LOCUS       sequence-469387-        6143 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Quantitative bacterial two-hybrid (qB2H). Expresses Asf1B. In fusion
            cloning to rpoA using BsaI based golden gate..
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 6143)
  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 6143)
  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: VN1897
            pB2Hws_v28_Ddcm_wkKanR_TIR51_pLWeakRBS_...
FEATURES             Location/Qualifiers
     source          1..6143
                     /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             2802..3617
                     /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             2802..3617
                     /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(3853..3874)
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     4065..4084
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     terminator      complement(4236..4263)
                     /label=T7Te terminator
                     /note="phage T7 early transcription terminator"
     rep_origin      4333..4878
                     /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     4779..4798
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     promoter        join(5118..5123,5124..5141,5142..5148)
                     /label=lac UV5 promoter
                     /note="E. coli lac promoter with an ""up"" mutation"
     protein_bind    5156..5172
                     /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     5161..5183
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     primer_bind     5180..5196
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     5180..5196
                     /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 gatttgacag cggcaggtac gatgagccat attcaacggg
     2821 aaacgtcttg ctctaggccg cgattaaatt ccaacatgga tgctgattta tatgggtata
     2881 aatgggcccg cgataatgtc gggcaatcag gtgcgacaat ctatcgattg tatgggaagc
     2941 ccgatgcgcc agagttgttt ctgaaacatg gcaaaggtag cgttgccaat gatgttacag
     3001 atgagatggt cagactaaac tggctgacgg aatttatgcc tcttccgacc atcaagcatt
     3061 ttatccgtac tcctgatgat gcatggttac tcaccactgc gatccccggg aaaacagcat
     3121 tccaggtatt agaagaatat cctgattcag gtgaaaatat tgttgatgcg ctggcagtgt
     3181 tcctgcgccg gttgcattcg attcctgttt gtaattgtcc ttttaacagc gatcgcgtat
     3241 ttcgtctcgc tcaggcgcaa tcacgaatga ataacggttt ggttgatgcg agtgattttg
     3301 atgacgagcg taatggctgg cctgttgaac aagtctggaa agaaatgcat aaacttttgc
     3361 cattctcacc ggattcagtc gtcactcatg gtgatttctc acttgataac cttatttttg
     3421 acgaggggaa attaataggt tgtattgatg ttggacgagt cggaatcgca gaccgatacc
     3481 aggatcttgc catcctatgg aactgcctcg gtgagttttc tccttcatta cagaaacggc
     3541 tttttcaaaa atatggtatt gataatcctg atatgaataa attgcagttt catttgatgc
     3601 tcgatgagtt tttctaagac tagtgaatta attcatgagc ggatacatat ttgaatgtat
     3661 ttagaaaaat aaacaaatag gggttccgcg cacatttccc cgaaaagtgc cacctaaatt
     3721 gtaagcgtta atattttgtt aaaattcgcg ttaaattttt gttaaatcag ctcatttttt
     3781 aaccaatagg ccgaaatcgg caaaatccct tataaatcaa aagaatagac cgagataggg
     3841 ttgagtgttg ttccagtttg gaacaagagt ccactattaa agaacgtgga ctccaacgtc
     3901 aaagggcgaa aaaccgtcta tcagggcgat ggcccactac gtgaaccatc accctaatca
     3961 agttttttgg ggtcgaggtg ccgtaaagca ctaaattgga accctaaagg gagcccccga
     4021 tttagagctt gacggggaaa gccggcgaac gtggcgagaa aggaagggaa gaaagcgaaa
     4081 ggagcgggcg ctagggcgct ggcaagtgta gcggtcacgc tgcgcgtaac caccacaccc
     4141 gccgcgctta atgcgccgct acagggcgca gatctaaata ataaaaaagc cggattaata
     4201 atctggcttt ttatattctc tctctagtat ataaacgcag aaaggcccac ccgaaggtga
     4261 gccagtgtga cgaacccccg cctcagagtg ggaaagctag aaatatttta tctgattaat
     4321 aagatgatct tcttgagatc gttttggtct gcgcgtaatc tcttgctctg aaaacgaaaa
     4381 aaccgccttg cagggcggtt tttcgaaggt tctctgagct accaactctt tgaaccgagg
     4441 taactggctt ggaggagcgc agtcaccaaa acttgtcctt tcagtttagc cttaaccggc
     4501 gcatgacttc aagactaact cctctaaatc aattaccagt ggctgctgcc agtggtgctt
     4561 ttgcatgtct ttccgggttg gactcaagac gatagttacc ggataaggcg cagcggtcgg
     4621 actgaacggg gggttcgtgc atacagtcca gcttggagcg aactgcctac ccggaactga
     4681 gtgtcaggcg tggaatgaga caaacgcggc cataacagcg gaatgacacc ggtaaaccga
     4741 aaggcaggaa caggagagcg cacgagggag ccgccagggg gaaacgcctg gtatctttat
     4801 agtcctgtcg ggtttcgcca ccactgattt gagcgtcaga tttcgtgatg cttgtcaggg
     4861 gggcggagcc tatggaaaaa cggctttgcc gcggccctct cacttccctg ttaagtatct
     4921 tcctggcatc ttccaggaaa tctccgcccc gttcgtaagc catttccgct ctccgcagtc
     4981 gaacgaccga gcgtagcgag tcagtgcgaa cgatcaaaaa taagtgcctt cccatcaaaa
     5041 aaatattgac aacataaaaa actttgtgtt atacttgtaa cgctacatgg agattaactc
     5101 aatctagcta gagaggcttt acactttatg cttccggctc gtataatgtg tggaattgtg
     5161 agcggataac aatttcacac aggaaacagc tatgaccatg attacggatt cactggaact
     5221 ctagaccaaa gagaggacac aatgcagggt tctgtgacag agtttctaaa accgcgcctg
     5281 gttgatatcg agcaagtgag ttcgacgcac gccaaggtga cccttgagcc tttagagcgt
     5341 ggctttggcc atactctggg taacgcactg cgccgtattc tgctctcatc gatgccgggt
     5401 tgcgcggtga ccgaggttga gattgatggt gtactacatg agtacagcac caaagaaggc
     5461 gttcaggaag atatcctgga aatcctgctc aacctgaaag ggctggcggt gagagttcag
     5521 ggcaaagatg aagttattct taccttgaat aaatctggca ttggccctgt gactgcagcc
     5581 gatatcaccc acgacggtga tgtcgaaatc gtcaagccgc agcacgtgat ctgccacctg
     5641 accgatgaga acgcgtctat tagcatgcgt atcaaagttc agcgcggtcg tggttatgtg
     5701 ccggcttcta cccgaattca ttcggaagaa gatgagcgcc caatcggccg tctgctggtc
     5761 gacgcatgct acagccctgt ggagcgtatt gcctacaatg ttgaagcagc gcgtgtagaa
     5821 cagcgtaccg acctggacaa gctggtcatc gaaatggaaa ccaacggcac aatcgatcct
     5881 gaagaggcga ttcgtcgtgc ggcaaccatt ctggctgaac aactggaagc tttcgttgac
     5941 ttacgtgatg tacgtcagcc tgaagtgaaa gaagagaaac cagaggcagc atgagaccat
     6001 cgatcgatgg tctcgaatag atcctctacg ccggacgcac tcggttccaa aaaaaaaaaa
     6061 aaagacgctg aaaagcgtct tttttttttt tggtccccac tggagcacct caaaaacacc
     6121 atcatacact aaatcagtaa gtt
//
