LOCUS       sequence-469039-        6156 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Quantitative bacterial two-hybrid (qB2H). Expresses Asf1A..
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 6156)
  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 6156)
  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: VN550 pAC-cI_E34P-Asf1A_Term_f1-pOL2-62_L-eGFP...
FEATURES             Location/Qualifiers
     source          1..6156
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     CDS             complement(1302..2012)
                     /codon_start=1
                     /gene="cI"
                     /product="phage lambda repressor"
                     /label=lambda repressor
                     /translation="MSTKKKPLTQEQLEDARRLKAIYEKKKNELGLSQPSVADKMGMGQ
                     SGVGALFNGINALNAYNAALLAKILKVSVEEFSPSIAREIYEMYEAVSMQPSLRSEYEY
                     PVFSHVQAGMFSPELRTFTKGDAERWVSTTKKASDSAFWLEVEGNSMTAPTGSKPSFPD
                     GMLILVDPEQAVEPGDFCIARLGGDEFTFKKLIRDSGQVFLQPLNPQYPMIPCNESCSV
                     VGKVIASQWPEETFG"
     primer_bind     complement(2022..2044)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    2033..2049
                     /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(2057..2063,2064..2081,2082..2087))
                     /label=lac UV5 promoter
                     /note="E. coli lac promoter with an ""up"" mutation"
     CDS             complement(2240..2284)
                     /codon_start=1
                     /product="affinity and epitope tag derived from pancreatic 
                     ribonuclease A"
                     /label=S-Tag
                     /translation="KETAAAKFERQHMDS"
     CDS             join(2492..2494,2495..2497,2498..3208)
                     /codon_start=1
                     /product="enhanced GFP"
                     /label=EGFP
                     /translation="MVSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTL
                     KFICTTGKLPVPWPTLVTTLTYGVQCFSRYPDHMKQHDFFKSAMPEGYVQERTIFFKDD
                     GNYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIK
                     VNFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSALSKDPNEKRDHMVLL
                     EFVTAAGITLGMDELYK"
     CDS             3240..4055
                     /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             3240..4055
                     /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(3313..3332)
                     /label=Kan-R
                     /note="Kanamycin resistance gene, reverse primer"
     primer_bind     complement(4284..4305)
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     4496..4515
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     terminator      complement(4667..4694)
                     /label=T7Te terminator
                     /note="phage T7 early transcription terminator"
     rep_origin      4764..5309
                     /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     5210..5229
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     5426..5443
                     /label=L4440
                     /note="L4440 vector, forward primer"
     promoter        5835..5937
                     /label=cat promoter
                     /note="promoter of the E. coli cat gene encoding 
                     chloramphenicol acetyltransferase"
     CDS             join(5938..6156,1..441)
                     /codon_start=1
                     /gene="cat"
                     /product="chloramphenicol acetyltransferase"
                     /label=CmR
                     /note="confers resistance to chloramphenicol"
                     /translation="MEKKITGYTTVDISQWHRKEHFEAFQSVAQCTYNQTVQLDITAFL
                     KTVKKNKHKFYPAFIHILARLMNAHPEFRMAMKDGELVIWDSVHPCYTVFHEQTETFSS
                     LWSEYHDDFRQFLHIYSQDVACYGENLAYFPKGFIENMFFVSANPWVSFTSFDLNVANM
                     DNFFAPVFTMGKYYTQGDKVLMPLAIQVHHAVCDGFHVGRMLNELQQYCDEWQGGA"
     primer_bind     complement(6004..6023)
                     /label=CAT-R
                     /note="Chloramphenicol resistance gene, reverse primer"
ORIGIN
        1 cgtatggcaa tgaaagacgg tgagctggtg atatgggata gtgttcaccc ttgttacacc
       61 gttttccatg agcaaactga aacgttttca tcgctctgga gtgaatacca cgacgatttc
      121 cggcagtttc tacacatata ttcgcaagat gtggcgtgtt acggtgaaaa cctggcctat
      181 ttccctaaag ggtttattga gaatatgttt ttcgtctcag ccaatccctg ggtgagtttc
      241 accagttttg atttaaacgt ggccaatatg gacaacttct tcgcccccgt tttcaccatg
      301 ggcaaatatt atacgcaagg cgacaaggtg ctgatgccgc tggcgattca ggttcatcat
      361 gccgtttgtg atggcttcca tgtcggcaga atgcttaatg aattacaaca gtactgcgat
      421 gagtggcagg gcggggcgta atttttttaa ggcagttatt ggtgccctta aacgcctggt
      481 tgctacgcct gaataagtga taataagcgg atgaatggca gaaattcgaa agcaaattcg
      541 acccggtcgt cggttcaggg cagggtcgtt aaatagccgc ttatgtctat tgctggttta
      601 ccggtttatt gactaccgga agcagtgtga ccgtgtgctt ctcaaatgcc tgaggccagt
      661 ttgctcaggc tctccccgtg gaggtaataa ttgacgatat gatcatttat tctgcctccc
      721 agagcctgat aaaaacggtt agcgcttcgt taatacagat gtaggtgttc cacagggtag
      781 ccagcagcat cctgcgatgc agatctgtaa ggtaaggatc cttatcagtt atcttcccaa
      841 ttaatgtgga atcttgtgac cctgggatta gatgccaaaa tattcctttg aagcttagaa
      901 aagtctggtt ttactggtgg attttccctt aattctgtct cagtatattc attatttaca
      961 taatagccaa ctctaataaa ttcttgtcct cgataggtac aagtaattag cacaacagtt
     1021 acgcctactg catctgcatc tggaatgagt cctggattag gtgcatcagc ctgaaataca
     1081 aacatatgcc ttcctgcggg aacaggaccc actaaaacag agtctaaaac ttgatcgtat
     1141 tcttcacttt ctgcagagcc cacatagata attttccatt ccaagtcttc agacaggtcc
     1201 tcgatgcact cgaaggtgat ctcgaactgg aacgggttgt agaaaggaga agggttatcc
     1261 agcaccacta cattgttcac ctgaaccttt gctgcggccg cgccaaacgt ctcttcaggc
     1321 cactgactag cgataacttt ccccacaacg gaacaactct cattgcatgg gatcattggg
     1381 tactgtgggt ttagtggttg taaaaacacc tgaccgctat ccctgatcag tttcttgaag
     1441 gtaaactcat cacccccaag tctggctatg cagaaatcac ctggctcaac agcctgctca
     1501 gggtcaacga gaattaacat tccgtcagga aagcttggct tggagcctgt tggtgcggtc
     1561 atggaattac cttcaacctc aagccagaat gcagaatcac tggctttttt ggttgtgctt
     1621 acccatctct ccgcatcacc tttggtaaag gttctaagct caggtgagaa catccctgcc
     1681 tgaacatgag aaaaaacagg gtactcatac tcacttctaa gtgacggctg catactaacc
     1741 gcttcataca tctcgtagat ttctctggcg attgaagggc taaattcttc aacgctaact
     1801 ttgagaattt ttgcaagcaa tgcggcgtta taagcattta atgcattgat gccattaaat
     1861 aaagcaccaa cgcctgactg ccccatcccc atcttgtctg cgacagacgg ctgggataag
     1921 ccaagttcat ttttcttttt ttcataaatt gctttaaggc gacgtgcgtc ctcaagctgc
     1981 tcttgtgtta atggtttctt ttttgtgctc atacgctgtt tcctgtgtga aattgttatc
     2041 cgctcacaat tccacacatt atacgagccg gaagcataaa gtgtaaagcc tggggtgcct
     2101 aatgagtgag aattagctta tcgatgataa gctgtcaaac atgagaatta caacttatat
     2161 cgtatggggc tgacttcagg tgctacattt gaagagataa attgcactga aatctagatg
     2221 tctccagacc gctagtagac gagtccatgt gctggcgttc aaatttcgca gcagcggttt
     2281 ctttaccaga ctcgagagcg aggctcttca agctcgcaaa attgctcttc aagctcgcaa
     2341 aaaaccccgc ccctgacagg gcggggtttt ttcgcgttcg atcccgcgaa atttaattaa
     2401 ccaacaccgc cagagataca ttaggcaccc caggcttgac actttatgct tccggctcgg
     2461 atactgtgtg gaattgaaag aggaacgtac gatggtcagt aaaggcgaag agttgtttac
     2521 gggtgtagta ccgattttag tggaactcga tggagatgtg aatgggcata aattttcggt
     2581 ttcaggtgaa ggtgaaggag atgccaccta tggcaaactg acgctgaagt tcatctgtac
     2641 gacaggcaaa ctgcctgttc cgtggccaac actggttact acgctgacct atggcgttca
     2701 atgcttttcg cgttatccgg accacatgaa acagcatgac ttctttaaat ccgcgatgcc
     2761 tgaaggttac gtacaagaac gcaccatctt cttcaaagat gacgggaact acaaaacccg
     2821 tgcagaggtc aaatttgagg gtgacaccct tgtgaatcgg attgagctga aaggcattga
     2881 ctttaaagaa gatggcaaca ttctgggtca taaactggag tacaactaca acagccataa
     2941 cgtgtatatc atggcggaca aacagaagaa tgggatcaaa gtgaatttca agattcgcca
     3001 caacattgaa gatggtagtg tgcagttagc cgaccactat cagcagaata ccccgattgg
     3061 cgatggccca gtcttgctgc cggataatca ttaccttagc acccaatctg cgttaagcaa
     3121 agatcccaac gaaaagcgcg atcacatggt cctcctggaa tttgtgactg ctgcaggcat
     3181 cactttgggt atggatgaac tgtataagta aacgcgttaa acaggaaaca gaagtccaaa
     3241 tgagccatat tcaacgggaa acgtcttgct ctaggccgcg attaaattcc aacatggatg
     3301 ctgatttata tgggtataaa tgggctcgcg ataatgtcgg gcaatcaggt gcgacaatct
     3361 atcgattgta tgggaagccc gatgcgccag agttgtttct gaaacatggc aaaggtagcg
     3421 ttgccaatga tgttacagat gagatggtca gactaaactg gctgacggaa tttatgcctc
     3481 ttccgaccat caagcatttt atccgtactc ctgatgatgc atggttactc accactgcga
     3541 tccccgggaa aacagcattc caggtattag aagaatatcc tgattcaggt gaaaatattg
     3601 ttgatgcgct ggcagtgttc ctgcgccggt tgcattcgat tcctgtttgt aattgtcctt
     3661 ttaacagcga tcgcgtattt cgtctcgctc aggcgcaatc acgaatgaat aacggtttgg
     3721 ttgatgcgag tgattttgat gacgagcgta atggctggcc tgttgaacaa gtctggaaag
     3781 aaatgcataa acttttgcca ttctcaccgg attcagtcgt cactcatggt gatttctcac
     3841 ttgataacct tatttttgac gaggggaaat taataggttg tattgatgtt ggacgagtcg
     3901 gaatcgcaga ccgataccag gatcttgcca tcctatggaa ctgcctcggt gagttttctc
     3961 cttcattaca gaaacggctt tttcaaaaat atggtattga taatcctgat atgaataaat
     4021 tgcagtttca tttgatgctc gatgagtttt tctaagaatt aattcatgag cggatacata
     4081 tttgaatgta tttagaaaaa taaacaaata ggggttccgc gcacatttcc ccgaaaagtg
     4141 ccacctaaat tgtaagcgtt aatattttgt taaaattcgc gttaaatttt tgttaaatca
     4201 gctcattttt taaccaatag gccgaaatcg gcaaaatccc ttataaatca aaagaataga
     4261 ccgagatagg gttgagtgtt gttccagttt ggaacaagag tccactatta aagaacgtgg
     4321 actccaacgt caaagggcga aaaaccgtct atcagggcga tggcccacta cgtgaaccat
     4381 caccctaatc aagttttttg gggtcgaggt gccgtaaagc actaaattgg aaccctaaag
     4441 ggagcccccg atttagagct tgacggggaa agccggcgaa cgtggcgaga aaggaaggga
     4501 agaaagcgaa aggagcgggc gctagggcgc tggcaagtgt agcggtcacg ctgcgcgtaa
     4561 ccaccacacc cgccgcgctt aatgcgccgc tacagggcgc agatctaaat aataaaaaag
     4621 ccggattaat aatctggctt tttatattct ctctctagta tataaacgca gaaaggccca
     4681 cccgaaggtg agccagtgtg acgaaccccc gcctcagagt gggaaagcta gaaatatttt
     4741 atctgattaa taagatgatc ttcttgagat cgttttggtc tgcgcgtaat ctcttgctct
     4801 gaaaacgaaa aaaccgcctt gcagggcggt ttttcgaagg ttctctgagc taccaactct
     4861 ttgaaccgag gtaactggct tggaggagcg cagtcaccaa aacttgtcct ttcagtttag
     4921 ccttaaccgg cgcatgactt caagactaac tcctctaaat caattaccag tggctgctgc
     4981 cagtggtgct tttgcatgtc tttccgggtt ggactcaaga cgatagttac cggataaggc
     5041 gcagcggtcg gactgaacgg ggggttcgtg catacagtcc agcttggagc gaactgccta
     5101 cccggaactg agtgtcaggc gtggaatgag acaaacgcgg ccataacagc ggaatgacac
     5161 cggtaaaccg aaaggcagga acaggagagc gcacgaggga gccgccaggg ggaaacgcct
     5221 ggtatcttta tagtcctgtc gggtttcgcc accactgatt tgagcgtcag atttcgtgat
     5281 gcttgtcagg ggggcggagc ctatggaaaa acggctttgc cgcggccctc tcacttccct
     5341 gttaagtatc ttcctggcat cttccaggaa atctccgccc cgttcgtaag ccatttccgc
     5401 tcgccgcagt cgaacgaccg agcgtagcga gtcagtgagc gaggaagcgg aatatatcct
     5461 gtatcacata ttctgctgac gcaccggtgc agcctttttt ctcctgccac atgaagcact
     5521 tcactgacac cctcatcagt gccaacatag taagccagta tacactccgc tagcgctgat
     5581 gtccggcggt gcttttgccg ttacgcacca ccccgtcagt agctgaacag gagggacagc
     5641 tgatagaaac agaagccact ggagcacctc aaaaacacca tcatacacta aatcagtaag
     5701 ttggcagcat cacccgacgc actttgcgcc gaataaatac ctgtgacgga agatcacttc
     5761 gcagaataaa taaatcctgg tgtccctgtt gataccggga agccctgggc caacttttgg
     5821 cgaaaatgag acgttgatcg gcacgtaaga ggttccaact ttcaccataa tgaaataaga
     5881 tcactaccgg gcgtattttt tgagttatcg agattttcag gagctaagga agctaaaatg
     5941 gagaaaaaaa tcactggata taccaccgtt gatatatccc aatggcatcg taaagaacat
     6001 tttgaggcat ttcagtcagt tgctcaatgt acctataacc agaccgttca gctggatatt
     6061 acggcctttt taaagaccgt aaagaaaaat aagcacaagt tttatccggc ctttattcac
     6121 attcttgccc gcctgatgaa tgctcatccg gaattc
//
