LOCUS       sequence-254960-        5443 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  expresses H258A mutant of PfeE in E.coli.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 5443)
  AUTHORS   Perraud Q, Moynie L, Gasser V, Munier M, Godet J, Hoegy F, Mely Y, 
            Mislin GLA, Naismith JH, Schalk IJ
  TITLE     A Key Role for the Periplasmic PfeE Esterase in Iron Acquisition via
            the Siderophore Enterobactin in Pseudomonas aeruginosa.
  JOURNAL   ACS Chem Biol. 2018 Sep 21;13(9):2603-2614. doi: 
            10.1021/acschembio.8b00543. Epub 2018 Aug 22.
  PUBMED    30086222
REFERENCE   2  (bases 1 to 5443)
  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"; doi: 
            "10.1021/acschembio.8b00543"; journalName: "ACS Chem Biol"; date: 
            "2018-09-21- 21"; volume: "13"; issue: "9"; pages: "2603-2614"
COMMENT     Sequence Label: pBADHISTEV_PfeEH258A
FEATURES             Location/Qualifiers
     source          1..5443
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     CDS             1..18
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     CDS             40..60
                     /codon_start=1
                     /product="tobacco etch virus (TEV) protease recognition and
                     cleavage site"
                     /label=TEV site
                     /translation="ENLYFQG"
     primer_bind     complement(970..987)
                     /label=pBAD Reverse
                     /note="For vectors with E. coli araBAD promoter, reverse 
                     primer"
     terminator      1140..1226
                     /gene="Escherichia coli rrnB"
                     /label=rrnB T1 terminator
                     /note="transcription terminator T1 from the E. coli rrnB 
                     gene"
     terminator      1318..1345
                     /label=rrnB T2 terminator
                     /note="transcription terminator T2 from the E. coli rrnB 
                     gene"
     promoter        1364..1455
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(1456..1524,1525..2316)
                     /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(1674..1693)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      2358..2813
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(2445..2464)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     2655..2676
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     rep_origin      2924..3512
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     3413..3432
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     3666..3683
                     /label=L4440
                     /note="L4440 vector, forward primer"
     primer_bind     3849..3871
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     primer_bind     4009..4028
                     /label=pBRrevBam
                     /note="pBR322 vectors, tet region, downstream of BamHI, 
                     reverse primer"
     CDS             complement(4208..5086)
                     /codon_start=1
                     /gene="araC"
                     /product="L-arabinose regulatory protein"
                     /label=araC
                     /translation="MAEAQNDPLLPGYSFNAHLVAGLTPIEANGYLDFFIDRPLGMKGY
                     ILNLTIRGQGVVKNQGREFVCRPGDILLFPPGEIHHYGRHPEAREWYHQWVYFRPRAYW
                     HEWLNWPSIFANTGFFRPDEAHQPHFSDLFGQIINAGQGEGRYSELLAINLLEQLLLRR
                     MEAINESLHPPMDNRVREACQYISDHLADSNFDIASVAQHVCLSPSRLSHLFRQQLGIS
                     VLSWREDQRISQAKLLLSTTRMPIATVGRNVGFDDQLYFSRVFKKCTGASPSEFRAGCE
                     EKVNDVAVKLS"
     promoter        5113..5397
                     /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     5320..5339
                     /label=pBAD Forward
                     /note="For vectors with E. coli araBAD promoter, forward 
                     primer"
ORIGIN
        1 catcaccatc accatcacga ttacgacatc ccaacgaccg aaaacctgta ttttcagggc
       61 gccatgaatc cggaccccga ggcgaccatg gaccgcagcc tgctgcaacg ccaggacctg
      121 ccctatcgct tcagcgccgt ggacctggac tccgtcgatg gccagcggca ttaccggctg
      181 tggctcggcc gaccgctgca ggcaccgccc gccgccggct acccggtggt ctggatgctc
      241 gacggcaacg ccgccgtcgg cgccctcgac gagtcaaccc tgagacgcct ggccgacggc
      301 gacgcaccgc tactggtcgc catcggctac cgcacgccgc tgcgcatcga ccgcgccggg
      361 cgtaccttcg actacacccc cgcgagtccc ggccaggccg atcagcgcga cccgctcaac
      421 ggcctgccca gcggtggcgc cgacgccttc ctcgacctgc tgcgcgacgg catgcgcccg
      481 gccgtggcgg cgcaggcgcc tctggacact gcgcgacaaa ccctctgggg acactcctac
      541 ggcggcttgc tggtgctcca cgcactgttc acccgtcccg gcgagttcgc ccgctacgcc
      601 gcggccagcc cgtcgctgtg gtggcgcgac ggggcgatcc tcggcgaacg agcggggctc
      661 gaacagcgcc tgcgcggaaa gcgcgcggaa ctcctgctct ggcgcggcag cgccgaaccg
      721 gccagcccgc gcggctcgct gaaagccgag ccgggccagg cgatggcgcg gctggtagat
      781 gacctgcgcc gggttgccgg gctcacgctg gacttccagc cgctcgacgg cctgggtgca
      841 ggcgaaacgc tgggggcctc gttgcgcctt ctgctcgccc gtccggcggt ggaacgccag
      901 cgctgaggat ccgaattcga gctccgtcga caagcttggc tgttttggcg gatgagagaa
      961 gattttcagc ctgatacaga ttaaatcaga acgcagaagc ggtctgataa aacagaattt
     1021 gcctggcggc agtagcgcgg tggtcccacc tgaccccatg ccgaactcag aagtgaaacg
     1081 ccgtagcgcc gatggtagtg tggggtctcc ccatgcgaga gtagggaact gccaggcatc
     1141 aaataaaacg aaaggctcag tcgaaagact gggcctttcg ttttatctgt tgtttgtcgg
     1201 tgaacgctct cctgagtagg acaaatccgc cgggagcgga tttgaacgtt gcgaagcaac
     1261 ggcccggagg gtggcgggca ggacgcccgc cataaactgc caggcatcaa attaagcaga
     1321 aggccatcct gacggatggc ctttttgcgt ttctacaaac tcttttgttt atttttctaa
     1381 atacattcaa atatgtatcc gctcatgaga caataaccct gataaatgct tcaataatat
     1441 tgaaaaagga agagtatgag tattcaacat ttccgtgtcg cccttattcc cttttttgcg
     1501 gcattttgcc ttcctgtttt tgctcaccca gaaacgctgg tgaaagtaaa agatgctgaa
     1561 gatcagttgg gtgcacgagt gggttacatc gaactggatc tcaacagcgg taagatcctt
     1621 gagagttttc gccccgaaga acgttttcca atgatgagca cttttaaagt tctgctatgt
     1681 ggcgcggtat tatcccgtgt tgacgccggg caagagcaac tcggtcgccg catacactat
     1741 tctcagaatg acttggttga gtactcacca gtcacagaaa agcatcttac ggatggcatg
     1801 acagtaagag aattatgcag tgctgccata accatgagtg ataacactgc ggccaactta
     1861 cttctgacaa cgatcggagg accgaaggag ctaaccgctt ttttgcacaa catgggggat
     1921 catgtaactc gccttgatcg ttgggaaccg gagctgaatg aagccatacc aaacgacgag
     1981 cgtgacacca cgatgcctgt agcaatggca acaacgttgc gcaaactatt aactggcgaa
     2041 ctacttactc tagcttcccg gcaacaatta atagactgga tggaggcgga taaagttgca
     2101 ggaccacttc tgcgctcggc ccttccggct ggctggttta ttgctgataa atctggagcc
     2161 ggtgagcgtg ggtctcgcgg tatcattgca gcactggggc cagatggtaa gccctcccgt
     2221 atcgtagtta tctacacgac ggggagtcag gcaactatgg atgaacgaaa tagacagatc
     2281 gctgagatag gtgcctcact gattaagcat tggtaactgt cagaccaagt ttactcatat
     2341 atactttaga ttgatttacg cgccctgtag cggcgcatta agcgcggcgg gtgtggtggt
     2401 tacgcgcagc gtgaccgcta cacttgccag cgccctagcg cccgctcctt tcgctttctt
     2461 cccttccttt ctcgccacgt tcgccggctt tccccgtcaa gctctaaatc gggggctccc
     2521 tttagggttc cgatttagtg ctttacggca cctcgacccc aaaaaacttg atttgggtga
     2581 tggttcacgt agtgggccat cgccctgata gacggttttt cgccctttga cgttggagtc
     2641 cacgttcttt aatagtggac tcttgttcca aactggaaca acactcaacc ctatctcggg
     2701 ctattctttt gatttataag ggattttgcc gatttcggcc tattggttaa aaaatgagct
     2761 gatttaacaa aaatttaacg cgaattttaa caaaatatta acgtttacaa tttaaaagga
     2821 tctaggtgaa gatccttttt gataatctca tgaccaaaat cccttaacgt gagttttcgt
     2881 tccactgagc gtcagacccc gtagaaaaga tcaaaggatc ttcttgagat cctttttttc
     2941 tgcgcgtaat ctgctgcttg caaacaaaaa aaccaccgct accagcggtg gtttgtttgc
     3001 cggatcaaga gctaccaact ctttttccga aggtaactgg cttcagcaga gcgcagatac
     3061 caaatactgt ccttctagtg tagccgtagt taggccacca cttcaagaac tctgtagcac
     3121 cgcctacata cctcgctctg ctaatcctgt taccagtggc tgctgccagt ggcgataagt
     3181 cgtgtcttac cgggttggac tcaagacgat agttaccgga taaggcgcag cggtcgggct
     3241 gaacgggggg ttcgtgcaca cagcccagct tggagcgaac gacctacacc gaactgagat
     3301 acctacagcg tgagctatga gaaagcgcca cgcttcccga agggagaaag gcggacaggt
     3361 atccggtaag cggcagggtc ggaacaggag agcgcacgag ggagcttcca gggggaaacg
     3421 cctggtatct ttatagtcct gtcgggtttc gccacctctg acttgagcgt cgatttttgt
     3481 gatgctcgtc aggggggcgg agcctatgga aaaacgccag caacgcggcc tttttacggt
     3541 tcctggcctt ttgctggcct tttgctcaca tgttctttcc tgcgttatcc cctgattctg
     3601 tggataaccg tattaccgcc tttgagtgag ctgataccgc tcgccgcagc cgaacgaccg
     3661 agcgcagcga gtcagtgagc gaggaagcgg aagagcgcct gatgcggtat tttctcctta
     3721 cgcatctgtg cggtatttca caccgcatag ggtcatggct gcgccccgac acccgccaac
     3781 acccgctgac gcgccctgac gggcttgtct gctcccggca tccgcttaca gacaagctgt
     3841 gaccgtctcc gggagctgca tgtgtcagag gttttcaccg tcatcaccga aacgcgcgag
     3901 gcagaaggag atggcgccca acagtccccc ggccacgggg cctgccacca tacccacgcc
     3961 gaaacaagcg ctcatgagcc cgaagtggcg agcccgatct tccccatcgg tgatgtcggc
     4021 gatataggcg ccagcaaccg cacctgtggc gccggtgatg ccggccacga tgcgtccggc
     4081 gtagaggatc tgctcatgtt tgacagctta tcatcgatgc ataatgtgcc tgtcaaatgg
     4141 acgaagcagg gattctgcaa accctatgct actccgtcaa gccgtcaatt gtctgattcg
     4201 ttaccaatta tgacaacttg acggctacat cattcacttt ttcttcacaa ccggcacgga
     4261 actcgctcgg gctggccccg gtgcattttt taaatacccg cgagaaatag agttgatcgt
     4321 caaaaccaac attgcgaccg acggtggcga taggcatccg ggtggtgctc aaaagcagct
     4381 tcgcctggct gatacgttgg tcctcgcgcc agcttaagac gctaatccct aactgctggc
     4441 ggaaaagatg tgacagacgc gacggcgaca agcaaacatg ctgtgcgacg ctggcgatat
     4501 caaaattgct gtctgccagg tgatcgctga tgtactgaca agcctcgcgt acccgattat
     4561 ccatcggtgg atggagcgac tcgttaatcg cttccatgcg ccgcagtaac aattgctcaa
     4621 gcagatttat cgccagcagc tccgaatagc gcccttcccc ttgcccggcg ttaatgattt
     4681 gcccaaacag gtcgctgaaa tgcggctggt gcgcttcatc cgggcgaaag aaccccgtat
     4741 tggcaaatat tgacggccag ttaagccatt catgccagta ggcgcgcgga cgaaagtaaa
     4801 cccactggtg ataccattcg cgagcctccg gatgacgacc gtagtgatga atctctcctg
     4861 gcgggaacag caaaatatca cccggtcggc aaacaaattc tcgtccctga tttttcacca
     4921 ccccctgacc gcgaatggtg agattgagaa tataaccttt cattcccagc ggtcggtcga
     4981 taaaaaaatc gagataaccg ttggcctcaa tcggcgttaa acccgccacc agatgggcat
     5041 taaacgagta tcccggcagc aggggatcat tttgcgcttc agccatactt ttcatactcc
     5101 cgccattcag agaagaaacc aattgtccat attgcatcag acattgccgt cactgcgtct
     5161 tttactggct cttctcgcta accaaaccgg taaccccgct tattaaaagc attctgtaac
     5221 aaagcgggac caaagccatg acaaaaacgc gtaacaaaag tgtctataat cacggcagaa
     5281 aagtccacat tgattatttg cacggcgtca cactttgcta tgccatagca tttttatcca
     5341 taagattagc ggatcttacc tgacgctttt tatcgcaact ctctactgtt tctccatacc
     5401 cgtttttttg ggctagcagg aggaatacac catgagctac tac
//
