LOCUS       sequence-469059-        7080 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Quantitative bacterial two-hybrid (qB2H). Expresses Asf1B and 
            IP3mut3A..
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 7080)
  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 7080)
  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: VN999 pB2Hws_v9_cI-Asf1B+rpoA-IP3mut3A
FEATURES             Location/Qualifiers
     source          1..7080
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     promoter        134..236
                     /label=cat promoter
                     /note="promoter of the E. coli cat gene encoding 
                     chloramphenicol acetyltransferase"
     CDS             237..896
                     /codon_start=1
                     /gene="cat"
                     /product="chloramphenicol acetyltransferase"
                     /label=CmR
                     /note="confers resistance to chloramphenicol"
                     /translation="MEKKITGYTTVDISQWHRKEHFEAFQSVAQCTYNQTVQLDITAFL
                     KTVKKNKHKFYPAFIHILARLMNAHPEFRMAMKDGELVIWDSVHPCYTVFHEQTETFSS
                     LWSEYHDDFRQFLHIYSQDVACYGENLAYFPKGFIENMFFVSANPWVSFTSFDLNVANM
                     DNFFAPVFTMGKYYTQGDKVLMPLAIQVHHAVCDGFHVGRMLNELQQYCDEWQGGA"
     primer_bind     complement(303..322)
                     /label=CAT-R
                     /note="Chloramphenicol resistance gene, reverse primer"
     CDS             complement(1757..2467)
                     /codon_start=1
                     /gene="cI"
                     /product="phage lambda repressor"
                     /label=lambda repressor
                     /translation="MSTKKKPLTQEQLEDARRLKAIYEKKKNELGLSQPSVADKMGMGQ
                     SGVGALFNGINALNAYNAALLAKILKVSVEEFSPSIAREIYEMYEAVSMQPSLRSEYEY
                     PVFSHVQAGMFSPELRTFTKGDAERWVSTTKKASDSAFWLEVEGNSMTAPTGSKPSFPD
                     GMLILVDPEQAVEPGDFCIARLGGDEFTFKKLIRDSGQVFLQPLNPQYPMIPCNESCSV
                     VGKVIASQWPEETFG"
     protein_bind    2488..2504
                     /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)."
     promoter        complement(join(2512..2518,2519..2536,2537..2542))
                     /label=lac UV5 promoter
                     /note="E. coli lac promoter with an ""up"" mutation"
     CDS             complement(2695..2739)
                     /codon_start=1
                     /product="affinity and epitope tag derived from pancreatic 
                     ribonuclease A"
                     /label=S-Tag
                     /translation="KETAAAKFERQHMDS"
     CDS             join(2947..2949,2950..2952,2953..3663)
                     /codon_start=1
                     /product="enhanced GFP"
                     /label=EGFP
                     /translation="MVSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTL
                     KFICTTGKLPVPWPTLVTTLTYGVQCFSRYPDHMKQHDFFKSAMPEGYVQERTIFFKDD
                     GNYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIK
                     VNFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSALSKDPNEKRDHMVLL
                     EFVTAAGITLGMDELYK"
     CDS             3686..4498
                     /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             3686..4498
                     /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(3756..3775)
                     /label=Kan-R
                     /note="Kanamycin resistance gene, reverse primer"
     primer_bind     complement(4734..4755)
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     4946..4965
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     terminator      complement(5117..5144)
                     /label=T7Te terminator
                     /note="phage T7 early transcription terminator"
     rep_origin      5214..5759
                     /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     5660..5679
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     promoter        join(5999..6004,6005..6022,6023..6029)
                     /label=lac UV5 promoter
                     /note="E. coli lac promoter with an ""up"" mutation"
     protein_bind    6037..6053
                     /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     6061..6077
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     6061..6077
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
ORIGIN
        1 tggcagcatc acccgacgca ctttgcgccg aataaatacc tgtgacggaa gatcacttcg
       61 cagaataaat aaatcctggt gtccctgttg ataccgggaa gccctgggcc aacttttggc
      121 gaaaatgaga cgttgatcgg cacgtaagag gttccaactt tcaccataat gaaataagat
      181 cactaccggg cgtatttttt gagttatcga gattttcagg agctaaggaa gctaaaatgg
      241 agaaaaaaat cactggatat accaccgttg atatatccca atggcatcgt aaagaacatt
      301 ttgaggcatt tcagtcagtt gctcaatgta cctataacca gaccgttcag ctggatatta
      361 cggccttttt aaagaccgta aagaaaaata agcacaagtt ttatccggcc tttattcaca
      421 ttcttgcccg cctgatgaat gctcatccgg aattccgtat ggcaatgaaa gacggtgagc
      481 tggtgatatg ggatagtgtt cacccttgtt acaccgtttt ccatgagcaa actgaaacgt
      541 tttcatcgct ctggagtgaa taccacgacg atttccggca gtttctacac atatattcgc
      601 aagatgtggc gtgttacggt gaaaacctgg cctatttccc taaagggttt attgagaata
      661 tgtttttcgt ctcagccaat ccctgggtga gtttcaccag ttttgattta aacgtggcca
      721 atatggacaa cttcttcgcc cccgttttca ccatgggcaa atattatacg caaggcgaca
      781 aggtgctgat gccgctggcg attcaggttc atcatgccgt ttgtgatggc ttccatgtcg
      841 gcagaatgct taatgaatta caacagtact gcgatgagtg gcagggcggg gcgtaatttt
      901 tttaaggcag ttattggtgc ccttaaacgc ctggttgcta cgcctgaata agtgataata
      961 agcggatgaa tggcagaaat tcgaaagcaa attcgacccg gtcgtcggtt cagggcaggg
     1021 tcgttaaata gccgcttatg tctattgctg gtttaccggt ttattgacta ccggaagcag
     1081 tgtgaccgtg tgcttctcaa atgcctgagg ccagtttgct caggctctcc ccgtggaggt
     1141 aataattgac gatatgatca tttattctgc ctcccagagc ctgataaaaa cggttagcgc
     1201 ttcgttaata cagatgtagg tgttccacag ggtagccagc agcatcctgc gatgcagatc
     1261 tgtaaggtaa gagctcttat cagttatccc aattgatatg gaagcgggta acacgcggat
     1321 tggatgccag aatgttgcgt tgcagctgac taaaatccgg tttcatcggc ggattttcac
     1381 gcagttccgg gttcaggtat tcattgttaa cgtaataacc cacgcggatg aattcctggc
     1441 cgtgatacgt gcaggtaatc agaaccaccg tgacaccaac tgcgtcggtt tccggaatca
     1501 gcgacgggtt cggagcatct gcttggaaaa caaacatatg acgacctgcc gggaccgggc
     1561 ccaccaggac tgagtccagg atctgatcaa attcttcaga ttccgcgctg cccacataga
     1621 taattttcca ttccaggtca tcggccagcg cttcggaaca ttcaaacgag atttcaaagc
     1681 ggaacgggct atggaacggg gacggatttt ccagcactgc cacattcagc accgagactt
     1741 tcgccattgc ggtaccgcca aacgtctctt caggccactg actagcgata actttcccca
     1801 caacggaaca actctcattg catgggatca ttgggtactg tgggtttagt ggttgtaaaa
     1861 acacctgacc gctatccctg atcagtttct tgaaggtaaa ctcatcaccc ccaagtctgg
     1921 ctatgcagaa atcacctggc tcaacagcct gctcagggtc aacgagaatt aacattccgt
     1981 caggaaagct tggcttggag cctgttggtg cggtcatgga attaccttca acctcaagcc
     2041 agaatgcaga atcactggct tttttggttg tgcttaccca tctctccgca tcacctttgg
     2101 taaaggttct aagctcaggt gagaacatcc ctgcctgaac atgagaaaaa acagggtact
     2161 catactcact tctaagtgac ggctgcatac taaccgcttc atacatctcg tagatttctc
     2221 tggcgattga agggctaaat tcttcaacgc taactttgag aatttttgca agcaatgcgg
     2281 cgttataagc atttaatgca ttgatgccat taaataaagc accaacgcct gactgcccca
     2341 tccccatctt gtctgcgaca gacggctggg ataagccaag ttcatttttc tttttttcat
     2401 aaattgcttt aaggcgacgt gcgtcctcaa gctgctcttg tgttaatggt ttcttttttg
     2461 tgctcatacg ctgtttcctg tgtgaaattg ttatccgctc acaattccac acattatacg
     2521 agccggaagc ataaagtgta aagcctgggg tgcctaatga gtgagaatta gcttatcgat
     2581 gataagctgt caaacatgag aattacaact tatatcgtat ggggctgact tcaggtgcta
     2641 catttgaaga gataaattgc actgaaatct agatgtctcc agaccgctag tagacgagtc
     2701 catgtgctgg cgttcaaatt tcgcagcagc ggtttcttta ccagactcga gagcgaggct
     2761 cttcaagctc gcaaaattgc tcttcaagct cgcaaaaaac cccgcccctg acagggcggg
     2821 gttttttcgc gttcgatccc gcgaaattta attaaccaac accgccagag atacattagg
     2881 caccccaggc ttgacacttt atgcttccgg ctcggatact gtgtggaatt gaaagaggaa
     2941 cgtacgatgg tcagtaaagg cgaagagttg tttacgggtg tagtaccgat tttagtggaa
     3001 ctcgatggag atgtgaatgg gcataaattt tcggtttcag gtgaaggtga aggagatgcc
     3061 acctatggca aactgacgct gaagttcatc tgtacgacag gcaaactgcc tgttccgtgg
     3121 ccaacactgg ttactacgct gacctatggc gttcaatgct tttcgcgtta tccggaccac
     3181 atgaaacagc atgacttctt taaatccgcg atgcctgaag gttacgtaca agaacgcacc
     3241 atcttcttca aagatgacgg gaactacaaa acccgtgcag aggtcaaatt tgagggtgac
     3301 acccttgtga atcggattga gctgaaaggc attgacttta aagaagatgg caacattctg
     3361 ggtcataaac tggagtacaa ctacaacagc cataacgtgt atatcatggc ggacaaacag
     3421 aagaatggga tcaaagtgaa tttcaagatt cgccacaaca ttgaagatgg tagtgtgcag
     3481 ttagccgacc actatcagca gaataccccg attggcgatg gcccagtctt gctgccggat
     3541 aatcattacc ttagcaccca atctgcgtta agcaaagatc ccaacgaaaa gcgcgatcac
     3601 atggtcctcc tggaatttgt gactgctgca ggcatcactt tgggtatgga tgaactgtat
     3661 aagtgaaatt cctggtccag gaatttcaca tattcaacgg gaaacgtctt gctctaggcc
     3721 gcgattaaat tccaacatgg atgctgattt atatgggtat aaatgggctc gcgataatgt
     3781 cgggcaatca ggtgcgacaa tctatcgatt gtatgggaag cccgatgcgc cagagttgtt
     3841 tctgaaacat ggcaaaggta gcgttgccaa tgatgttaca gatgagatgg tcagactaaa
     3901 ctggctgacg gaatttatgc ctcttccgac catcaagcat tttatccgta ctcctgatga
     3961 tgcatggtta ctcaccactg cgatccccgg gaaaacagca ttccaggtat tagaagaata
     4021 tcctgattca ggtgaaaata ttgttgatgc gctggcagtg ttcctgcgcc ggttgcattc
     4081 gattcctgtt tgtaattgtc cttttaacag cgatcgcgta tttcgtctcg ctcaggcgca
     4141 atcacgaatg aataacggtt tggttgatgc gagtgatttt gatgacgagc gtaatggctg
     4201 gcctgttgaa caagtctgga aagaaatgca taaacttttg ccattctcac cggattcagt
     4261 cgtcactcat ggtgatttct cacttgataa ccttattttt gacgagggga aattaatagg
     4321 ttgtattgat gttggacgag tcggaatcgc agaccgatac caggatcttg ccatcctatg
     4381 gaactgcctc ggtgagtttt ctccttcatt acagaaacgg ctttttcaaa aatatggtat
     4441 tgataatcct gatatgaata aattgcagtt tcatttgatg ctcgatgagt ttttctaaga
     4501 ctagtgaatt aattcatgag cggatacata tttgaatgta tttagaaaaa taaacaaata
     4561 ggggttccgc gcacatttcc ccgaaaagtg ccacctaaat tgtaagcgtt aatattttgt
     4621 taaaattcgc gttaaatttt tgttaaatca gctcattttt taaccaatag gccgaaatcg
     4681 gcaaaatccc ttataaatca aaagaataga ccgagatagg gttgagtgtt gttccagttt
     4741 ggaacaagag tccactatta aagaacgtgg actccaacgt caaagggcga aaaaccgtct
     4801 atcagggcga tggcccacta cgtgaaccat caccctaatc aagttttttg gggtcgaggt
     4861 gccgtaaagc actaaattgg aaccctaaag ggagcccccg atttagagct tgacggggaa
     4921 agccggcgaa cgtggcgaga aaggaaggga agaaagcgaa aggagcgggc gctagggcgc
     4981 tggcaagtgt agcggtcacg ctgcgcgtaa ccaccacacc cgccgcgctt aatgcgccgc
     5041 tacagggcgc agatctaaat aataaaaaag ccggattaat aatctggctt tttatattct
     5101 ctctctagta tataaacgca gaaaggccca cccgaaggtg agccagtgtg acgaaccccc
     5161 gcctcagagt gggaaagcta gaaatatttt atctgattaa taagatgatc ttcttgagat
     5221 cgttttggtc tgcgcgtaat ctcttgctct gaaaacgaaa aaaccgcctt gcagggcggt
     5281 ttttcgaagg ttctctgagc taccaactct ttgaaccgag gtaactggct tggaggagcg
     5341 cagtcaccaa aacttgtcct ttcagtttag ccttaaccgg cgcatgactt caagactaac
     5401 tcctctaaat caattaccag tggctgctgc cagtggtgct tttgcatgtc tttccgggtt
     5461 ggactcaaga cgatagttac cggataaggc gcagcggtcg gactgaacgg ggggttcgtg
     5521 catacagtcc agcttggagc gaactgccta cccggaactg agtgtcaggc gtggaatgag
     5581 acaaacgcgg ccataacagc ggaatgacac cggtaaaccg aaaggcagga acaggagagc
     5641 gcacgaggga gccgccaggg ggaaacgcct ggtatcttta tagtcctgtc gggtttcgcc
     5701 accactgatt tgagcgtcag atttcgtgat gcttgtcagg ggggcggagc ctatggaaaa
     5761 acggctttgc cgcggccctc tcacttccct gttaagtatc ttcctggcat cttccaggaa
     5821 atctccgccc cgttcgtaag ccatttccgc tctccgcagt cgaacgaccg agcgtagcga
     5881 gtcagtgcga acgatcaaaa ataagtgcct tcccatcaaa aaaatattga caacataaaa
     5941 aactttgtgt tatacttgta acgctacatg gagattaact caatctagct agagaggctt
     6001 tacactttat gcttccggct cgtataatgt gtggaattgt gagcggataa caatttcaca
     6061 caggaaacag ctatgaccat gattacggat tcactggaac tctagaccaa agagaggaca
     6121 caatgcaggg ttctgtgaca gagtttctaa aaccgcgcct ggttgatatc gagcaagtga
     6181 gttcgacgca cgccaaggtg acccttgagc ctttagagcg tggctttggc catactctgg
     6241 gtaacgcact gcgccgtatt ctgctctcat cgatgccggg ttgcgcggtg accgaggttg
     6301 agattgatgg tgtactacat gagtacagca ccaaagaagg cgttcaggaa gatatcctgg
     6361 aaatcctgct caacctgaaa gggctggcgg tgagagttca gggcaaagat gaagttattc
     6421 ttaccttgaa taaatctggc attggccctg tgactgcagc cgatatcacc cacgacggtg
     6481 atgtcgaaat cgtcaagccg cagcacgtga tctgccacct gaccgatgag aacgcgtcta
     6541 ttagcatgcg tatcaaagtt cagcgcggtc gtggttatgt gccggcttct acccgaattc
     6601 attcggaaga agatgagcgc ccaatcggcc gtctgctggt cgacgcatgc tacagccctg
     6661 tggagcgtat tgcctacaat gttgaagcag cgcgtgtaga acagcgtacc gacctggaca
     6721 agctggtcat cgaaatggaa accaacggca caatcgatcc tgaagaggcg attcgtcgtg
     6781 cggcaaccat tctggctgaa caactggaag ctttcgttga cttacgtgat gtacgtcagc
     6841 ctgaagtgaa agaagagaaa ccagaggcgg ccgcagcaag caccgaaaga aaatgggcag
     6901 aagcagcacg tcgtgcacgt ggtgccggtg gtgttaccct caatggtgca ggttgaggat
     6961 cctctacgcc ggacgcactc ggttccaaaa aaaaaaaaaa agacgctgaa aagcgtcttt
     7021 tttttttttg gtccccactg gagcacctca aaaacaccat catacactaa atcagtaagt
//
