LOCUS       sequence-469190-        6936 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Quantitative bacterial two-hybrid (qB2H). Expresses Asf1B and IP3..
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 6936)
  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 6936)
  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: VN1315
            pB2Hws_v30_pLWeakRBS_TIR371_cI-Asf1B+rp...
FEATURES             Location/Qualifiers
     source          1..6936
                     /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(1756..2466)
                     /codon_start=1
                     /gene="cI"
                     /product="phage lambda repressor"
                     /label=lambda repressor
                     /translation="MSTKKKPLTQEQLEDARRLKAIYEKKKNELGLSQPSVADKMGMGQ
                     SGVGALFNGINALNAYNAALLAKILKVSVEEFSPSIAREIYEMYEAVSMQPSLRSEYEY
                     PVFSHVQAGMFSPELRTFTKGDAERWVSTTKKASDSAFWLEVEGNSMTAPTGSKPSFPD
                     GMLILVDPEQAVEPGDFCIARLGGDEFTFKKLIRDSGQVFLQPLNPQYPMIPCNESCSV
                     VGKVIASQWPEETFG"
     protein_bind    2508..2526
                     /gene="tetO"
                     /label=tet operator
                     /bound_moiety="tetracycline repressor TetR"
                     /note="bacterial operator O2 for the tetR and tetA genes"
     CDS             join(2802..2804,2805..2807,2808..3518)
                     /codon_start=1
                     /product="enhanced GFP"
                     /label=EGFP
                     /translation="MVSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTL
                     KFICTTGKLPVPWPTLVTTLTYGVQCFSRYPDHMKQHDFFKSAMPEGYVQERTIFFKDD
                     GNYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIK
                     VNFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSALSKDPNEKRDHMVLL
                     EFVTAAGITLGMDELYK"
     CDS             3541..4353
                     /codon_start=1
                     /product="aminoglycoside phosphotransferase"
                     /label=KanR
                     /note="confers resistance to kanamycin in bacteria or G418 
                     (Geneticin(R)) in eukaryotes"
                     /translation="SHIQRETSCSRPRLNSNMDADLYGYKWARDNVGQSGATIYRLYGK
                     PDAPELFLKHGKGSVANDVTDEMVRLNWLTEFMPLPTIKHFIRTPDDAWLLTTAIPGKT
                     AFQVLEEYPDSGENIVDALAVFLRRLHSIPVCNCPFNSDRVFRLAQAQSRMNNGLVDAS
                     DFDDERNGWPVEQVWKEMHKLLPFSPDSVVTHGDFSLDNLIFDEGKLIGCIDVGRVGIA
                     DRYQDLAILWNCLGEFSPSLQKRLFQKYGIDNPDMNKLQFHLMLDEFF"
     CDS             3541..4353
                     /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="SHIQRETSCSRPRLNSNMDADLYGYKWARDNVGQSGATIYRLYGK
                     PDAPELFLKHGKGSVANDVTDEMVRLNWLTEFMPLPTIKHFIRTPDDAWLLTTAIPGKT
                     AFQVLEEYPDSGENIVDALAVFLRRLHSIPVCNCPFNSDRVFRLAQAQSRMNNGLVDAS
                     DFDDERNGWPVEQVWKEMHKLLPFSPDSVVTHGDFSLDNLIFDEGKLIGCIDVGRVGIA
                     DRYQDLAILWNCLGEFSPSLQKRLFQKYGIDNPDMNKLQFHLMLDEFF"
     primer_bind     complement(3611..3630)
                     /label=Kan-R
                     /note="Kanamycin resistance gene, reverse primer"
     primer_bind     complement(4589..4610)
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     4801..4820
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     terminator      complement(4972..4999)
                     /label=T7Te terminator
                     /note="phage T7 early transcription terminator"
     rep_origin      5069..5614
                     /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     5515..5534
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     promoter        join(5854..5859,5860..5877,5878..5884)
                     /label=lac UV5 promoter
                     /note="E. coli lac promoter with an ""up"" mutation"
     protein_bind    5892..5908
                     /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     5897..5919
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     primer_bind     5916..5932
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     5916..5932
                     /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 agttatccca attgatatgg aagcgggtaa cacgcggatt
     1321 ggatgccaga atgttgcgtt gcagctgact aaaatccggt ttcatcggcg gattttcacg
     1381 cagttccggg ttcaggtatt cattgttaac gtaataaccc acgcggatga attcctggcc
     1441 gtgatacgtg caggtaatca gaaccaccgt gacaccaact gcgtcggttt ccggaatcag
     1501 cgacgggttc ggagcatctg cttggaaaac aaacatatga cgacctgccg ggaccgggcc
     1561 caccaggact gagtccagga tctgatcaaa ttcttcagat tccgcgctgc ccacatagat
     1621 aattttccat tccaggtcat cggccagcgc ttcggaacat tcaaacgaga tttcaaagcg
     1681 gaacgggcta tggaacgggg acggattttc cagcactgcc acattcagca ccgagacttt
     1741 cgccattgcg gtaccgccaa acgtctcttc aggccactga ctagcgataa ctttccccac
     1801 aacggaacaa ctctcattgc atgggatcat tgggtactgt gggtttagtg gttgtaaaaa
     1861 cacctgaccg ctatccctga tcagtttctt gaaggtaaac tcatcacccc caagtctggc
     1921 tatgcagaaa tcacctggct caacagcctg ctcagggtca acgagaatta acattccgtc
     1981 aggaaagctt ggcttggagc ctgttggtgc ggtcatggaa ttaccttcaa cctcaagcca
     2041 gaatgcagaa tcactggctt ttttggttgt gcttacccat ctctccgcat cacctttggt
     2101 aaaggttcta agctcaggtg agaacatccc tgcctgaaca tgagaaaaaa cagggtactc
     2161 atactcactt ctaagtgacg gctgcatact aaccgcttca tacatctcgt agatttctct
     2221 ggcgattgaa gggctaaatt cttcaacgct aactttgaga atttttgcaa gcaatgcggc
     2281 gttataagca tttaatgcat tgatgccatt aaataaagca ccaacgcctg actgccccat
     2341 ccccatcttg tctgcgacag acggctggga taagccaagt tcatttttct ttttttcata
     2401 aattgcttta aggcgacgtg cgtcctcaag ctgctcttgt gttaatggtt tcttttttgt
     2461 gctcattata tgacctagcg agctggtcta cttaagtgct agcagtatct ctatcactga
     2521 tagggatgtc aagcgcgctt gaagagataa attgcactga aatctagatg tctccagacc
     2581 gctagtagac gagtccatgt ctcgagagcg aggctcttca agctcgcaaa attgctcttc
     2641 aagctcgcaa aaaaccccgc ccctgacagg gcggggtttt ttcgcgttcg atcccgcgaa
     2701 atttaattaa ccaacaccgc cagagataca ttaggcaccg gcggcttgac actttatgct
     2761 tccggctcgg atactgtgtg gaattgaaag aggaacgtac gatggtcagt aaaggcgaag
     2821 agttgtttac gggtgtagta ccgattttag tggaactcga tggagatgtg aatgggcata
     2881 aattttcggt ttcaggtgaa ggtgaaggag atgccaccta tggcaaactg acgctgaagt
     2941 tcatctgtac gacaggcaaa ctgcctgttc cgtggccaac actggttact acgctgacct
     3001 atggcgttca atgcttttcg cgttatccgg accacatgaa acagcatgac ttctttaaat
     3061 ccgcgatgcc tgaaggttac gtacaagaac gcaccatctt cttcaaagat gacgggaact
     3121 acaaaacccg tgcagaggtc aaatttgagg gtgacaccct tgtgaatcgg attgagctga
     3181 aaggcattga ctttaaagaa gatggcaaca ttctgggtca taaactggag tacaactaca
     3241 acagccataa cgtgtatatc atggcggaca aacagaagaa tgggatcaaa gtgaatttca
     3301 agattcgcca caacattgaa gatggtagtg tgcagttagc cgaccactat cagcagaata
     3361 ccccgattgg cgatggccca gtcttgctgc cggataatca ttaccttagc acccaatctg
     3421 cgttaagcaa agatcccaac gaaaagcgcg atcacatggt cctcctggaa tttgtgactg
     3481 ctgcaggcat cactttgggt atggatgaac tgtataagtg aaattcctgg tccaggaatt
     3541 tcacatattc aacgggaaac gtcttgctct aggccgcgat taaattccaa catggatgct
     3601 gatttatatg ggtataaatg ggctcgcgat aatgtcgggc aatcaggtgc gacaatctat
     3661 cgattgtatg ggaagcccga tgcgccagag ttgtttctga aacatggcaa aggtagcgtt
     3721 gccaatgatg ttacagatga gatggtcaga ctaaactggc tgacggaatt tatgcctctt
     3781 ccgaccatca agcattttat ccgtactcct gatgatgcat ggttactcac cactgcgatc
     3841 cccgggaaaa cagcattcca ggtattagaa gaatatcctg attcaggtga aaatattgtt
     3901 gatgcgctgg cagtgttcct gcgccggttg cattcgattc ctgtttgtaa ttgtcctttt
     3961 aacagcgatc gcgtatttcg tctcgctcag gcgcaatcac gaatgaataa cggtttggtt
     4021 gatgcgagtg attttgatga cgagcgtaat ggctggcctg ttgaacaagt ctggaaagaa
     4081 atgcataaac ttttgccatt ctcaccggat tcagtcgtca ctcatggtga tttctcactt
     4141 gataacctta tttttgacga ggggaaatta ataggttgta ttgatgttgg acgagtcgga
     4201 atcgcagacc gataccagga tcttgccatc ctatggaact gcctcggtga gttttctcct
     4261 tcattacaga aacggctttt tcaaaaatat ggtattgata atcctgatat gaataaattg
     4321 cagtttcatt tgatgctcga tgagtttttc taagactagt gaattaattc atgagcggat
     4381 acatatttga atgtatttag aaaaataaac aaataggggt tccgcgcaca tttccccgaa
     4441 aagtgccacc taaattgtaa gcgttaatat tttgttaaaa ttcgcgttaa atttttgtta
     4501 aatcagctca ttttttaacc aataggccga aatcggcaaa atcccttata aatcaaaaga
     4561 atagaccgag atagggttga gtgttgttcc agtttggaac aagagtccac tattaaagaa
     4621 cgtggactcc aacgtcaaag ggcgaaaaac cgtctatcag ggcgatggcc cactacgtga
     4681 accatcaccc taatcaagtt ttttggggtc gaggtgccgt aaagcactaa attggaaccc
     4741 taaagggagc ccccgattta gagcttgacg gggaaagccg gcgaacgtgg cgagaaagga
     4801 agggaagaaa gcgaaaggag cgggcgctag ggcgctggca agtgtagcgg tcacgctgcg
     4861 cgtaaccacc acacccgccg cgcttaatgc gccgctacag ggcgcagatc taaataataa
     4921 aaaagccgga ttaataatct ggctttttat attctctctc tagtatataa acgcagaaag
     4981 gcccacccga aggtgagcca gtgtgacgaa cccccgcctc agagtgggaa agctagaaat
     5041 attttatctg attaataaga tgatcttctt gagatcgttt tggtctgcgc gtaatctctt
     5101 gctctgaaaa cgaaaaaacc gccttgcagg gcggtttttc gaaggttctc tgagctacca
     5161 actctttgaa ccgaggtaac tggcttggag gagcgcagtc accaaaactt gtcctttcag
     5221 tttagcctta accggcgcat gacttcaaga ctaactcctc taaatcaatt accagtggct
     5281 gctgccagtg gtgcttttgc atgtctttcc gggttggact caagacgata gttaccggat
     5341 aaggcgcagc ggtcggactg aacggggggt tcgtgcatac agtccagctt ggagcgaact
     5401 gcctacccgg aactgagtgt caggcgtgga atgagacaaa cgcggccata acagcggaat
     5461 gacaccggta aaccgaaagg caggaacagg agagcgcacg agggagccgc cagggggaaa
     5521 cgcctggtat ctttatagtc ctgtcgggtt tcgccaccac tgatttgagc gtcagatttc
     5581 gtgatgcttg tcaggggggc ggagcctatg gaaaaacggc tttgccgcgg ccctctcact
     5641 tccctgttaa gtatcttcct ggcatcttcc aggaaatctc cgccccgttc gtaagccatt
     5701 tccgctctcc gcagtcgaac gaccgagcgt agcgagtcag tgcgaacgat caaaaataag
     5761 tgccttccca tcaaaaaaat attgacaaca taaaaaactt tgtgttatac ttgtaacgct
     5821 acatggagat taactcaatc tagctagaga ggctttacac tttatgcttc cggctcgtat
     5881 aatgtgtgga attgtgagcg gataacaatt tcacacagga aacagctatg accatgatta
     5941 cggattcact ggaactctag accaaagaga ggacacaatg cagggttctg tgacagagtt
     6001 tctaaaaccg cgcctggttg atatcgagca agtgagttcg acgcacgcca aggtgaccct
     6061 tgagccttta gagcgtggct ttggccatac tctgggtaac gcactgcgcc gtattctgct
     6121 ctcatcgatg ccgggttgcg cggtgaccga ggttgagatt gatggtgtac tacatgagta
     6181 cagcaccaaa gaaggcgttc aggaagatat cctggaaatc ctgctcaacc tgaaagggct
     6241 ggcggtgaga gttcagggca aagatgaagt tattcttacc ttgaataaat ctggcattgg
     6301 ccctgtgact gcagccgata tcacccacga cggtgatgtc gaaatcgtca agccgcagca
     6361 cgtgatctgc cacctgaccg atgagaacgc gtctattagc atgcgtatca aagttcagcg
     6421 cggtcgtggt tatgtgccgg cttctacccg aattcattcg gaagaagatg agcgcccaat
     6481 cggccgtctg ctggtcgacg catgctacag ccctgtggag cgtattgcct acaatgttga
     6541 agcagcgcgt gtagaacagc gtaccgacct ggacaagctg gtcatcgaaa tggaaaccaa
     6601 cggcacaatc gatcctgaag aggcgattcg tcgtgcggca accattctgg ctgaacaact
     6661 ggaagctttc gttgacttac gtgatgtacg tcagcctgaa gtgaaagaag agaaaccaga
     6721 ggcggccgca gcaagcaccg aacgcaaatg ggcagaactg gcacgtcgta ttcgtggtgc
     6781 aggcggtgtt accctcaatg gttttggttg aggatcctct acgccggacg cactcggttc
     6841 caaaaaaaaa aaaaaagacg ctgaaaagcg tctttttttt ttttggtccc cactggagca
     6901 cctcaaaaac accatcatac actaaatcag taagtt
//
