LOCUS       sequence-469023-        5179 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Quantitative bacterial two-hybrid (qB2H). Expresses IP3..
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 5179)
  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 5179)
  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: VN517 pBR-rpoA-IP3
FEATURES             Location/Qualifiers
     source          1..5179
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     promoter        join(235..240,241..258,259..265)
                     /label=lac UV5 promoter
                     /note="E. coli lac promoter with an ""up"" mutation"
     protein_bind    273..289
                     /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     278..300
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     primer_bind     297..313
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     297..313
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(1256..1275)
                     /label=pBRrevBam
                     /note="pBR322 vectors, tet region, downstream of BamHI, 
                     reverse primer"
     CDS             2733..2924
                     /codon_start=1
                     /gene="rop"
                     /product="Rop protein, which maintains plasmids at low copy
                     number"
                     /label=rop
                     /translation="MTKQEKTALNMARFIRSQTLTLLEKLNELDADEQADICESLHDHA
                     DELYRSCLARFGDDGENL"
     primer_bind     complement(2918..2940)
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     misc_feature    3026..3166
                     /label=bom
                     /note="basis of mobility region from pBR322"
     primer_bind     3081..3100
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     complement(3181..3198)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(3352..3940)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(3432..3451)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(4111..4902,4903..4971))
                     /codon_start=1
                     /gene="bla"
                     /product="beta-lactamase"
                     /label=AmpR
                     /note="confers resistance to ampicillin, carbenicillin, and
                     related antibiotics"
                     /translation="MSIQHFRVALIPFFAAFCLPVFAHPETLVKVKDAEDQLGARVGYI
                     ELDLNSGKILESFRPEERFPMMSTFKVLLCGAVLSRVDAGQEQLGRRIHYSQNDLVEYS
                     PVTEKHLTDGMTVRELCSAAITMSDNTAANLLLTTIGGPKELTAFLHNMGDHVTRLDRW
                     EPELNEAIPNDERDTTMPAAMATTLRKLLTGELLTLASRQQLIDWMEADKVAGPLLRSA
                     LPAGWFIADKSGAGERGSRGIIAALGPDGKPSRIVVIYTTGSQATMDERNRQIAEIGAS
                     LIKHW"
     primer_bind     4734..4753
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(4972..5076)
                     /gene="bla"
                     /label=AmpR promoter
     primer_bind     5144..5162
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
ORIGIN
        1 gcaataaaaa atcaaatcgg atttcactat ataatctcac tttatctaag atgaatccga
       61 tggaagcatc ctgttttctc tcaatttttt tatctaaaac ccagcgttcg atgcttcttt
      121 gagcgaacga tcaaaaataa gtgccttccc atcaaaaaaa tattgacaac ataaaaaact
      181 ttgtgttata cttgtaacgc tacatggaga ttaactcaat ctagctagag aggctttaca
      241 ctttatgctt ccggctcgta taatgtgtgg aattgtgagc ggataacaat ttcacacagg
      301 aaacagctat gaccatgatt acggattcac tggaactcta gaccaaagag aggacacaat
      361 gcagggttct gtgacagagt ttctaaaacc gcgcctggtt gatatcgagc aagtgagttc
      421 gacgcacgcc aaggtgaccc ttgagccttt agagcgtggc tttggccata ctctgggtaa
      481 cgcactgcgc cgtattctgc tctcatcgat gccgggttgc gcggtgaccg aggttgagat
      541 tgatggtgta ctacatgagt acagcaccaa agaaggcgtt caggaagata tcctggaaat
      601 cctgctcaac ctgaaagggc tggcggtgag agttcagggc aaagatgaag ttattcttac
      661 cttgaataaa tctggcattg gccctgtgac tgcagccgat atcacccacg acggtgatgt
      721 cgaaatcgtc aagccgcagc acgtgatctg ccacctgacc gatgagaacg cgtctattag
      781 catgcgtatc aaagttcagc gcggtcgtgg ttatgtgccg gcttctaccc gaattcattc
      841 ggaagaagat gagcgcccaa tcggccgtct gctggtcgac gcatgctaca gccctgtgga
      901 gcgtattgcc tacaatgttg aagcagcgcg tgtagaacag cgtaccgacc tggacaagct
      961 ggtcatcgaa atggaaacca acggcacaat cgatcctgaa gaggcgattc gtcgtgcggc
     1021 aaccattctg gctgaacaac tggaagcttt cgttgactta cgtgatgtac gtcagcctga
     1081 agtgaaagaa gagaaaccag aggcggccgc agcaagcacc gaacgcaaat gggcagaact
     1141 ggcacgtcgt attcgtggtg caggcggtgt taccctcaat ggttttggtt gaggatcctc
     1201 tacgccggac gcatcgtggc cggcatcacc ggcgccacag gtgcggttgc tggcgcctat
     1261 atcgccgaca tcaccgatgg ggaagatcgg gctcgccact tcgggctcat gagcgcttgt
     1321 ttcggcgtgg gtatggtggc aggccccgtg gccgggggac tgttgggcgc catctccttg
     1381 catgcaccat tccttgcggc ggcggtgctc aacggcctca acctactact gggctgcttc
     1441 ctaatgcagg agtcgcataa gggagagcgt cgaccgatgc ccttgagagc cttcaaccca
     1501 gtcagctcct tccggtgggc gcggggcatg actatcgtcg ccgcacttat gactgtcttc
     1561 tttatcatgc aactcgtagg acaggtgccg gcagcgctct gggtcatttt cggcgaggac
     1621 cgctttcgct ggagcgcgac gatgatcggc ctgtcgcttg cggtattcgg aatcttgcac
     1681 gccctcgctc aagccttcgt cactggtccc gccaccaaac gtttcggcga gaagcaggcc
     1741 attatcgccg gcatggcggc cgacgcgctg ggctacgtct tgctggcgtt cgcgacgcga
     1801 ggctggatgg ccttccccat tatgattctt ctcgcttccg gcggcatcgg gatgcccgcg
     1861 ttgcaggcca tgctgtccag gcaggtagat gacgaccatc agggacagct tcaaggatcg
     1921 ctcgcggctc ttaccagcct aacttcgatc attggaccgc tgatcgtcac ggcgatttat
     1981 gccgcctcgg cgagcacatg gaacgggttg gcatggattg taggcgccgc cctatacctt
     2041 gtctgcctcc ccgcgttgcg tcgcggtgca tggagccggg ccacctcgac ctgaatggaa
     2101 gccggcggca cctcgctaac ggattcacca ctccaagaat tggagccaat caattcttgc
     2161 ggagaactgt gaatgcgcaa accaaccctt ggcagaacat atccatcgcg tccgccatct
     2221 ccagcagccg cacgcggcgc atctcgggca gcgttgggtc ctggccacgg gtgcgcatga
     2281 tcgtgctcct gtcgttgagg acccggctag gctggcgggg ttgccttact ggttagcaga
     2341 atgaatcacc gatacgcgag cgaacgtgaa gcgactgctg ctgcaaaacg tctgcgacct
     2401 gagcaacaac atgaatggtc ttcggtttcc gtgtttcgta aagtctggaa acgcggaagt
     2461 cagcgccctg caccattatg ttccggatct gcatcgcagg atgctgctgg ctaccctgtg
     2521 gaacacctac atctgtatta acgaagcgct ggcattgacc ctgagtgatt tttctctggt
     2581 cccgccgcat ccataccgcc agttgtttac cctcacaacg ttccagtaac cgggcatgtt
     2641 catcatcagt aacccgtatc gtgagcatcc tctctcgttt catcggtatc attaccccca
     2701 tgaacagaaa tcccccttac acggaggcat cagtgaccaa acaggaaaaa accgccctta
     2761 acatggcccg ctttatcaga agccagacat taacgcttct ggagaaactc aacgagctgg
     2821 acgcggatga acaggcagac atctgtgaat cgcttcacga ccacgctgat gagctttacc
     2881 gcagctgcct cgcgcgtttc ggtgatgacg gtgaaaacct ctgacacatg cagctcccgg
     2941 agacggtcac agcttgtctg taagcggatg ccgggagcag acaagcccgt cagggcgcgt
     3001 cagcgggtgt tggcgggtgt cggggcgcag ccatgaccca gtcacgtagc gatagcggag
     3061 tgtatactgg cttaactatg cggcatcaga gcagattgta ctgagagtgc accatatgcg
     3121 gtgtgaaata ccgcacagat gcgtaaggag aaaataccgc atcaggcgct cttccgcttc
     3181 ctcgctcact gactcgctgc gctcggtcgt tcggctgcgg cgagcggtat cagctcactc
     3241 aaaggcggta atacggttat ccacagaatc aggggataac gcaggaaaga acatgtgagc
     3301 aaaaggccag caaaaggcca ggaaccgtaa aaaggccgcg ttgctggcgt ttttccatag
     3361 gctccgcccc cctgacgagc atcacaaaaa tcgacgctca agtcagaggt ggcgaaaccc
     3421 gacaggacta taaagatacc aggcgtttcc ccctggaagc tccctcgtgc gctctcctgt
     3481 tccgaccctg ccgcttaccg gatacctgtc cgcctttctc ccttcgggaa gcgtggcgct
     3541 ttctcatagc tcacgctgta ggtatctcag ttcggtgtag gtcgttcgct ccaagctggg
     3601 ctgtgtgcac gaaccccccg ttcagcccga ccgctgcgcc ttatccggta actatcgtct
     3661 tgagtccaac ccggtaagac acgacttatc gccactggca gcagccactg gtaacaggat
     3721 tagcagagcg aggtatgtag gcggtgctac agagttcttg aagtggtggc ctaactacgg
     3781 ctacactaga aggacagtat ttggtatctg cgctctgctg aagccagtta ccttcggaaa
     3841 aagagttggt agctcttgat ccggcaaaca aaccaccgct ggtagcggtg gtttttttgt
     3901 ttgcaagcag cagattacgc gcagaaaaaa aggatctcaa gaagatcctt tgatcttttc
     3961 tacggggtct gacgctcagt ggaacgaaaa ctcacgttaa gggattttgg tcatgagatt
     4021 atcaaaaagg atcttcacct agatcctttt aaattaaaaa tgaagtttta aatcaatcta
     4081 aagtatatat gagtaaactt ggtctgacag ttaccaatgc ttaatcagtg aggcacctat
     4141 ctcagcgatc tgtctatttc gttcatccat agttgcctga ctccccgtcg tgtagataac
     4201 tacgatacgg gagggcttac catctggccc cagtgctgca atgataccgc gagacccacg
     4261 ctcaccggct ccagatttat cagcaataaa ccagccagcc ggaagggccg agcgcagaag
     4321 tggtcctgca actttatccg cctccatcca gtctattaat tgttgccggg aagctagagt
     4381 aagtagttcg ccagttaata gtttgcgcaa cgttgttgcc attgctgcag gcatcgtggt
     4441 gtcacgctcg tcgtttggta tggcttcatt cagctccggt tcccaacgat caaggcgagt
     4501 tacatgatcc cccatgttgt gcaaaaaagc ggttagctcc ttcggtcctc cgatcgttgt
     4561 cagaagtaag ttggccgcag tgttatcact catggttatg gcagcactgc ataattctct
     4621 tactgtcatg ccatccgtaa gatgcttttc tgtgactggt gagtactcaa ccaagtcatt
     4681 ctgagaatag tgtatgcggc gaccgagttg ctcttgcccg gcgtcaacac gggataatac
     4741 cgcgccacat agcagaactt taaaagtgct catcattgga aaacgttctt cggggcgaaa
     4801 actctcaagg atcttaccgc tgttgagatc cagttcgatg taacccactc gtgcacccaa
     4861 ctgatcttca gcatctttta ctttcaccag cgtttctggg tgagcaaaaa caggaaggca
     4921 aaatgccgca aaaaagggaa taagggcgac acggaaatgt tgaatactca tactcttcct
     4981 ttttcaatat tattgaagca tttatcaggg ttattgtctc atgagcggat acatatttga
     5041 atgtatttag aaaaataaac aaataggggt tccgcgcaca tttccccgaa aagtgccacc
     5101 tgacgtctaa gaaaccatta ttatcatgac attaacctat aaaaataggc gtatcacgag
     5161 gccctttgga taaccagaa
//
