LOCUS       sequence-469388-        5777 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Quantitative bacterial two-hybrid (qB2H). Expresses IP3..
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 5777)
  AUTHORS   Guyot A, Maillard E, Ferreira-Pinto K, Plancon-Arnould L, 
            Nadaradjane AA, Ochsenbein F, Guerois R, Martin L, Ramos OHP
  TITLE     Optimized quantitative bacterial two-hybrid (qB2H) for 
            protein-protein interaction assessment
  JOURNAL   bioRxiv 2025.10.29.680610
REFERENCE   2  (bases 1 to 5777)
  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: "bioRxiv 
            2025.10.29.680610"
COMMENT     Sequence Label: VN1272
            pB2Hws_v28_Ddcm_wkKanR_TIR51_pLWeakRBS_...
FEATURES             Location/Qualifiers
     source          1..5777
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     promoter        133..235
                     /label=cat promoter
                     /note="promoter of the E. coli cat gene encoding 
                     chloramphenicol acetyltransferase"
     CDS             236..895
                     /codon_start=1
                     /gene="cat"
                     /product="chloramphenicol acetyltransferase"
                     /label=CmR
                     /note="confers resistance to chloramphenicol"
                     /translation="MEKKITGYTTVDISQWHRKEHFEAFQSVAQCTYNQTVQLDITAFL
                     KTVKKNKHKFYPAFIHILARLMNAHPEFRMAMKDGELVIWDSVHPCYTVFHEQTETFSS
                     LWSEYHDDFRQFLHIYSQDVACYGENLAYFPKGFIENMFFVSANPWVSFTSFDLNVANM
                     DNFFAPVFTMGKYYTQGDKVLMPLAIQVHHAVCDGFHVGRMLNELQQYCDEWQGGA"
     primer_bind     complement(302..321)
                     /label=CAT-R
                     /note="Chloramphenicol resistance gene, reverse primer"
     CDS             complement(1333..2043)
                     /codon_start=1
                     /gene="cI"
                     /product="phage lambda repressor"
                     /label=lambda repressor
                     /translation="MSTKKKPLTQEQLEDARRLKAIYEKKKNELGLSQPSVADKMGMGQ
                     SGVGALFNGINALNAYNAALLAKILKVSVEEFSPSIAREIYEMYEAVSMQPSLRSEYEY
                     PVFSHVQAGMFSPELRTFTKGDAERWVSTTKKASDSAFWLEVEGNSMTAPTGSKPSFPD
                     GMLILVDPEQAVEPGDFCIARLGGDEFTFKKLIRDSGQVFLQPLNPQYPMIPCNESCSV
                     VGKVIASQWPEETFG"
     protein_bind    2085..2103
                     /gene="tetO"
                     /label=tet operator
                     /bound_moiety="tetracycline repressor TetR"
                     /note="bacterial operator O2 for the tetR and tetA genes"
     CDS             2379..3194
                     /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             2379..3194
                     /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(3430..3451)
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     3642..3661
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     terminator      complement(3813..3840)
                     /label=T7Te terminator
                     /note="phage T7 early transcription terminator"
     rep_origin      3910..4455
                     /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     4356..4375
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     promoter        join(4695..4700,4701..4718,4719..4725)
                     /label=lac UV5 promoter
                     /note="E. coli lac promoter with an ""up"" mutation"
     protein_bind    4733..4749
                     /label=lac operator
                     /bound_moiety="lac repressor encoded by lacI"
                     /note="The lac repressor binds to the lac operator to 
                     inhibit transcription in E. coli. This inhibition can be 
                     relieved by adding lactose or 
                     isopropyl-beta-D-thiogalactopyranoside (IPTG)."
     primer_bind     4738..4760
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     primer_bind     4757..4773
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     4757..4773
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
ORIGIN
        1 ggcagcatca cccgacgcac tttgcgccga ataaatacct gtgacggaag atcacttcgc
       61 agaataaata aatcctggtg tccctgttga taccgggaag ccctgggcca acttttggcg
      121 aaaatgagac gttgatcggc acgtaagagg ttccaacttt caccataatg aaataagatc
      181 actaccgggc gtattttttg agttatcgag attttcagga gctaaggaag ctaaaatgga
      241 gaaaaaaatc actggatata ccaccgttga tatatcccaa tggcatcgta aagaacattt
      301 tgaggcattt cagtcagttg ctcaatgtac ctataaccag accgttcagc tggatattac
      361 ggccttttta aagaccgtaa agaaaaataa gcacaagttt tatccggcct ttattcacat
      421 tcttgcccgc ctgatgaatg ctcatccgga attccgtatg gcaatgaaag acggtgagct
      481 ggtgatatgg gatagtgttc acccttgtta caccgttttc catgagcaaa ctgaaacgtt
      541 ttcatcgctc tggagtgaat accacgacga tttccggcag tttctacaca tatattcgca
      601 agatgtggcg tgttacggtg aaaacctggc ctatttccct aaagggttta ttgagaatat
      661 gtttttcgtc tcagccaatc cctgggtgag tttcaccagt tttgatttaa acgtggccaa
      721 tatggacaac ttcttcgccc ccgttttcac catgggcaaa tattatacgc aaggcgacaa
      781 ggtgctgatg ccgctggcga ttcaggttca tcatgccgtt tgtgatggct tccatgtcgg
      841 cagaatgctt aatgaattac aacagtactg cgatgagtgg cagggcgggg cgtaattttt
      901 ttaaggcagt tattggtgcc cttaaacgcc tggttgctac gcctgaataa gtgataataa
      961 gcggatgaat ggcagaaatt cgaaagcaaa ttcgacccgg tcgtcggttc agggcagggt
     1021 cgttaaatag ccgcttatgt ctattgctgg tttaccggtt tattgactac cggaagcagt
     1081 gtgaccgtgt gcttctcaaa tgcctgaggc cagtttgctc aggctctccc cgtggaggta
     1141 ataattgacg atatgatcat ttattctgcc tcccagagcc tgataaaaac ggttagcgct
     1201 tcgttaatac agatgtaggt gttccacagg gtagccagca gcatcctgcg atgcagatct
     1261 gtaaggtaag agctcttatc aagatccgag accgacttcc cagagttcct gacggtctca
     1321 catggcggta ccgccaaacg tctcttcagg ccactgacta gcgataactt tccccacaac
     1381 ggaacaactc tcattgcatg ggatcattgg gtactgtggg tttagtggtt gtaaaaacac
     1441 ctgaccgcta tccctgatca gtttcttgaa ggtaaactca tcacccccaa gtctggctat
     1501 gcagaaatca cctggctcaa cagcctgctc agggtcaacg agaattaaca ttccgtcagg
     1561 aaagcttggc ttggagcctg ttggtgcggt catggaatta ccttcaacct caagccagaa
     1621 tgcagaatca ctggcttttt tggttgtgct tacccatctc tccgcatcac ctttggtaaa
     1681 ggttctaagc tcaggtgaga acatccctgc ctgaacatga gaaaaaacag ggtactcata
     1741 ctcacttcta agtgacggct gcatactaac cgcttcatac atctcgtaga tttctctggc
     1801 gattgaaggg ctaaattctt caacgctaac tttgagaatt tttgcaagca atgcggcgtt
     1861 ataagcattt aatgcattga tgccattaaa taaagcacca acgcctgact gccccatccc
     1921 catcttgtct gcgacagacg gctgggataa gccaagttca tttttctttt tttcataaat
     1981 tgctttaagg cgacgtgcgt cctcaagctg ctcttgtgtt aatggtttct tttttgtgct
     2041 cattatatga cctagcgagc tggtctactt aagtgctagc agtatctcta tcactgatag
     2101 ggatgtcaag cgcgcttgaa gagataaatt gcactgaaat ctagatgtct ccagaccgct
     2161 agtagacgag tccatgtctc gagagcgagg ctcttcaagc tcgcaaaatt gctcttcaag
     2221 ctcgcaaaaa accccgcccc tgacagggcg gggttttttc gcgttcgatc ccgcgaaatt
     2281 taattaacca acaccgccag agatacatta ggcaccggcg gcttgacact ttatgcttcc
     2341 ggctcggata ctgtgtggat ttgacagcgg caggtacgat gagccatatt caacgggaaa
     2401 cgtcttgctc taggccgcga ttaaattcca acatggatgc tgatttatat gggtataaat
     2461 gggcccgcga taatgtcggg caatcaggtg cgacaatcta tcgattgtat gggaagcccg
     2521 atgcgccaga gttgtttctg aaacatggca aaggtagcgt tgccaatgat gttacagatg
     2581 agatggtcag actaaactgg ctgacggaat ttatgcctct tccgaccatc aagcatttta
     2641 tccgtactcc tgatgatgca tggttactca ccactgcgat ccccgggaaa acagcattcc
     2701 aggtattaga agaatatcct gattcaggtg aaaatattgt tgatgcgctg gcagtgttcc
     2761 tgcgccggtt gcattcgatt cctgtttgta attgtccttt taacagcgat cgcgtatttc
     2821 gtctcgctca ggcgcaatca cgaatgaata acggtttggt tgatgcgagt gattttgatg
     2881 acgagcgtaa tggctggcct gttgaacaag tctggaaaga aatgcataaa cttttgccat
     2941 tctcaccgga ttcagtcgtc actcatggtg atttctcact tgataacctt atttttgacg
     3001 aggggaaatt aataggttgt attgatgttg gacgagtcgg aatcgcagac cgataccagg
     3061 atcttgccat cctatggaac tgcctcggtg agttttctcc ttcattacag aaacggcttt
     3121 ttcaaaaata tggtattgat aatcctgata tgaataaatt gcagtttcat ttgatgctcg
     3181 atgagttttt ctaagactag tgaattaatt catgagcgga tacatatttg aatgtattta
     3241 gaaaaataaa caaatagggg ttccgcgcac atttccccga aaagtgccac ctaaattgta
     3301 agcgttaata ttttgttaaa attcgcgtta aatttttgtt aaatcagctc attttttaac
     3361 caataggccg aaatcggcaa aatcccttat aaatcaaaag aatagaccga gatagggttg
     3421 agtgttgttc cagtttggaa caagagtcca ctattaaaga acgtggactc caacgtcaaa
     3481 gggcgaaaaa ccgtctatca gggcgatggc ccactacgtg aaccatcacc ctaatcaagt
     3541 tttttggggt cgaggtgccg taaagcacta aattggaacc ctaaagggag cccccgattt
     3601 agagcttgac ggggaaagcc ggcgaacgtg gcgagaaagg aagggaagaa agcgaaagga
     3661 gcgggcgcta gggcgctggc aagtgtagcg gtcacgctgc gcgtaaccac cacacccgcc
     3721 gcgcttaatg cgccgctaca gggcgcagat ctaaataata aaaaagccgg attaataatc
     3781 tggcttttta tattctctct ctagtatata aacgcagaaa ggcccacccg aaggtgagcc
     3841 agtgtgacga acccccgcct cagagtggga aagctagaaa tattttatct gattaataag
     3901 atgatcttct tgagatcgtt ttggtctgcg cgtaatctct tgctctgaaa acgaaaaaac
     3961 cgccttgcag ggcggttttt cgaaggttct ctgagctacc aactctttga accgaggtaa
     4021 ctggcttgga ggagcgcagt caccaaaact tgtcctttca gtttagcctt aaccggcgca
     4081 tgacttcaag actaactcct ctaaatcaat taccagtggc tgctgccagt ggtgcttttg
     4141 catgtctttc cgggttggac tcaagacgat agttaccgga taaggcgcag cggtcggact
     4201 gaacgggggg ttcgtgcata cagtccagct tggagcgaac tgcctacccg gaactgagtg
     4261 tcaggcgtgg aatgagacaa acgcggccat aacagcggaa tgacaccggt aaaccgaaag
     4321 gcaggaacag gagagcgcac gagggagccg ccagggggaa acgcctggta tctttatagt
     4381 cctgtcgggt ttcgccacca ctgatttgag cgtcagattt cgtgatgctt gtcagggggg
     4441 cggagcctat ggaaaaacgg ctttgccgcg gccctctcac ttccctgtta agtatcttcc
     4501 tggcatcttc caggaaatct ccgccccgtt cgtaagccat ttccgctctc cgcagtcgaa
     4561 cgaccgagcg tagcgagtca gtgcgaacga tcaaaaataa gtgccttccc atcaaaaaaa
     4621 tattgacaac ataaaaaact ttgtgttata cttgtaacgc tacatggaga ttaactcaat
     4681 ctagctagag aggctttaca ctttatgctt ccggctcgta taatgtgtgg aattgtgagc
     4741 ggataacaat ttcacacagg aaacagctat gaccatgatt acggattcac tggaactcta
     4801 gaccaaagag aggacacaat gcagggttct gtgacagagt ttctaaaacc gcgcctggtt
     4861 gatatcgagc aagtgagttc gacgcacgcc aaggtgaccc ttgagccttt agagcgtggc
     4921 tttggccata ctctgggtaa cgcactgcgc cgtattctgc tctcatcgat gccgggttgc
     4981 gcggtgaccg aggttgagat tgatggtgta ctacatgagt acagcaccaa agaaggcgtt
     5041 caggaagata tcctggaaat cctgctcaac ctgaaagggc tggcggtgag agttcagggc
     5101 aaagatgaag ttattcttac cttgaataaa tctggcattg gccctgtgac tgcagccgat
     5161 atcacccacg acggtgatgt cgaaatcgtc aagccgcagc acgtgatctg ccacctgacc
     5221 gatgagaacg cgtctattag catgcgtatc aaagttcagc gcggtcgtgg ttatgtgccg
     5281 gcttctaccc gaattcattc ggaagaagat gagcgcccaa tcggccgtct gctggtcgac
     5341 gcatgctaca gccctgtgga gcgtattgcc tacaatgttg aagcagcgcg tgtagaacag
     5401 cgtaccgacc tggacaagct ggtcatcgaa atggaaacca acggcacaat cgatcctgaa
     5461 gaggcgattc gtcgtgcggc aaccattctg gctgaacaac tggaagcttt cgttgactta
     5521 cgtgatgtac gtcagcctga agtgaaagaa gagaaaccag aggcggccgc agcaagcacc
     5581 gaacgcaaat gggcagaact ggcacgtcgt attcgtggtg caggcggtgt taccctcaat
     5641 ggttttggtt gaggatcctc tacgccggac gcactcggtt ccaaaaaaaa aaaaaaagac
     5701 gctgaaaagc gtcttttttt tttttggtcc ccactggagc acctcaaaaa caccatcata
     5761 cactaaatca gtaagtt
//
