domain of human FRAP"
                     /label=FRB
                     /translation="ILWHEMWHEGLEEASRLYFGERNVKGMFEVLEPLHAMMERGPQTL
                     KETSFNQAYGRDLMEAQEWCRKYMKSGNVKDLTQAWDLYYHVFRRISK"
     CDS             269..538
                     /codon_start=1
                     /product="FKBP-rapamycin binding domain of human FRAP"
                     /label=FRB
                     /translation="EMWHEGLEEASRLYFGERNVKGMFEVLEPLHAMMERGPQTLKETS
                     FNQAYGRDLMEAQEWCRKYMKSGNVKDLTQAWDLYYHVFRRISKQ"
     CDS             1028..1045
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     primer_bind     complement(1098..1116)
                     /label=T7 Term
                     /note="T7 terminator, reverse primer"
     terminator      1134..1220
                     /gene="Escherichia coli rrnB"
                     /label=rrnB T1 terminator
                     /note="transcription terminator T1 from the E. coli rrnB 
                     gene"
     rep_origin      complement(1385..1973)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(1465..1484)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     terminator      complement(2061..2155)
                     /label=lambda t0 terminator
                     /note="transcription terminator from phage lambda"
     CDS             complement(2161..2787)
                     /codon_start=1
                     /gene="tetR"
                     /product="tetracycline repressor TetR"
                     /label=TetR
                     /note="TetR binds to the tetracycline operator tetO to 
                     inhibit transcription. This inhibition can be relieved by 
                     adding tetracycline or doxycycline."
                     /translation="MMSRLDKSKVINSALELLNEVGIEGLTTRKLTQKLGVEQPTLYWH
                     VKNKRALLDALAIEMLDRHHTHFCPLEGESWQDFLRNNAKSFRCALLSHRDGAKVHLGT
                     RPTEKQYETLENQLAFLCQQGFSLENALYALSAVGHFTLGCVLEDQEHQVAKEERETPT
                     TDSMPPLLRQAIELFDHQGAEPAFLFGLELIIYGLEKQLKCESGS"
     CDS             complement(2825..3616)
                     /codon_start=1
                     /gene="aadA"
                     /product="aminoglycoside adenylyltransferase (Murphy, 
                     1985)"
                     /label=SmR
                     /note="confers resistance to spectinomycin and 
                     streptomycin"
                     /translation="MREAVIAEVSTQLSEVVGVIERHLEPTLLAVHLYGSAVDGGLKPH
                     SDIDLLVTVTVRLDETTRRALINDLLETSASPGESEILRAVEVTIVVHDDIIPWRYPAK
                     RELQFGEWQRNDILAGIFEPATIDIDLAILLTKAREHSVALVGPAAEELFDPVPEQDLF
                     EALNETLTLWNSPPDWAGDERNVVLTLSRIWYSAVTGKIAPKDVAADWAMERLPAQYQP
                     VILEARQAYLGQEEDRLASRADQLEEFVHYVKGEITKVVGK"
     primer_bind     join(3971..3987,1..2)
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
ORIGIN
        1 gccctttcgt cttcacctcg agtccctatc agtgatagag attgacatcc ctatcagtga
       61 tagagatact gagcacatca gcaggacgca ctgaccgagt tcattaaaga ggagaaagat
      121 acccatgggc gatttaccag acgaccatta cctgtcgaca caaactatcc tttcgaaaga
      181 tctcaacgga actgacgttg ggtctggcgg tggttccggt ggtggaggtt ctcatatgga
      241 gctgatccga gtggccatcc tctggcatga gatgtggcat gaaggcctgg aagaggcatc
      301 tcgtttgtac tttggggaaa ggaacgtgaa aggcatgttt gaggtgctgg agcccttgca
      361 tgctatgatg gaacggggcc cccagactct gaaggaaaca tcctttaatc aggcctatgg
      421 tcgagattta atggaggccc aagagtggtg caggaagtac atgaaatcag ggaatgtcaa
      481 ggacctcacc caagcctggg acctctatta tcatgtgttc cgacgaatct caaagcagta
      541 aggtacctaa ctaagtaatt aaaggaaaca gacgatgact agtggagtgc aggtggaaac
      601 catctcccca ggaaacgggc gcaccttccc caagcgcggc cagacctgcg tggtgcacta
      661 caccgggatg cttgaagatg gaaagaaatt tgattcctcc cgggacagaa acaagccctt
      721 taagtttatg ctaggcaagc aggaggtgat ccgaggctgg gaagaagggg ttgcccagat
      781 gagtgtgggt cagagagcca aactgactat atcacctgac tatgcatacg gagctacagg
      841 tcatcctgga attattccac ctcacgctac tctcgtcttc gatgtggagc ttctaaaact
      901 ggaaggatcc ggtggcggtg gttcaggtgg agggtctggt ggcggatcaa catctgaaaa
      961 gcgtgaccac atggtccttc ttgagtatgt aactgctgct gggattacag atgcatctta
     1021 actcgagcac caccaccacc accactgaga tccggctgct aacaaagccc gaaaggaagc
     1081 tgagttggct gctgccaccg ctgagcaata actagcataa cctctagagg catcaaataa
     1141 aacgaaaggc tcagtcgaaa gactgggcct ttcgttttat ctgttgtttg tcggtgaacg
     1201 ctctcctgag taggacaaat ccgccgccct agacctaggg cgttcggctg cggcgagcgg
     1261 tatcagctca ctcaaaggcg gtaatacggt tatccacaga atcaggggat aacgcaggaa
     1321 agaacatgtg agcaaaaggc cagcaaaagg ccaggaaccg taaaaaggcc gcgttgctgg
     1381 cgtttttcca taggctccgc ccccctgacg agcatcacaa aaatcgacgc tcaagtcaga
     1441 ggtggcgaaa cccgacagga ctataaagat accaggcgtt tccccctgga agctccctcg
     1501 tgcgctctcc tgttccgacc ctgccgctta ccggatacct gtccgccttt ctcccttcgg
     1561 gaagcgtggc gctttctcat agctcacgct gtaggtatct cagttcggtg taggtcgttc
     1621 gctccaagct gggctgtgtg cacgaacccc ccgttcagcc cgaccgctgc gccttatccg
     1681 gtaactatcg tcttgagtcc aacccggtaa gacacgactt atcgccactg gcagcagcca
     1741 ctggtaacag gattagcaga gcgaggtatg taggcggtgc tacagagttc ttgaagtggt
     1801 ggcctaacta cggctacact agaaggacag tatttggtat ctgcgctctg ctgaagccag
     1861 ttaccttcgg aaaaagagtt ggtagctctt gatccggcaa acaaaccacc gctggtagcg
     1921 gtggtttttt tgtttgcaag cagcagatta cgcgcagaaa aaaaggatct caagaagatc
     1981 ctttgatctt ttctacgggg tctgacgctc agtggaacga aaactcacgt taagggattt
     2041 tggtcatgac tagcgcttgg attctcacca ataaaaaacg cccggcggca accgagcgtt
     2101 ctgaacaaat ccagatggag ttctgaggtc attactggat ctatcaacag gagtccaagc
     2161 ttaagaccca ctttcacatt taagttgttt ttctaatccg tatatgatca attcaaggcc
     2221 gaataagaag gctggctctg caccttggtg atcaaataat tcgatagctt gtcgtaataa
     2281 tggcggcata ctatcagtag taggtgtttc cctttcttct ttagcgactt gatgctcttg
     2341 atcttccaat acgcaaccta aagtaaaatg ccccacagcg ctgagtgcat ataatgcatt
     2401 ctctagtgaa aaaccttgtt ggcataaaaa ggctaattga ttttcgagag tttcatactg
     2461 tttttctgta ggccgtgtac ctaaatgtac ttttgctcca tcgcgatgac ttagtaaagc
     2521 acatctaaaa cttttagcgt tattacgtaa aaaatcttgc cagctttccc cttctaaagg
     2581 gcaaaagtga gtatggtgcc tatctaacat ctcaatggcc aaggcgtcga gcaatgcccg
     2641 cttatttttt acatgccaat acaatgtagg ctgctctaca cctagcttct gggtgagttt
     2701 acgggttgtt aaaccttcga ttccgacctc attaagcagc tctaatgcgc tgttaatcac
     2761 tttactttta tctaatctgg acatcattaa tgtttattga gctctcgaac cccagagtcc
     2821 cgcattattt gccgactacc ttggtgatct cgcctttcac gtagtggaca aattcttcca
     2881 actgatctgc gcgcgaggcc aagcgatctt cttcttgtcc aagataagcc tgtctagctt
     2941 caagtatgac gggctgatac tgggccggca ggcgctccat tgcccagtcg gcagcgacat
     3001 ccttcggcgc gattttgccg gttactgcgc tgtaccaaat gcgggacaac gtaagcacta
     3061 catttcgctc atcgccagcc cagtcgggcg gcgagttcca tagcgttaag gtttcattta
     3121 gcgcctcaaa tagatcctgt tcaggaaccg gatcaaagag ttcctccgcc gctggaccta
     3181 ccaaggcaac gctatgttct cttgcttttg tcagcaagat agccagatca atgtcgatcg
     3241 tggctggctc gaagatacct gcaagaatgt cattgcgctg ccattctcca aattgcagtt
     3301 cgcgcttagc tggataacgc cacggaatga tgtcgtcgtg cacaacaatg gtgacttcta
     3361 cagcgcggag aatctcgctc tctccagggg aagccgaagt ttccaaaagg tcgttgatca
     3421 aagctcgccg cgttgtttca tcaagcctta cggtcaccgt aaccagcaaa tcaatatcac
     3481 tgtgtggctt caggccgcca tccactgcgg agccgtacaa atgtacggcc agcaacgtcg
     3541 gttcgagatg gcgctcgatg acgccaacta cctctgatag ttgagtcgat acttcggcga
     3601 tcaccgcttc cctcatgatg tttaactttg ttttagggcg actgccctgc tgcgtaacat
     3661 cgttgctgct ccataacatc gaacatcgac ccacggcgta acgcgcttgc tgcttggatg
     3721 cccgaggcat agactgtacc ccaaaaaaac agtcataaca agccatgaaa accgccactg
     3781 cgccgttacc atgcgaaacg atcctcatcc tgtctcttga tcagatcttg atcccctgcg
     3841 ccatcagatc cttggcggca agaaagccat ccagtttact ttgcagggct tcccaacctt
     3901 accagagggc gccccagctg gcaattccga cgtctaagaa accattatta tcatgacatt
     3961 aacctataaa aataggcgta tcacgag
//
