LOCUS       sequence-434784-        6696 bp    DNA     circular SYN 15-APR-2025
DEFINITION  express E. coli E69 (O9a:K30) Wzc with Y715E, Y717E, Y718E and N711Y
            mutations.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 6696)
  TITLE     Molecular basis for the phosphorylation of bacterial tyrosine kinase
            Wzc
  JOURNAL   Unpublished
REFERENCE   2  (bases 1 to 6696)
  AUTHORS   .
  TITLE     Direct Submission
  JOURNAL   Exported Apr 15, 2025 from SnapGene Server 8.0.1
            https://www.snapgene.com
COMMENT     SGRef: number: 1; type: "Journal Article"; journalName: 
            "Unpublished"
COMMENT     Sequence Label: pBN413
FEATURES             Location/Qualifiers
     source          1..6696
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     CDS             1889..1906
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     primer_bind     complement(1960..1977)
                     /label=pBAD Reverse
                     /note="For vectors with E. coli araBAD promoter, reverse 
                     primer"
     terminator      2130..2216
                     /gene="Escherichia coli rrnB"
                     /label=rrnB T1 terminator
                     /note="transcription terminator T1 from the E. coli rrnB 
                     gene"
     terminator      2308..2335
                     /label=rrnB T2 terminator
                     /note="transcription terminator T2 from the E. coli rrnB 
                     gene"
     promoter        2354..2445
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(2446..2514,2515..3306)
                     /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(2664..2683)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      3348..3803
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(3435..3454)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     3645..3666
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     rep_origin      3914..4502
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     4403..4422
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     4656..4673
                     /label=L4440
                     /note="L4440 vector, forward primer"
     primer_bind     4839..4861
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     primer_bind     4999..5018
                     /label=pBRrevBam
                     /note="pBR322 vectors, tet region, downstream of BamHI, 
                     reverse primer"
     CDS             complement(5198..6076)
                     /codon_start=1
                     /gene="araC"
                     /product="L-arabinose regulatory protein"
                     /label=araC
                     /translation="MAEAQNDPLLPGYSFNAHLVAGLTPIEANGYLDFFIDRPLGMKGY
                     ILNLTIRGQGVVKNQGREFVCRPGDILLFPPGEIHHYGRHPEAREWYHQWVYFRPRAYW
                     HEWLNWPSIFANTGFFRPDEAHQPHFSDLFGQIINAGQGEGRYSELLAINLLEQLLLRR
                     MEAINESLHPPMDNRVREACQYISDHLADSNFDIASVAQHVCLSPSRLSHLFRQQLGIS
                     VLSWREDQRISQAKLLLSTTRMPIATVGRNVGFDDQLYFSRVFKKCTGASPSEFRAGCE
                     EKVNDVAVKLS"
     promoter        6103..6387
                     /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     6310..6329
                     /label=pBAD Forward
                     /note="For vectors with E. coli araBAD promoter, forward 
                     primer"
ORIGIN
        1 gctacaatct cgaatgattc ttggcaaaac tatagatgat ttaaaccttc agattcaaat
       61 tgaacaaaaa tattttcctg ttattggccg tgggttagca agattaatgg gagaaaaacc
      121 aggtaatatt gatattactc ggttatattt gcctgatagt gacgatatta gtaataatac
      181 tcctagtata atattaacag taaaagataa agaaaattat tcgatcaata gcgatggtat
      241 tcaactcaat ggggttgtag ggacgttatt aaatgaaaaa gggatatcat tactggttaa
      301 cgaaatcgat gctaaacctg gagatcaatt tgttattaca cagttgcctc gattaaaggc
      361 catttctgat cttttaaaaa gttttagcgt tgctgatttg ggtaaagata cgggaatgtt
      421 aacgttgact cttactggag ataacccaaa aagaatatct catattttgg acagtatcag
      481 tcagaactat cttgcacaga acattgctag acaagctgcg caggatgcta aaagtctcga
      541 gtttttaaat caacaattac ccaaagttcg tgccgaatta gattctgcgg aagacaagtt
      601 aaatgcttat cgtaagcaaa aagactctgt tgacttgaat atggaagcta aatcagtact
      661 tgatcaaata gttaacgttg ataatcaact aaatgaatta accttccgtg aggcagaagt
      721 ttcacagcta tatactaagg aacatcctac ttataaggcg ttaatggaaa aacggcaaac
      781 acttcaagaa gaaaaatcta aacttaataa gcgcgttagt agtatgccct cgacccagca
      841 ggaggttctg cgattaagtc gagatgttga atctggtcgt gctgtatatt tgcaattgct
      901 aaatcggcaa caagaattaa acattgccaa atcaagtgct attggcaacg ttagaattat
      961 tgataatgct gtaacagatc ctaatcctgt aagaccaaag aagactatca taattgtaat
     1021 aggtgttgtt ttagggctaa tcgtttcggt tgtactggta ttgttccaag tgtttctacg
     1081 ccgtggtatc gaatccccag agcaactcga agaaatcgga ataaacgtgt atgcaagtat
     1141 tccaatatct gaatggctta caaaaaatgc tcgtcagtca gggaaagtca gaaaaaatca
     1201 gtcagacaca ttgcttgctg ttggtaatcc tgccgattta gctgttgagg ccattcgtgg
     1261 tttacgcaca agtttgcatt ttgcaatgat ggaagcaaaa aataatgtcc taatgatatc
     1321 gggtgctagt ccaagcgcag gtaaaacttt tattagcagt aacttggcag ccactatagc
     1381 aattacaggg aaaaaagttt tattcattga tgctgacctt cgtaagggat atgctcataa
     1441 aatgtttggt cataaaaacg ataaaggatt atctgagttt ttatctggac aagccgctgc
     1501 tgaaatgatc attgataaag tagaaggggg gggatttgat tacattggtc gtggacaaat
     1561 tcctccaaac ccagctgaat tacttatgca tccgcgtttt gaacaattat taaattgggc
     1621 ctcacaaaat tatgatttaa ttataataga tacacctcca attttggcag tgacagatgc
     1681 tgcgattatt ggtcgatatg ctggaacatg tctattagtc gctcgctttg aaaaaaatac
     1741 tgttaaagaa attgatgtaa gtatgaagcg gtttgagcaa agtggtgtgg tagtcaaagg
     1801 ctgtatactt aacggtgttg taaagaaagc tagcagctac tatagatatg gccattatca
     1861 ttatggcgag tctgaggaag ataaaaaaca ccaccaccac caccactgac tgcaggcatg
     1921 caagcttggc tgttttggcg gatgagagaa gattttcagc ctgatacaga ttaaatcaga
     1981 acgcagaagc ggtctgataa aacagaattt gcctggcggc agtagcgcgg tggtcccacc
     2041 tgaccccatg ccgaactcag aagtgaaacg ccgtagcgcc gatggtagtg tggggtctcc
     2101 ccatgcgaga gtagggaact gccaggcatc aaataaaacg aaaggctcag tcgaaagact
     2161 gggcctttcg ttttatctgt tgtttgtcgg tgaacgctct cctgagtagg acaaatccgc
     2221 cgggagcgga tttgaacgtt gcgaagcaac ggcccggagg gtggcgggca ggacgcccgc
     2281 cataaactgc caggcatcaa attaagcaga aggccatcct gacggatggc ctttttgcgt
     2341 ttctacaaac tcttttgttt atttttctaa atacattcaa atatgtatcc gctcatgaga
     2401 caataaccct gataaatgct tcaataatat tgaaaaagga agagtatgag tattcaacat
     2461 ttccgtgtcg cccttattcc cttttttgcg gcattttgcc ttcctgtttt tgctcaccca
     2521 gaaacgctgg tgaaagtaaa agatgctgaa gatcagttgg gtgcacgagt gggttacatc
     2581 gaactggatc tcaacagcgg taagatcctt gagagttttc gccccgaaga acgttttcca
     2641 atgatgagca cttttaaagt tctgctatgt ggcgcggtat tatcccgtgt tgacgccggg
     2701 caagagcaac tcggtcgccg catacactat tctcagaatg acttggttga gtactcacca
     2761 gtcacagaaa agcatcttac ggatggcatg acagtaagag aattatgcag tgctgccata
     2821 accatgagtg ataacactgc ggccaactta cttctgacaa cgatcggagg accgaaggag
     2881 ctaaccgctt ttttgcacaa catgggggat catgtaactc gccttgatcg ttgggaaccg
     2941 gagctgaatg aagccatacc aaacgacgag cgtgacacca cgatgcctgt agcaatggca
     3001 acaacgttgc gcaaactatt aactggcgaa ctacttactc tagcttcccg gcaacaatta
     3061 atagactgga tggaggcgga taaagttgca ggaccacttc tgcgctcggc ccttccggct
     3121 ggctggttta ttgctgataa atctggagcc ggtgagcgtg ggtctcgcgg tatcattgca
     3181 gcactggggc cagatggtaa gccctcccgt atcgtagtta tctacacgac ggggagtcag
     3241 gcaactatgg atgaacgaaa tagacagatc gctgagatag gtgcctcact gattaagcat
     3301 tggtaactgt cagaccaagt ttactcatat atactttaga ttgatttacg cgccctgtag
     3361 cggcgcatta agcgcggcgg gtgtggtggt tacgcgcagc gtgaccgcta cacttgccag
     3421 cgccctagcg cccgctcctt tcgctttctt cccttccttt ctcgccacgt tcgccggctt
     3481 tccccgtcaa gctctaaatc gggggctccc tttagggttc cgatttagtg ctttacggca
     3541 cctcgacccc aaaaaacttg atttgggtga tggttcacgt agtgggccat cgccctgata
     3601 gacggttttt cgccctttga cgttggagtc cacgttcttt aatagtggac tcttgttcca
     3661 aactggaaca acactcaacc ctatctcggg ctattctttt gatttataag ggattttgcc
     3721 gatttcggcc tattggttaa aaaatgagct gatttaacaa aaatttaacg cgaattttaa
     3781 caaaatatta acgtttacaa tttaaaagga tctaggtgaa gatccttttt gataatctca
     3841 tgaccaaaat cccttaacgt gagttttcgt tccactgagc gtcagacccc gtagaaaaga
     3901 tcaaaggatc ttcttgagat cctttttttc tgcgcgtaat ctgctgcttg caaacaaaaa
     3961 aaccaccgct accagcggtg gtttgtttgc cggatcaaga gctaccaact ctttttccga
     4021 aggtaactgg cttcagcaga gcgcagatac caaatactgt ccttctagtg tagccgtagt
     4081 taggccacca cttcaagaac tctgtagcac cgcctacata cctcgctctg ctaatcctgt
     4141 taccagtggc tgctgccagt ggcgataagt cgtgtcttac cgggttggac tcaagacgat
     4201 agttaccgga taaggcgcag cggtcgggct gaacgggggg ttcgtgcaca cagcccagct
     4261 tggagcgaac gacctacacc gaactgagat acctacagcg tgagctatga gaaagcgcca
     4321 cgcttcccga agggagaaag gcggacaggt atccggtaag cggcagggtc ggaacaggag
     4381 agcgcacgag ggagcttcca gggggaaacg cctggtatct ttatagtcct gtcgggtttc
     4441 gccacctctg acttgagcgt cgatttttgt gatgctcgtc aggggggcgg agcctatgga
     4501 aaaacgccag caacgcggcc tttttacggt tcctggcctt ttgctggcct tttgctcaca
     4561 tgttctttcc tgcgttatcc cctgattctg tggataaccg tattaccgcc tttgagtgag
     4621 ctgataccgc tcgccgcagc cgaacgaccg agcgcagcga gtcagtgagc gaggaagcgg
     4681 aagagcgcct gatgcggtat tttctcctta cgcatctgtg cggtatttca caccgcatag
     4741 ggtcatggct gcgccccgac acccgccaac acccgctgac gcgccctgac gggcttgtct
     4801 gctcccggca tccgcttaca gacaagctgt gaccgtctcc gggagctgca tgtgtcagag
     4861 gttttcaccg tcatcaccga aacgcgcgag gcagaaggag atggcgccca acagtccccc
     4921 ggccacgggg cctgccacca tacccacgcc gaaacaagcg ctcatgagcc cgaagtggcg
     4981 agcccgatct tccccatcgg tgatgtcggc gatataggcg ccagcaaccg cacctgtggc
     5041 gccggtgatg ccggccacga tgcgtccggc gtagaggatc tgctcatgtt tgacagctta
     5101 tcatcgatgc ataatgtgcc tgtcaaatgg acgaagcagg gattctgcaa accctatgct
     5161 actccgtcaa gccgtcaatt gtctgattcg ttaccaatta tgacaacttg acggctacat
     5221 cattcacttt ttcttcacaa ccggcacgga actcgctcgg gctggccccg gtgcattttt
     5281 taaatacccg cgagaaatag agttgatcgt caaaaccaac attgcgaccg acggtggcga
     5341 taggcatccg ggtggtgctc aaaagcagct tcgcctggct gatacgttgg tcctcgcgcc
     5401 agcttaagac gctaatccct aactgctggc ggaaaagatg tgacagacgc gacggcgaca
     5461 agcaaacatg ctgtgcgacg ctggcgatat caaaattgct gtctgccagg tgatcgctga
     5521 tgtactgaca agcctcgcgt acccgattat ccatcggtgg atggagcgac tcgttaatcg
     5581 cttccatgcg ccgcagtaac aattgctcaa gcagatttat cgccagcagc tccgaatagc
     5641 gcccttcccc ttgcccggcg ttaatgattt gcccaaacag gtcgctgaaa tgcggctggt
     5701 gcgcttcatc cgggcgaaag aaccccgtat tggcaaatat tgacggccag ttaagccatt
     5761 catgccagta ggcgcgcgga cgaaagtaaa cccactggtg ataccattcg cgagcctccg
     5821 gatgacgacc gtagtgatga atctctcctg gcgggaacag caaaatatca cccggtcggc
     5881 aaacaaattc tcgtccctga tttttcacca ccccctgacc gcgaatggtg agattgagaa
     5941 tataaccttt cattcccagc ggtcggtcga taaaaaaatc gagataaccg ttggcctcaa
     6001 tcggcgttaa acccgccacc agatgggcat taaacgagta tcccggcagc aggggatcat
     6061 tttgcgcttc agccatactt ttcatactcc cgccattcag agaagaaacc aattgtccat
     6121 attgcatcag acattgccgt cactgcgtct tttactggct cttctcgcta accaaaccgg
     6181 taaccccgct tattaaaagc attctgtaac aaagcgggac caaagccatg acaaaaacgc
     6241 gtaacaaaag tgtctataat cacggcagaa aagtccacat tgattatttg cacggcgtca
     6301 cactttgcta tgccatagca tttttatcca taagattagc ggatcctacc tgacgctttt
     6361 tatcgcaact ctctactgtt tctccatacc cgtttttttg ggctagcagg aggaattcac
     6421 catgacttca gtcacatcta agcaatcaac gatactagga tctgatgaaa tagatttagg
     6481 tcgcgttatc ggtgagctaa ttgatcatcg taagcttatt atatctataa catcggtttt
     6541 taccttattt gcaatactct atgcattatt agcgactcct atctatgaga cggatgcgct
     6601 aattcaaata gagcagaagc aaggtaatgc tatactaagc agtctaagtc aggttttacc
     6661 tgatggccaa ccccaatctg cacctgaaac agcttt
//
