LOCUS       sequence-469072-        7080 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 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: VN990 pB2Hws_v9_cI-Asf1B+rpoA-IP3
FEATURES             Location/Qualifiers
     source          1..7080
                     /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    2487..2503
                     /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(2511..2517,2518..2535,2536..2541))
                     /label=lac UV5 promoter
                     /note="E. coli lac promoter with an ""up"" mutation"
     CDS             complement(2694..2738)
                     /codon_start=1
                     /product="affinity and epitope tag derived from pancreatic 
                     ribonuclease A"
                     /label=S-Tag
                     /translation="KETAAAKFERQHMDS"
     CDS             join(2946..2948,2949..2951,2952..3662)
                     /codon_start=1
                     /product="enhanced GFP"
                     /label=EGFP
                     /translation="MVSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTL
                     KFICTTGKLPVPWPTLVTTLTYGVQCFSRYPDHMKQHDFFKSAMPEGYVQERTIFFKDD
                     GNYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIK
                     VNFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSALSKDPNEKRDHMVLL
                     EFVTAAGITLGMDELYK"
     CDS             3685..4497
                     /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             3685..4497
                     /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(3755..3774)
                     /label=Kan-R
                     /note="Kanamycin resistance gene, reverse primer"
     primer_bind     complement(4733..4754)
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     4945..4964
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     terminator      complement(5116..5143)
                     /label=T7Te terminator
                     /note="phage T7 early transcription terminator"
     rep_origin      5213..5758
                     /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     5659..5678
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     promoter        join(5998..6003,6004..6021,6022..6028)
                     /label=lac UV5 promoter
                     /note="E. coli lac promoter with an ""up"" mutation"
     protein_bind    6036..6052
                     /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     6060..6076
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     6060..6076
                     /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 gctcatacgc tgtttcctgt gtgaaattgt tatccgctca caattccaca cattatacga
     2521 gccggaagca taaagtgtaa agcctggggt gcctaatgag tgagaattag cttatcgatg
     2581 ataagctgtc aaacatgaga attacaactt atatcgtatg gggctgactt caggtgctac
     2641 atttgaagag ataaattgca ctgaaatcta gatgtctcca gaccgctagt agacgagtcc
     2701 atgtgctggc gttcaaattt cgcagcagcg gtttctttac cagactcgag agcgaggctc
     2761 ttcaagctcg caaaattgct cttcaagctc gcaaaaaacc ccgcccctga cagggcgggg
     2821 ttttttcgcg ttcgatcccg cgaaatttaa ttaaccaaca ccgccagaga tacattaggc
     2881 accccaggct tgacacttta tgcttccggc tcggatactg tgtggaattg aaagaggaac
     2941 gtacgatggt cagtaaaggc gaagagttgt ttacgggtgt agtaccgatt ttagtggaac
     3001 tcgatggaga tgtgaatggg cataaatttt cggtttcagg tgaaggtgaa ggagatgcca
     3061 cctatggcaa actgacgctg aagttcatct gtacgacagg caaactgcct gttccgtggc
     3121 caacactggt tactacgctg acctatggcg ttcaatgctt ttcgcgttat ccggaccaca
     3181 tgaaacagca tgacttcttt aaatccgcga tgcctgaagg ttacgtacaa gaacgcacca
     3241 tcttcttcaa agatgacggg aactacaaaa cccgtgcaga ggtcaaattt gagggtgaca
     3301 cccttgtgaa tcggattgag ctgaaaggca ttgactttaa agaagatggc aacattctgg
     3361 gtcataaact ggagtacaac tacaacagcc ataacgtgta tatcatggcg gacaaacaga
     3421 agaatgggat caaagtgaat ttcaagattc gccacaacat tgaagatggt agtgtgcagt
     3481 tagccgacca ctatcagcag aataccccga ttggcgatgg cccagtcttg ctgccggata
     3541 atcattacct tagcacccaa tctgcgttaa gcaaagatcc caacgaaaag cgcgatcaca
     3601 tggtcctcct ggaatttgtg actgctgcag gcatcacttt gggtatggat gaactgtata
     3661 agtgaaattc ctggtccagg aatttcacat attcaacggg aaacgtcttg ctctaggccg
     3721 cgattaaatt ccaacatgga tgctgattta tatgggtata aatgggctcg cgataatgtc
     3781 gggcaatcag gtgcgacaat ctatcgattg tatgggaagc ccgatgcgcc agagttgttt
     3841 ctgaaacatg gcaaaggtag cgttgccaat gatgttacag atgagatggt cagactaaac
     3901 tggctgacgg aatttatgcc tcttccgacc atcaagcatt ttatccgtac tcctgatgat
     3961 gcatggttac tcaccactgc gatccccggg aaaacagcat tccaggtatt agaagaatat
     4021 cctgattcag gtgaaaatat tgttgatgcg ctggcagtgt tcctgcgccg gttgcattcg
     4081 attcctgttt gtaattgtcc ttttaacagc gatcgcgtat ttcgtctcgc tcaggcgcaa
     4141 tcacgaatga ataacggttt ggttgatgcg agtgattttg atgacgagcg taatggctgg
     4201 cctgttgaac aagtctggaa agaaatgcat aaacttttgc cattctcacc ggattcagtc
     4261 gtcactcatg gtgatttctc acttgataac cttatttttg acgaggggaa attaataggt
     4321 tgtattgatg ttggacgagt cggaatcgca gaccgatacc aggatcttgc catcctatgg
     4381 aactgcctcg gtgagttttc tccttcatta cagaaacggc tttttcaaaa atatggtatt
     4441 gataatcctg atatgaataa attgcagttt catttgatgc tcgatgagtt tttctaagac
     4501 tagtgaatta attcatgagc ggatacatat ttgaatgtat ttagaaaaat aaacaaatag
     4561 gggttccgcg cacatttccc cgaaaagtgc cacctaaatt gtaagcgtta atattttgtt
     4621 aaaattcgcg ttaaattttt gttaaatcag ctcatttttt aaccaatagg ccgaaatcgg
     4681 caaaatccct tataaatcaa aagaatagac cgagataggg ttgagtgttg ttccagtttg
     4741 gaacaagagt ccactattaa agaacgtgga ctccaacgtc aaagggcgaa aaaccgtcta
     4801 tcagggcgat ggcccactac gtgaaccatc accctaatca agttttttgg ggtcgaggtg
     4861 ccgtaaagca ctaaattgga accctaaagg gagcccccga tttagagctt gacggggaaa
     4921 gccggcgaac gtggcgagaa aggaagggaa gaaagcgaaa ggagcgggcg ctagggcgct
     4981 ggcaagtgta gcggtcacgc tgcgcgtaac caccacaccc gccgcgctta atgcgccgct
     5041 acagggcgca gatctaaata ataaaaaagc cggattaata atctggcttt ttatattctc
     5101 tctctagtat ataaacgcag aaaggcccac ccgaaggtga gccagtgtga cgaacccccg
     5161 cctcagagtg ggaaagctag aaatatttta tctgattaat aagatgatct tcttgagatc
     5221 gttttggtct gcgcgtaatc tcttgctctg aaaacgaaaa aaccgccttg cagggcggtt
     5281 tttcgaaggt tctctgagct accaactctt tgaaccgagg taactggctt ggaggagcgc
     5341 agtcaccaaa acttgtcctt tcagtttagc cttaaccggc gcatgacttc aagactaact
     5401 cctctaaatc aattaccagt ggctgctgcc agtggtgctt ttgcatgtct ttccgggttg
     5461 gactcaagac gatagttacc ggataaggcg cagcggtcgg actgaacggg gggttcgtgc
     5521 atacagtcca gcttggagcg aactgcctac ccggaactga gtgtcaggcg tggaatgaga
     5581 caaacgcggc cataacagcg gaatgacacc ggtaaaccga aaggcaggaa caggagagcg
     5641 cacgagggag ccgccagggg gaaacgcctg gtatctttat agtcctgtcg ggtttcgcca
     5701 ccactgattt gagcgtcaga tttcgtgatg cttgtcaggg gggcggagcc tatggaaaaa
     5761 cggctttgcc gcggccctct cacttccctg ttaagtatct tcctggcatc ttccaggaaa
     5821 tctccgcccc gttcgtaagc catttccgct ctccgcagtc gaacgaccga gcgtagcgag
     5881 tcagtgcgaa cgatcaaaaa taagtgcctt cccatcaaaa aaatattgac aacataaaaa
     5941 actttgtgtt atacttgtaa cgctacatgg agattaactc aatctagcta gagaggcttt
     6001 acactttatg cttccggctc gtataatgtg tggaattgtg agcggataac aatttcacac
     6061 aggaaacagc tatgaccatg attacggatt cactggaact ctagaccaaa gagaggacac
     6121 aatgcagggt tctgtgacag agtttctaaa accgcgcctg gttgatatcg agcaagtgag
     6181 ttcgacgcac gccaaggtga cccttgagcc tttagagcgt ggctttggcc atactctggg
     6241 taacgcactg cgccgtattc tgctctcatc gatgccgggt tgcgcggtga ccgaggttga
     6301 gattgatggt gtactacatg agtacagcac caaagaaggc gttcaggaag atatcctgga
     6361 aatcctgctc aacctgaaag ggctggcggt gagagttcag ggcaaagatg aagttattct
     6421 taccttgaat aaatctggca ttggccctgt gactgcagcc gatatcaccc acgacggtga
     6481 tgtcgaaatc gtcaagccgc agcacgtgat ctgccacctg accgatgaga acgcgtctat
     6541 tagcatgcgt atcaaagttc agcgcggtcg tggttatgtg ccggcttcta cccgaattca
     6601 ttcggaagaa gatgagcgcc caatcggccg tctgctggtc gacgcatgct acagccctgt
     6661 ggagcgtatt gcctacaatg ttgaagcagc gcgtgtagaa cagcgtaccg acctggacaa
     6721 gctggtcatc gaaatggaaa ccaacggcac aatcgatcct gaagaggcga ttcgtcgtgc
     6781 ggcaaccatt ctggctgaac aactggaagc tttcgttgac ttacgtgatg tacgtcagcc
     6841 tgaagtgaaa gaagagaaac cagaggcggc cgcagcaagc accgaacgca aatgggcaga
     6901 actggcacgt cgtattcgtg gtgcaggcgg tgttaccctc aatggttttg gttgaggatc
     6961 ctctacgccg gacgcactcg gttccaaaaa aaaaaaaaaa gacgctgaaa agcgtctttt
     7021 ttttttttgg tccccactgg agcacctcaa aaacaccatc atacactaaa tcagtaagtt
//
