LOCUS       sequence-254940-        5818 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  expresses Pseudomonas aeruginosa PiuA in E. coli.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 5818)
  AUTHORS   Moynie L, Luscher A, Rolo D, Pletzer D, Tortajada A, Weingart H, 
            Braun Y, Page MG, Naismith JH, Kohler T
  TITLE     Structure and Function of the PiuA and PirA Siderophore-Drug 
            Receptors from Pseudomonas aeruginosa and Acinetobacter baumannii.
  JOURNAL   Antimicrob Agents Chemother. 2017 Mar 24;61(4). pii: AAC.02531-16. 
            doi: 10.1128/AAC.02531-16. Print 2017 Apr.
  PUBMED    28137795
REFERENCE   2  (bases 1 to 5818)
  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: "Antimicrob 
            Agents Chemother."; date: "2017-03-24"; volume: "61(4). pii: 
            AAC.02531-16. doi"; pages: " 10.1128/AAC.02531-16. Print 2017 Apr"
COMMENT     Sequence Label: pET20b_Pa-PiuA
FEATURES             Location/Qualifiers
     source          1..5818
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     1..20
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        1..19
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     RBS             51..73
                     /note="efficient ribosome binding site from bacteriophage 
                     T7 gene 10 (Olins and Rangwala, 1989)"
     sig_peptide     81..146
                     /codon_start=1
                     /gene="pelB (fragment)"
                     /product="leader peptide for secretion"
                     /label=pelB signal sequence
                     /translation="MKYLLPTAAAGLLLLAAQPAMA"
     CDS             2319..2336
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     primer_bind     complement(2389..2407)
                     /label=T7 Term
                     /note="T7 terminator, reverse primer"
     terminator      2403..2450
                     /label=T7 terminator
                     /note="transcription terminator for bacteriophage T7 RNA 
                     polymerase"
     rep_origin      2487..2942
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(2574..2593)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     2784..2805
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     promoter        2968..3072
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(3073..3141,3142..3933)
                     /codon_start=1
                     /gene="bla"
                     /product="beta-lactamase"
                     /label=AmpR
                     /note="confers resistance to ampicillin, carbenicillin, and
                     related antibiotics"
                     /translation="MSIQHFRVALIPFFAAFCLPVFAHPETLVKVKDAEDQLGARVGYI
                     ELDLNSGKILESFRPEERFPMMSTFKVLLCGAVLSRIDAGQEQLGRRIHYSQNDLVEYS
                     PVTEKHLTDGMTVRELCSAAITMSDNTAANLLLTTIGGPKELTAFLHNMGDHVTRLDRW
                     EPELNEAIPNDERDTTMPAAMATTLRKLLTGELLTLASRQQLIDWMEADKVAGPLLRSA
                     LPAGWFIADKSGAGERGSRGIIAALGPDGKPSRIVVIYTTGSQATMDERNRQIAEIGAS
                     LIKHW"
     primer_bind     complement(3291..3310)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      4104..4692
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     4593..4612
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     4846..4863
                     /label=L4440
                     /note="L4440 vector, forward primer"
     misc_feature    4878..5017
                     /label=bom
                     /note="basis of mobility region from pBR322"
     primer_bind     complement(4943..4962)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     5103..5125
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     CDS             complement(5119..5310)
                     /codon_start=1
                     /gene="rop"
                     /product="Rop protein, which maintains plasmids at low copy
                     number"
                     /label=rop
                     /translation="MTKQEKTALNMARFIRSQTLTLLEKLNELDADEQADICESLHDHA
                     DELYRSCLARFGDDGENL"
ORIGIN
        1 taatacgact cactataggg agaccacaac ggtttccctc tagaaataat tttgtttaac
       61 tttaagaagg agatatacat atgaaatacc tgctgccgac cgctgctgct ggtctgctgc
      121 tcctcgctgc ccagccggcg atggccatga acgaggccgg gcagaaaaag accgacaagg
      181 accgcgtact gtcgctggat gcggctacca tcgtcggcga gcagcaggac gaaaccacct
      241 ataacgtcga ccgctcggcg tcgaagaagt acaccgcgcc gctgctggat acgccgaaga
      301 ccgtgaccgt gatcccgcag caggtgatca aggacactgg cgccctgacc ctggccgacg
      361 cactgcgcac caccccgggt atcaccttcg gcgcgggcga gggcggcaac cccgcaggcg
      421 accggccatt catccgtggc ttcaacgccg agagcgatac gttcctcgac ggtatgcgcg
      481 acgtcgcctc gcaaacccgc gaggtgttca acgtcgagca gatcgaggtc agcaagggcc
      541 cgggctcggc ctacaccggc gccggctcca ccggtggcag cctgaacctg atcagcaaga
      601 ccgccaagca ggacaacttc accgacgccg gcttcacctg gggctcagac cagacccgcc
      661 gcactactct cgacgtgaac cggatgattg gcgacaacgc cgccttccgc ctcaacctga
      721 tgaagcacga tgcccacgtc gcagggcgcg acgaagtcag cgtcagccgc tggggcgtgg
      781 ctccgaccgt taccttcggc ttcgacacgc cgacccgcgc caccctgtcc tattaccatc
      841 tcagcaccga tgatatgccg gactacggcc tgccgctgac caacgtcaac cgcagcaaag
      901 ccaatccgag caagccggcc tcggtggatc gcgataactt ctatggcctg aaggaccgcg
      961 actatcgcaa gagcaccacc gacagcggta ccttccgcat cgagcatgac ctgaacgaca
     1021 acctgaccct gtcgaatagc acccgcctgg tgcgcacgac gctcgactac atcgtcagca
     1081 accccgacga cagccgtggc aacgttgcca acggctacgt ctaccgttcg gcgaaaagcc
     1141 gcaattcgac ctcgaagggc tgggtcaacc agaccgacct gaaggccaac ttcgaaaccg
     1201 gcttcatcaa gcacaccctg gtgaccggcc tggagttcag ctacgaggac gtgcacaacc
     1261 gtccgtacgc cattacttcc ggtggcggcg ccggcaacac ctgcaatgca cgattgctcg
     1321 ccagtggcga ctgcaccagc ctgaatcgcc cgactccggg cgacaactgg accggcagca
     1381 tcaccgacgg cctggcctat accgataccg acaccaagac cagcgcggct tatgtgttcg
     1441 ataccctgaa gctaagcgaa caatgggagc tgaatctcgg tctgcgctac gacgacttcg
     1501 ataccaagtc cagcggctac cagacggctg gccgcaacgg tcccgccggc tacttcaagc
     1561 gcgagaacaa cagccacttc tggaactacc agaccggcct ggtctacaaa ccggcgccca
     1621 acggcagcat ctacctggcc tggtcgacct cgagcaaccc caccggcgag accggcggcg
     1681 agggccaggc ggatatcagc gtaggcaaca acgggttgga tccggagcgt aaccgtaacc
     1741 tcgaactggg taccaagtgg gccttcttcg acgatgctct gtcgctgaac gccgcgcttt
     1801 tccgcaccga caagaccaat gcccgggtcg cttcgccgga tgtctccact cttcaggtac
     1861 tggacggcga gcagcgggtg cagggcgtcg agctgggctt caatggcaag ctgaccgaga
     1921 agtggaaggt cttcggcggc tacacctacc tcgacagcga aatccgcaag tcgacggtga
     1981 agagcgacga aggcaacaag atgccgcaga ccgcgcagaa caacttcact ctgtggacca
     2041 cctacgatct gttgcagaac ttcactatcg gcggtggtac cacctatgtg gacaagcagt
     2101 acggcaacac tgcgaactct acctatatcc cgtcctactg gcgctatgac gccatggcca
     2161 gctacaaggt gagcaagaac gtcgacctgc aactgaacgt gcagaacctg accgacaagc
     2221 gctacttcga ccaggtctac agcacgcaca tggcccacgt cgctccgggc cgcacggcat
     2281 tgctcggggt gaacttccac ttctcggccg cactcgagca ccaccaccac caccactgag
     2341 atccggctgc taacaaagcc cgaaaggaag ctgagttggc tgctgccacc gctgagcaat
     2401 aactagcata accccttggg gcctctaaac gggtcttgag gggttttttg ctgaaaggag
     2461 gaactatatc cggattggcg aatgggacgc gccctgtagc ggcgcattaa gcgcggcggg
     2521 tgtggtggtt acgcgcagcg tgaccgctac acttgccagc gccctagcgc ccgctccttt
     2581 cgctttcttc ccttcctttc tcgccacgtt cgccggcttt ccccgtcaag ctctaaatcg
     2641 ggggctccct ttagggttcc gatttagtgc tttacggcac ctcgacccca aaaaacttga
     2701 ttagggtgat ggttcacgta gtgggccatc gccctgatag acggtttttc gccctttgac
     2761 gttggagtcc acgttcttta atagtggact cttgttccaa actggaacaa cactcaaccc
     2821 tatctcggtc tattcttttg atttataagg gattttgccg atttcggcct attggttaaa
     2881 aaatgagctg atttaacaaa aatttaacgc gaattttaac aaaatattaa cgcttacaat
     2941 ttaggtggca cttttcgggg aaatgtgcgc ggaaccccta tttgtttatt tttctaaata
     3001 cattcaaata tgtatccgct catgagacaa taaccctgat aaatgcttca ataatattga
     3061 aaaaggaaga gtatgagtat tcaacatttc cgtgtcgccc ttattccctt ttttgcggca
     3121 ttttgccttc ctgtttttgc tcacccagaa acgctggtga aagtaaaaga tgctgaagat
     3181 cagttgggtg cacgagtggg ttacatcgaa ctggatctca acagcggtaa gatccttgag
     3241 agttttcgcc ccgaagaacg ttttccaatg atgagcactt ttaaagttct gctatgtggc
     3301 gcggtattat cccgtattga cgccgggcaa gagcaactcg gtcgccgcat acactattct
     3361 cagaatgact tggttgagta ctcaccagtc acagaaaagc atcttacgga tggcatgaca
     3421 gtaagagaat tatgcagtgc tgccataacc atgagtgata acactgcggc caacttactt
     3481 ctgacaacga tcggaggacc gaaggagcta accgcttttt tgcacaacat gggggatcat
     3541 gtaactcgcc ttgatcgttg ggaaccggag ctgaatgaag ccataccaaa cgacgagcgt
     3601 gacaccacga tgcctgcagc aatggcaaca acgttgcgca aactattaac tggcgaacta
     3661 cttactctag cttcccggca acaattaata gactggatgg aggcggataa agttgcagga
     3721 ccacttctgc gctcggccct tccggctggc tggtttattg ctgataaatc tggagccggt
     3781 gagcgtgggt ctcgcggtat cattgcagca ctggggccag atggtaagcc ctcccgtatc
     3841 gtagttatct acacgacggg gagtcaggca actatggatg aacgaaatag acagatcgct
     3901 gagataggtg cctcactgat taagcattgg taactgtcag accaagttta ctcatatata
     3961 ctttagattg atttaaaact tcatttttaa tttaaaagga tctaggtgaa gatccttttt
     4021 gataatctca tgaccaaaat cccttaacgt gagttttcgt tccactgagc gtcagacccc
     4081 gtagaaaaga tcaaaggatc ttcttgagat cctttttttc tgcgcgtaat ctgctgcttg
     4141 caaacaaaaa aaccaccgct accagcggtg gtttgtttgc cggatcaaga gctaccaact
     4201 ctttttccga aggtaactgg cttcagcaga gcgcagatac caaatactgt ccttctagtg
     4261 tagccgtagt taggccacca cttcaagaac tctgtagcac cgcctacata cctcgctctg
     4321 ctaatcctgt taccagtggc tgctgccagt ggcgataagt cgtgtcttac cgggttggac
     4381 tcaagacgat agttaccgga taaggcgcag cggtcgggct gaacgggggg ttcgtgcaca
     4441 cagcccagct tggagcgaac gacctacacc gaactgagat acctacagcg tgagctatga
     4501 gaaagcgcca cgcttcccga agggagaaag gcggacaggt atccggtaag cggcagggtc
     4561 ggaacaggag agcgcacgag ggagcttcca gggggaaacg cctggtatct ttatagtcct
     4621 gtcgggtttc gccacctctg acttgagcgt cgatttttgt gatgctcgtc aggggggcgg
     4681 agcctatgga aaaacgccag caacgcggcc tttttacggt tcctggcctt ttgctggcct
     4741 tttgctcaca tgttctttcc tgcgttatcc cctgattctg tggataaccg tattaccgcc
     4801 tttgagtgag ctgataccgc tcgccgcagc cgaacgaccg agcgcagcga gtcagtgagc
     4861 gaggaagcgg aagagcgcct gatgcggtat tttctcctta cgcatctgtg cggtatttca
     4921 caccgcaatg gtgcactctc agtacaatct gctctgatgc cgcatagtta agccagtata
     4981 cactccgcta tcgctacgtg actgggtcat ggctgcgccc cgacacccgc caacacccgc
     5041 tgacgcgccc tgacgggctt gtctgctccc ggcatccgct tacagacaag ctgtgaccgt
     5101 ctccgggagc tgcatgtgtc agaggttttc accgtcatca ccgaaacgcg cgaggcagct
     5161 gcggtaaagc tcatcagcgt ggtcgtgaag cgattcacag atgtctgcct gttcatccgc
     5221 gtccagctcg ttgagtttct ccagaagcgt taatgtctgg cttctgataa agcgggccat
     5281 gttaagggcg gttttttcct gtttggtcac tgatgcctcc gtgtaagggg gatttctgtt
     5341 catgggggta atgataccga tgaaacgaga gaggatgctc acgatacggg ttactgatga
     5401 tgaacatgcc cggttactgg aacgttgtga gggtaaacaa ctggcggtat ggatgcggcg
     5461 ggaccagaga aaaatcactc agggtcaatg ccagcgcttc gttaatacag atgtaggtgt
     5521 tccacagggt agccagcagc atcctgcgat gcagatccgg aacataatgg tgcagggcgc
     5581 tgacttccgc gtttccagac tttacgaaac acggaaaccg aagaccattc atgttgttgc
     5641 tcaggtcgca gacgttttgc agcagcagtc gcttcacgtt cgctcgcgta tcggtgattc
     5701 attctgctaa ccagtaaggc aaccccgcca gcctagccgg gtcctcaacg acaggagcac
     5761 gatcatgcgc acccgtggcc aggacccaac gctgcccgag atctcgatcc cgcgaaat
//
