LOCUS       sequence-434432-        6981 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Study the interaction of FeoB in P. aeruginosa, specifically 
            focusing on the variant in which outer loop 4 (OL4) is substituted 
            with OL3 in its interaction with exogenous peptides..
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 6981)
  AUTHORS   Sharifian Gh M, Norouzi F, Sorci M, Zaidi TS, Pier GB, Achimovich A,
            Ongwae GM, Liang B, Ryan M, Lemke M, Belfort G, Gadjeva M, Gahlmann 
            A, Pires MM, Venter H, Harris TE, Laurie GW
  TITLE     Lacritin cleavage-potentiated targeting of iron - respiratory 
            reciprocity promotes bacterial death.
  JOURNAL   J Biol Chem. 2025 May;301(5):108455. doi: 10.1016/j.jbc.2025.108455.
            Epub 2025 Mar 26.
  PUBMED    40154612
REFERENCE   2  (bases 1 to 6981)
  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: "J Biol 
            Chem."; date: "2025-05"; volume: "301(5):108455. doi"; pages: " 
            10.1016/j.jbc.2025.108455. Epub 2025 Mar 26"
COMMENT     Sequence Label: pET41-FeoB-OL3/4sub
FEATURES             Location/Qualifiers
     source          1..6981
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     CDS             1006..1023
                     /codon_start=1
                     /product="thrombin recognition and cleavage site"
                     /label=thrombin site
                     /translation="LVPRGS"
     CDS             1048..1071
                     /codon_start=1
                     /product="8xHis affinity tag"
                     /label=8xHis
                     /translation="HHHHHHHH"
     primer_bind     complement(1134..1152)
                     /label=T7 Term
                     /note="T7 terminator, reverse primer"
     terminator      1148..1195
                     /label=T7 terminator
                     /note="transcription terminator for bacteriophage T7 RNA 
                     polymerase"
     rep_origin      1232..1687
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(1319..1338)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     1529..1550
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     CDS             complement(1779..2594)
                     /codon_start=1
                     /product="aminoglycoside phosphotransferase"
                     /label=KanR
                     /note="confers resistance to kanamycin in bacteria or G418 
                     (Geneticin(R)) in eukaryotes"
                     /translation="MSHIQRETSCSRPRLNSNMDADLYGYKWARDNVGQSGATIYRLYG
                     KPDAPELFLKHGKGSVANDVTDEMVRLNWLTEFMPLPTIKHFIRTPDDAWLLTTAIPGK
                     TAFQVLEEYPDSGENIVDALAVFLRRLHSIPVCNCPFNSDRVFRLAQAQSRMNNGLVDA
                     SDFDDERNGWPVEQVWKEMHKLLPFSPDSVVTHGDFSLDNLIFDEGKLIGCIDVGRVGI
                     ADRYQDLAILWNCLGEFSPSLQKRLFQKYGIDNPDMNKLQFHLMLDEFF"
     CDS             complement(1779..2594)
                     /codon_start=1
                     /gene="aph(3')-Ia"
                     /product="aminoglycoside phosphotransferase"
                     /label=KanR
                     /note="confers resistance to kanamycin in bacteria or G418 
                     (Geneticin(R)) in eukaryotes"
                     /translation="MSHIQRETSCSRPRLNSNMDADLYGYKWARDNVGQSGATIYRLYG
                     KPDAPELFLKHGKGSVANDVTDEMVRLNWLTEFMPLPTIKHFIRTPDDAWLLTTAIPGK
                     TAFQVLEEYPDSGENIVDALAVFLRRLHSIPVCNCPFNSDRVFRLAQAQSRMNNGLVDA
                     SDFDDERNGWPVEQVWKEMHKLLPFSPDSVVTHGDFSLDNLIFDEGKLIGCIDVGRVGI
                     ADRYQDLAILWNCLGEFSPSLQKRLFQKYGIDNPDMNKLQFHLMLDEFF"
     primer_bind     2502..2521
                     /label=Kan-R
                     /note="Kanamycin resistance gene, reverse primer"
     primer_bind     2586..2605
                     /label=pENTR-R
                     /note="pENTR vectors, reverse primer"
     rep_origin      2716..3304
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     3205..3224
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     3458..3475
                     /label=L4440
                     /note="L4440 vector, forward primer"
     misc_feature    3490..3629
                     /label=bom
                     /note="basis of mobility region from pBR322"
     primer_bind     complement(3555..3574)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     3715..3737
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     CDS             complement(3731..3922)
                     /codon_start=1
                     /gene="rop"
                     /product="Rop protein, which maintains plasmids at low copy
                     number"
                     /label=rop
                     /translation="MTKQEKTALNMARFIRSQTLTLLEKLNELDADEQADICESLHDHA
                     DELYRSCLARFGDDGENL"
     CDS             complement(4494..5576)
                     /codon_start=1
                     /gene="lacI"
                     /product="lac repressor"
                     /label=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)."
                     /translation="MKPVTLYDVAEYAGVSYQTVSRVVNQASHVSAKTREKVEAAMAEL
                     NYIPNRVAQQLAGKQSLLIGVATSSLALHAPSQIVAAIKSRADQLGASVVVSMVERSGV
                     EACKAAVHNLLAQRVSGLIINYPLDDQDAIAVEAACTNVPALFLDVSDQTPINSIIFSH
                     EDGTRLGVEHLVALGHQQIALLAGPLSSVSARLRLAGWHKYLTRNQIQPIAEREGDWSA
                     MSGFQQTMQMLNEGIVPTAMLVANDQMALGAMRAITESGLRVGADISVVGYDDTEDSSC
                     YIPPLTTIKQDFRLLGQTSVDRLLQLSQGQAVKGNQLLPVSLVKRKTTLAPNTQTASPR
                     ALADSLMQLARQVSRLESGQ"
     primer_bind     5538..5557
                     /label=LacI-R
                     /note="LacI, reverse primer"
     promoter        complement(5577..5654)
                     /gene="lacI"
                     /label=lacI promoter
     primer_bind     5860..5879
                     /label=pBRrevBam
                     /note="pBR322 vectors, tet region, downstream of BamHI, 
                     reverse primer"
     primer_bind     5967..5986
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        5967..5985
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     protein_bind    5986..6010
                     /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)."
     RBS             6025..6047
                     /note="efficient ribosome binding site from bacteriophage 
                     T7 gene 10 (Olins and Rangwala, 1989)"
ORIGIN
        1 ggctcgtcgg cgatcttcat cgacggcatc cagtggctgg gtatccgctt cggcctgccc
       61 gattggctca cggtgttcct cgcccagggc atcggtggcg gggtcaacac ggtgttgccg
      121 ctggtgccgc agatcggctt gatgtacctg ttcctctcgc tgctcgagga ttccggctac
      181 atggcacgcg cggccttcgt catggaccgc ctgatgcagg cgctgggcct gccgggcaag
      241 tcgttcgtgc cgctgatcgt cggtttcggc tgcaacgtac cgtcgatcat gggcgcgcgg
      301 accctggacg cgcagcgcga gcggctgatt accatcatga tggcgccgtt catgtcctgc
      361 ggcgcgcgcc tggcgatctt cgccgtattc gccggggcct tcttcggcca gggcggcgcg
      421 ctggtgatct tctccctgta cctgctcggc atcgtcgtgg cgatcctcac cgggctgttg
      481 ctcaagcaca ccttgatgcg cggcgaggcc tcgccgttcg tcatggaact gccgctgtac
      541 catgtgccac accttaagag cctgctgctg cagacctggt cgcgcctgcg cggcttcgtg
      601 gtgcgggccg gcaaggtgat catcctggtc agcctggtga tcggcggcct gaacagcatc
      661 ggggccttct tcggccaggg cggcgcggcg gcctacagct atctggtctt cgtcctgctc
      721 tacgtgccct gcgtgaccgc catgggcgcg atcgcccggg aaagcagcaa gggctggatg
      781 gctttctcgg tgctctgggg gctgaacgtc gcctactcgc tggcgaccct ctgctaccag
      841 gtcgccacct tcgccgcgca ccccgagcgt agcgtcctga ccatcgccgt ggtgctgctc
      901 ttcaacctga tcctgatgac ctgcctgcgt ctgttcggcc gtgagcaggt actgcaactg
      961 ccaggccgaa tggccgatgc cccgagcggg cagggaggat gtcacctggt gccacgcggt
     1021 agtgggaagc ttgcggccgc actcgagcac caccaccacc accaccacca ctaattgatt
     1081 aatacctagg ctgctaaaca aagcccgaaa ggaagctgag ttggctgctg ccaccgctga
     1141 gcaataacta gcataacccc ttggggcctc taaacgggtc ttgaggggtt ttttgctgaa
     1201 aggaggaact atatccggat tggcgaatgg gacgcgccct gtagcggcgc attaagcgcg
     1261 gcgggtgtgg tggttacgcg cagcgtgacc gctacacttg ccagcgccct agcgcccgct
     1321 cctttcgctt tcttcccttc ctttctcgcc acgttcgccg gctttccccg tcaagctcta
     1381 aatcgggggc tccctttagg gttccgattt agtgctttac ggcacctcga ccccaaaaaa
     1441 cttgattagg gtgatggttc acgtagtggg ccatcgccct gatagacggt ttttcgccct
     1501 ttgacgttgg agtccacgtt ctttaatagt ggactcttgt tccaaactgg aacaacactc
     1561 aaccctatct cggtctattc ttttgattta taagggattt tgccgatttc ggcctattgg
     1621 ttaaaaaatg agctgattta acaaaaattt aacgcgaatt ttaacaaaat attaacgctt
     1681 acaatttagg tggcactttt cggggaaatg tgcgcggaac ccctatttgt ttatttttct
     1741 aaatacattc aaatatgtat ccgctcatga attaattctt agaaaaactc atcgagcatc
     1801 aaatgaaact gcaatttatt catatcagga ttatcaatac catatttttg aaaaagccgt
     1861 ttctgtaatg aaggagaaaa ctcaccgagg cagttccata ggatggcaag atcctggtat
     1921 cggtctgcga ttccgactcg tccaacatca atacaaccta ttaatttccc ctcgtcaaaa
     1981 ataaggttat caagtgagaa atcaccatga gtgacgactg aatccggtga gaatggcaaa
     2041 agtttatgca tttctttcca gacttgttca acaggccagc cattacgctc gtcatcaaaa
     2101 tcactcgcat caaccaaacc gttattcatt cgtgattgcg cctgagcgag acgaaatacg
     2161 cgatcgctgt taaaaggaca attacaaaca ggaatcgaat gcaaccggcg caggaacact
     2221 gccagcgcat caacaatatt ttcacctgaa tcaggatatt cttctaatac ctggaatgct
     2281 gttttcccgg ggatcgcagt ggtgagtaac catgcatcat caggagtacg gataaaatgc
     2341 ttgatggtcg gaagaggcat aaattccgtc agccagttta gtctgaccat ctcatctgta
     2401 acatcattgg caacgctacc tttgccatgt ttcagaaaca actctggcgc atcgggcttc
     2461 ccatacaatc gatagattgt cgcacctgat tgcccgacat tatcgcgagc ccatttatac
     2521 ccatataaat cagcatccat gttggaattt aatcgcggcc tagagcaaga cgtttcccgt
     2581 tgaatatggc tcataacacc ccttgtatta ctgtttatgt aagcagacag ttttattgtt
     2641 catgaccaaa atcccttaac gtgagttttc gttccactga gcgtcagacc ccgtagaaaa
     2701 gatcaaagga tcttcttgag atcctttttt tctgcgcgta atctgctgct tgcaaacaaa
     2761 aaaaccaccg ctaccagcgg tggtttgttt gccggatcaa gagctaccaa ctctttttcc
     2821 gaaggtaact ggcttcagca gagcgcagat accaaatact gtccttctag tgtagccgta
     2881 gttaggccac cacttcaaga actctgtagc accgcctaca tacctcgctc tgctaatcct
     2941 gttaccagtg gctgctgcca gtggcgataa gtcgtgtctt accgggttgg actcaagacg
     3001 atagttaccg gataaggcgc agcggtcggg ctgaacgggg ggttcgtgca cacagcccag
     3061 cttggagcga acgacctaca ccgaactgag atacctacag cgtgagctat gagaaagcgc
     3121 cacgcttccc gaagggagaa aggcggacag gtatccggta agcggcaggg tcggaacagg
     3181 agagcgcacg agggagcttc cagggggaaa cgcctggtat ctttatagtc ctgtcgggtt
     3241 tcgccacctc tgacttgagc gtcgattttt gtgatgctcg tcaggggggc ggagcctatg
     3301 gaaaaacgcc agcaacgcgg cctttttacg gttcctggcc ttttgctggc cttttgctca
     3361 catgttcttt cctgcgttat cccctgattc tgtggataac cgtattaccg cctttgagtg
     3421 agctgatacc gctcgccgca gccgaacgac cgagcgcagc gagtcagtga gcgaggaagc
     3481 ggaagagcgc ctgatgcggt attttctcct tacgcatctg tgcggtattt cacaccgcaa
     3541 tggtgcactc tcagtacaat ctgctctgat gccgcatagt taagccagta tacactccgc
     3601 tatcgctacg tgactgggtc atggctgcgc cccgacaccc gccaacaccc gctgacgcgc
     3661 cctgacgggc ttgtctgctc ccggcatccg cttacagaca agctgtgacc gtctccggga
     3721 gctgcatgtg tcagaggttt tcaccgtcat caccgaaacg cgcgaggcag ctgcggtaaa
     3781 gctcatcagc gtggtcgtga agcgattcac agatgtctgc ctgttcatcc gcgtccagct
     3841 cgttgagttt ctccagaagc gttaatgtct ggcttctgat aaagcgggcc atgttaaggg
     3901 cggttttttc ctgtttggtc actgatgcct ccgtgtaagg gggatttctg ttcatggggg
     3961 taatgatacc gatgaaacga gagaggatgc tcacgatacg ggttactgat gatgaacatg
     4021 cccggttact ggaacgttgt gagggtaaac aactggcggt atggatgcgg cgggaccaga
     4081 gaaaaatcac tcagggtcaa tgccagcgct tcgttaatac agatgtaggt gttccacagg
     4141 gtagccagca gcatcctgcg atgcagatcc ggaacataat ggtgcagggc gctgacttcc
     4201 gcgtttccag actttacgaa acacggaaac cgaagaccat tcatgttgtt gctcaggtcg
     4261 cagacgtttt gcagcagcag tcgcttcacg ttcgctcgcg tatcggtgat tcattctgct
     4321 aaccagtaag gcaaccccgc cagcctagcc gggtcctcaa cgacaggagc acgatcatgc
     4381 tagtcatgcc ccgcgcccac cggaaggagc tgactgggtt gaaggctctc aagggcatcg
     4441 gtcgagatcc cggtgcctaa tgagtgagct aacttacatt aattgcgttg cgctcactgc
     4501 ccgctttcca gtcgggaaac ctgtcgtgcc agctgcatta atgaatcggc caacgcgcgg
     4561 ggagaggcgg tttgcgtatt gggcgccagg gtggtttttc ttttcaccag tgagacgggc
     4621 aacagctgat tgcccttcac cgcctggccc tgagagagtt gcagcaagcg gtccacgctg
     4681 gtttgcccca gcaggcgaaa atcctgtttg atggtggtta acggcgggat ataacatgag
     4741 ctgtcttcgg tatcgtcgta tcccactacc gagatgtccg caccaacgcg cagcccggac
     4801 tcggtaatgg cgcgcattgc gcccagcgcc atctgatcgt tggcaaccag catcgcagtg
     4861 ggaacgatgc cctcattcag catttgcatg gtttgttgaa aaccggacat ggcactccag
     4921 tcgccttccc gttccgctat cggctgaatt tgattgcgag tgagatattt atgccagcca
     4981 gccagacgca gacgcgccga gacagaactt aatgggcccg ctaacagcgc gatttgctgg
     5041 tgacccaatg cgaccagatg ctccacgccc agtcgcgtac cgtcttcatg ggagaaaata
     5101 atactgttga tgggtgtctg gtcagagaca tcaagaaata acgccggaac attagtgcag
     5161 gcagcttcca cagcaatggc atcctggtca tccagcggat agttaatgat cagcccactg
     5221 acgcgttgcg cgagaagatt gtgcaccgcc gctttacagg cttcgacgcc gcttcgttct
     5281 accatcgaca ccaccacgct ggcacccagt tgatcggcgc gagatttaat cgccgcgaca
     5341 atttgcgacg gcgcgtgcag ggccagactg gaggtggcaa cgccaatcag caacgactgt
     5401 ttgcccgcca gttgttgtgc cacgcggttg ggaatgtaat tcagctccgc catcgccgct
     5461 tccacttttt cccgcgtttt cgcagaaacg tggctggcct ggttcaccac gcgggaaacg
     5521 gtctgataag agacaccggc atactctgcg acatcgtata acgttactgg tttcacattc
     5581 accaccctga attgactctc ttccgggcgc tatcatgcca taccgcgaaa ggttttgcgc
     5641 cattcgatgg tgtccgggat ctcgacgctc tcccttatgc gactcctgca ttaggaagca
     5701 gcccagtagt aggttgaggc cgttgagcac cgccgccgca aggaatggtg catgcaagga
     5761 gatggcgccc aacagtcccc cggccacggg gcctgccacc atacccacgc cgaaacaagc
     5821 gctcatgagc ccgaagtggc gagcccgatc ttccccatcg gtgatgtcgg cgatataggc
     5881 gccagcaacc gcacctgtgg cgccggtgat gccggccacg atgcgtccgg cgtagaggat
     5941 cgagatcgat ctcgatcccg cgaaattaat acgactcact ataggggaat tgtgagcgga
     6001 taacaattcc cctctagaaa taattttgtt taactttaag aaggagatat acatatgacc
     6061 gcattgaccc tcggcctgat cggcaatccc aactccggca agaccaccct gttcaaccag
     6121 ctcaccggct cgcgccagcg ggtcggcaac tgggccggcg tcaccgtgga gcgcaaggag
     6181 ggtgcgttcc ataccgtccg tcacgcggtg cgcctggtcg acctgcccgg cacctattcg
     6241 ttgaccagcg tttccgccca ggcctcgctg gacgagcaga tcgcctgccg ctacatcgcc
     6301 agcggcgaag tggacgtgct ggtcaacgtg gtggatgcgg cgaacctgga gcgcaacctg
     6361 tacctcacgg tgcaactgcg ggagatgggc atcccctgca tcgtcgcgct gaacatgctc
     6421 gacatcgccc gtagccagcg catccgcatc gatatcgacg gtctggcgcg gcgtctcggc
     6481 tgcccggtgg tgccgctggt atccacccgc gccgatggca tcgacgaact gaaggcggcc
     6541 atcgactccc tacagctccc gcaggcggcg ctggcggtgg attacccacc ggccatccag
     6601 gcgcaggtcg gctaccttct ggaaacgcgc gctccggcgg cctcggccat cgagccgcgc
     6661 tggctggcgt tgcaggcgct ggaaggcgac atcttcaacg gcccggcgct gggcttgccg
     6721 cccgccaccc tggaacaggc gcgccggggc tgcggcgagg agcccgagct ggcgatcgtc
     6781 gatgcgcgct accggctgat cggcgagatc tgtgcggcgg tctgcgacca ccagcaggct
     6841 cagccgcacc ggctgaccca atggctcgac cgggtcgtcc tgaatcgctg gctgggtctg
     6901 ccgatcttcc tgctggtgat gtacctgatg ttcttcttcg ccatcaacat cggcggggcg
     6961 ctacagccga tcttcgacaa g
//
