LOCUS       sequence-434785-        6696 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  express E. coli E69 (O9a:K30) Wzc_K540M with Y715E, Y717E, Y718E and
            N711Y mutations.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 6696)
  AUTHORS   Yang Y, Batista M, Clarke BR, Agyare-Tabbi MR, Song H, Kuehfuss NM, 
            Le Bas A, Robinson CV, Whitfield C, Stansfeld PJ, Naismith JH, Liu J
  TITLE     Molecular basis for the phosphorylation of bacterial tyrosine kinase
            Wzc.
  JOURNAL   Nat Commun. 2025 Apr 11;16(1):3437. doi: 10.1038/s41467-025-58693-7.
  PUBMED    40210632
REFERENCE   2  (bases 1 to 6696)
  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: "Nat 
            Commun."; date: "2025-04-11"; volume: "16(1):3437. doi"; pages: " 
            10.1038/s41467-025-58693-7"
COMMENT     Sequence Label: pBN363
FEATURES             Location/Qualifiers
     source          1..6696
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     CDS             complement(96..974)
                     /codon_start=1
                     /gene="araC"
                     /product="L-arabinose regulatory protein"
                     /label=araC
                     /translation="MAEAQNDPLLPGYSFNAHLVAGLTPIEANGYLDFFIDRPLGMKGY
                     ILNLTIRGQGVVKNQGREFVCRPGDILLFPPGEIHHYGRHPEAREWYHQWVYFRPRAYW
                     HEWLNWPSIFANTGFFRPDEAHQPHFSDLFGQIINAGQGEGRYSELLAINLLEQLLLRR
                     MEAINESLHPPMDNRVREACQYISDHLADSNFDIASVAQHVCLSPSRLSHLFRQQLGIS
                     VLSWREDQRISQAKLLLSTTRMPIATVGRNVGFDDQLYFSRVFKKCTGASPSEFRAGCE
                     EKVNDVAVKLS"
     promoter        1001..1285
                     /gene="araBAD"
                     /label=araBAD promoter
                     /note="promoter of the L-arabinose operon of E. coli; the 
                     araC regulatory gene is transcribed in the opposite 
                     direction (Guzman et al., 1995)"
     primer_bind     1208..1227
                     /label=pBAD Forward
                     /note="For vectors with E. coli araBAD promoter, forward 
                     primer"
     CDS             3483..3500
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     primer_bind     complement(3554..3571)
                     /label=pBAD Reverse
                     /note="For vectors with E. coli araBAD promoter, reverse 
                     primer"
     terminator      3724..3810
                     /gene="Escherichia coli rrnB"
                     /label=rrnB T1 terminator
                     /note="transcription terminator T1 from the E. coli rrnB 
                     gene"
     terminator      3902..3929
                     /label=rrnB T2 terminator
                     /note="transcription terminator T2 from the E. coli rrnB 
                     gene"
     promoter        3948..4039
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(4040..4108,4109..4900)
                     /codon_start=1
                     /gene="bla"
                     /product="beta-lactamase"
                     /label=AmpR
                     /note="confers resistance to ampicillin, carbenicillin, and
                     related antibiotics"
                     /translation="MSIQHFRVALIPFFAAFCLPVFAHPETLVKVKDAEDQLGARVGYI
                     ELDLNSGKILESFRPEERFPMMSTFKVLLCGAVLSRVDAGQEQLGRRIHYSQNDLVEYS
                     PVTEKHLTDGMTVRELCSAAITMSDNTAANLLLTTIGGPKELTAFLHNMGDHVTRLDRW
                     EPELNEAIPNDERDTTMPVAMATTLRKLLTGELLTLASRQQLIDWMEADKVAGPLLRSA
                     LPAGWFIADKSGAGERGSRGIIAALGPDGKPSRIVVIYTTGSQATMDERNRQIAEIGAS
                     LIKHW"
     primer_bind     complement(4258..4277)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      4942..5397
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(5029..5048)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     5239..5260
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     rep_origin      5508..6096
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     5997..6016
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     6250..6267
                     /label=L4440
                     /note="L4440 vector, forward primer"
     primer_bind     6433..6455
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     primer_bind     6593..6612
                     /label=pBRrevBam
                     /note="pBR322 vectors, tet region, downstream of BamHI, 
                     reverse primer"
ORIGIN
        1 atcgatgcat aatgtgcctg tcaaatggac gaagcaggga ttctgcaaac cctatgctac
       61 tccgtcaagc cgtcaattgt ctgattcgtt accaattatg acaacttgac ggctacatca
      121 ttcacttttt cttcacaacc ggcacggaac tcgctcgggc tggccccggt gcatttttta
      181 aatacccgcg agaaatagag ttgatcgtca aaaccaacat tgcgaccgac ggtggcgata
      241 ggcatccggg tggtgctcaa aagcagcttc gcctggctga tacgttggtc ctcgcgccag
      301 cttaagacgc taatccctaa ctgctggcgg aaaagatgtg acagacgcga cggcgacaag
      361 caaacatgct gtgcgacgct ggcgatatca aaattgctgt ctgccaggtg atcgctgatg
      421 tactgacaag cctcgcgtac ccgattatcc atcggtggat ggagcgactc gttaatcgct
      481 tccatgcgcc gcagtaacaa ttgctcaagc agatttatcg ccagcagctc cgaatagcgc
      541 ccttcccctt gcccggcgtt aatgatttgc ccaaacaggt cgctgaaatg cggctggtgc
      601 gcttcatccg ggcgaaagaa ccccgtattg gcaaatattg acggccagtt aagccattca
      661 tgccagtagg cgcgcggacg aaagtaaacc cactggtgat accattcgcg agcctccgga
      721 tgacgaccgt agtgatgaat ctctcctggc gggaacagca aaatatcacc cggtcggcaa
      781 acaaattctc gtccctgatt tttcaccacc ccctgaccgc gaatggtgag attgagaata
      841 taacctttca ttcccagcgg tcggtcgata aaaaaatcga gataaccgtt ggcctcaatc
      901 ggcgttaaac ccgccaccag atgggcatta aacgagtatc ccggcagcag gggatcattt
      961 tgcgcttcag ccatactttt catactcccg ccattcagag aagaaaccaa ttgtccatat
     1021 tgcatcagac attgccgtca ctgcgtcttt tactggctct tctcgctaac caaaccggta
     1081 accccgctta ttaaaagcat tctgtaacaa agcgggacca aagccatgac aaaaacgcgt
     1141 aacaaaagtg tctataatca cggcagaaaa gtccacattg attatttgca cggcgtcaca
     1201 ctttgctatg ccatagcatt tttatccata agattagcgg atcctacctg acgcttttta
     1261 tcgcaactct ctactgtttc tccatacccg tttttttggg ctagcaggag gaattcacca
     1321 tgacttcagt cacatctaag caatcaacga tactaggatc tgatgaaata gatttaggtc
     1381 gcgttatcgg tgagctaatt gatcatcgta agcttattat atctataaca tcggttttta
     1441 ccttatttgc aatactctat gcattattag cgactcctat ctatgagacg gatgcgctaa
     1501 ttcaaataga gcagaagcaa ggtaatgcta tactaagcag tctaagtcag gttttacctg
     1561 atggccaacc ccaatctgca cctgaaacag ctttgctaca atctcgaatg attcttggca
     1621 aaactataga tgatttaaac cttcagattc aaattgaaca aaaatatttt cctgttattg
     1681 gccgtgggtt agcaagatta atgggagaaa aaccaggtaa tattgatatt actcggttat
     1741 atttgcctga tagtgacgat attagtaata atactcctag tataatatta acagtaaaag
     1801 ataaagaaaa ttattcgatc aatagcgatg gtattcaact caatggggtt gtagggacgt
     1861 tattaaatga aaaagggata tcattactgg ttaacgaaat cgatgctaaa cctggagatc
     1921 aatttgttat tacacagttg cctcgattaa aggccatttc tgatctttta aaaagtttta
     1981 gcgttgctga tttgggtaaa gatacgggaa tgttaacgtt gactcttact ggagataacc
     2041 caaaaagaat atctcatatt ttggacagta tcagtcagaa ctatcttgca cagaacattg
     2101 ctagacaagc tgcgcaggat gctaaaagtc tcgagttttt aaatcaacaa ttacccaaag
     2161 ttcgtgccga attagattct gcggaagaca agttaaatgc ttatcgtaag caaaaagact
     2221 ctgttgactt gaatatggaa gctaaatcag tacttgatca aatagttaac gttgataatc
     2281 aactaaatga attaaccttc cgtgaggcag aagtttcaca gctatatact aaggaacatc
     2341 ctacttataa ggcgttaatg gaaaaacggc aaacacttca agaagaaaaa tctaaactta
     2401 ataagcgcgt tagtagtatg ccctcgaccc agcaggaggt tctgcgatta agtcgagatg
     2461 ttgaatctgg tcgtgctgta tatttgcaat tgctaaatcg gcaacaagaa ttaaacattg
     2521 ccaaatcaag tgctattggc aacgttagaa ttattgataa tgctgtaaca gatcctaatc
     2581 ctgtaagacc aaagaagact atcataattg taataggtgt tgttttaggg ctaatcgttt
     2641 cggttgtact ggtattgttc caagtgtttc tacgccgtgg tatcgaatcc ccagagcaac
     2701 tcgaagaaat cggaataaac gtgtatgcaa gtattccaat atctgaatgg cttacaaaaa
     2761 atgctcgtca gtcagggaaa gtcagaaaaa atcagtcaga cacattgctt gctgttggta
     2821 atcctgccga tttagctgtt gaggccattc gtggtttacg cacaagtttg cattttgcaa
     2881 tgatggaagc aaaaaataat gtcctaatga tatcgggtgc tagtccaagc gcaggtatga
     2941 cttttattag cagtaacttg gcagccacta tagcaattac agggaaaaaa gttttattca
     3001 ttgatgctga ccttcgtaag ggatatgctc ataaaatgtt tggtcataaa aacgataaag
     3061 gattatctga gtttttatct ggacaagccg ctgctgaaat gatcattgat aaagtagaag
     3121 gggggggatt tgattacatt ggtcgtggac aaattcctcc aaacccagct gaattactta
     3181 tgcatccgcg ttttgaacaa ttattaaatt gggcctcaca aaattatgat ttaattataa
     3241 tagatacacc tccaattttg gcagtgacag atgctgcgat tattggtcga tatgctggaa
     3301 catgtctatt agtcgctcgc tttgaaaaaa atactgttaa agaaattgat gtaagtatga
     3361 agcggtttga gcaaagtggt gtggtagtca aaggctgtat acttaacggt gttgtaaaga
     3421 aagctagcag ctactataga tatggccatt atcattatgg cgagtctgag gaagataaaa
     3481 aacaccacca ccaccaccac tgactgcagg catgcaagct tggctgtttt ggcggatgag
     3541 agaagatttt cagcctgata cagattaaat cagaacgcag aagcggtctg ataaaacaga
     3601 atttgcctgg cggcagtagc gcggtggtcc cacctgaccc catgccgaac tcagaagtga
     3661 aacgccgtag cgccgatggt agtgtggggt ctccccatgc gagagtaggg aactgccagg
     3721 catcaaataa aacgaaaggc tcagtcgaaa gactgggcct ttcgttttat ctgttgtttg
     3781 tcggtgaacg ctctcctgag taggacaaat ccgccgggag cggatttgaa cgttgcgaag
     3841 caacggcccg gagggtggcg ggcaggacgc ccgccataaa ctgccaggca tcaaattaag
     3901 cagaaggcca tcctgacgga tggccttttt gcgtttctac aaactctttt gtttattttt
     3961 ctaaatacat tcaaatatgt atccgctcat gagacaataa ccctgataaa tgcttcaata
     4021 atattgaaaa aggaagagta tgagtattca acatttccgt gtcgccctta ttcccttttt
     4081 tgcggcattt tgccttcctg tttttgctca cccagaaacg ctggtgaaag taaaagatgc
     4141 tgaagatcag ttgggtgcac gagtgggtta catcgaactg gatctcaaca gcggtaagat
     4201 ccttgagagt tttcgccccg aagaacgttt tccaatgatg agcactttta aagttctgct
     4261 atgtggcgcg gtattatccc gtgttgacgc cgggcaagag caactcggtc gccgcataca
     4321 ctattctcag aatgacttgg ttgagtactc accagtcaca gaaaagcatc ttacggatgg
     4381 catgacagta agagaattat gcagtgctgc cataaccatg agtgataaca ctgcggccaa
     4441 cttacttctg acaacgatcg gaggaccgaa ggagctaacc gcttttttgc acaacatggg
     4501 ggatcatgta actcgccttg atcgttggga accggagctg aatgaagcca taccaaacga
     4561 cgagcgtgac accacgatgc ctgtagcaat ggcaacaacg ttgcgcaaac tattaactgg
     4621 cgaactactt actctagctt cccggcaaca attaatagac tggatggagg cggataaagt
     4681 tgcaggacca cttctgcgct cggcccttcc ggctggctgg tttattgctg ataaatctgg
     4741 agccggtgag cgtgggtctc gcggtatcat tgcagcactg gggccagatg gtaagccctc
     4801 ccgtatcgta gttatctaca cgacggggag tcaggcaact atggatgaac gaaatagaca
     4861 gatcgctgag ataggtgcct cactgattaa gcattggtaa ctgtcagacc aagtttactc
     4921 atatatactt tagattgatt tacgcgccct gtagcggcgc attaagcgcg gcgggtgtgg
     4981 tggttacgcg cagcgtgacc gctacacttg ccagcgccct agcgcccgct cctttcgctt
     5041 tcttcccttc ctttctcgcc acgttcgccg gctttccccg tcaagctcta aatcgggggc
     5101 tccctttagg gttccgattt agtgctttac ggcacctcga ccccaaaaaa cttgatttgg
     5161 gtgatggttc acgtagtggg ccatcgccct gatagacggt ttttcgccct ttgacgttgg
     5221 agtccacgtt ctttaatagt ggactcttgt tccaaactgg aacaacactc aaccctatct
     5281 cgggctattc ttttgattta taagggattt tgccgatttc ggcctattgg ttaaaaaatg
     5341 agctgattta acaaaaattt aacgcgaatt ttaacaaaat attaacgttt acaatttaaa
     5401 aggatctagg tgaagatcct ttttgataat ctcatgacca aaatccctta acgtgagttt
     5461 tcgttccact gagcgtcaga ccccgtagaa aagatcaaag gatcttcttg agatcctttt
     5521 tttctgcgcg taatctgctg cttgcaaaca aaaaaaccac cgctaccagc ggtggtttgt
     5581 ttgccggatc aagagctacc aactcttttt ccgaaggtaa ctggcttcag cagagcgcag
     5641 ataccaaata ctgtccttct agtgtagccg tagttaggcc accacttcaa gaactctgta
     5701 gcaccgccta catacctcgc tctgctaatc ctgttaccag tggctgctgc cagtggcgat
     5761 aagtcgtgtc ttaccgggtt ggactcaaga cgatagttac cggataaggc gcagcggtcg
     5821 ggctgaacgg ggggttcgtg cacacagccc agcttggagc gaacgaccta caccgaactg
     5881 agatacctac agcgtgagct atgagaaagc gccacgcttc ccgaagggag aaaggcggac
     5941 aggtatccgg taagcggcag ggtcggaaca ggagagcgca cgagggagct tccaggggga
     6001 aacgcctggt atctttatag tcctgtcggg tttcgccacc tctgacttga gcgtcgattt
     6061 ttgtgatgct cgtcaggggg gcggagccta tggaaaaacg ccagcaacgc ggccttttta
     6121 cggttcctgg ccttttgctg gccttttgct cacatgttct ttcctgcgtt atcccctgat
     6181 tctgtggata accgtattac cgcctttgag tgagctgata ccgctcgccg cagccgaacg
     6241 accgagcgca gcgagtcagt gagcgaggaa gcggaagagc gcctgatgcg gtattttctc
     6301 cttacgcatc tgtgcggtat ttcacaccgc atagggtcat ggctgcgccc cgacacccgc
     6361 caacacccgc tgacgcgccc tgacgggctt gtctgctccc ggcatccgct tacagacaag
     6421 ctgtgaccgt ctccgggagc tgcatgtgtc agaggttttc accgtcatca ccgaaacgcg
     6481 cgaggcagaa ggagatggcg cccaacagtc ccccggccac ggggcctgcc accataccca
     6541 cgccgaaaca agcgctcatg agcccgaagt ggcgagcccg atcttcccca tcggtgatgt
     6601 cggcgatata ggcgccagca accgcacctg tggcgccggt gatgccggcc acgatgcgtc
     6661 cggcgtagag gatctgctca tgtttgacag cttatc
//
