LOCUS       sequence-434431-        6969 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 OL5 in its interaction with exogenous peptides..
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 6969)
  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 6969)
  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-OL5/4sub
FEATURES             Location/Qualifiers
     source          1..6969
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     RBS             1773..1795
                     /note="efficient ribosome binding site from bacteriophage 
                     T7 gene 10 (Olins and Rangwala, 1989)"
     protein_bind    1810..1834
                     /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)."
     primer_bind     complement(1834..1853)
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        complement(1835..1853)
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     primer_bind     complement(1941..1960)
                     /label=pBRrevBam
                     /note="pBR322 vectors, tet region, downstream of BamHI, 
                     reverse primer"
     promoter        2166..2243
                     /gene="lacI"
                     /label=lacI promoter
     CDS             2244..3326
                     /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     complement(2263..2282)
                     /label=LacI-R
                     /note="LacI, reverse primer"
     CDS             3898..4089
                     /codon_start=1
                     /gene="rop"
                     /product="Rop protein, which maintains plasmids at low copy
                     number"
                     /label=rop
                     /translation="MTKQEKTALNMARFIRSQTLTLLEKLNELDADEQADICESLHDHA
                     DELYRSCLARFGDDGENL"
     primer_bind     complement(4083..4105)
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     misc_feature    4191..4330
                     /label=bom
                     /note="basis of mobility region from pBR322"
     primer_bind     4246..4265
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     complement(4345..4362)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(4516..5104)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(4596..4615)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     complement(5215..5234)
                     /label=pENTR-R
                     /note="pENTR vectors, reverse primer"
     CDS             5226..6041
                     /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             5226..6041
                     /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     complement(5299..5318)
                     /label=Kan-R
                     /note="Kanamycin resistance gene, reverse primer"
     rep_origin      complement(6133..6588)
                     /direction=LEFT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(6270..6291)
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     6482..6501
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     terminator      complement(6625..6672)
                     /label=T7 terminator
                     /note="transcription terminator for bacteriophage T7 RNA 
                     polymerase"
     primer_bind     6668..6686
                     /label=T7 Term
                     /note="T7 terminator, reverse primer"
     CDS             complement(6749..6772)
                     /codon_start=1
                     /product="8xHis affinity tag"
                     /label=8xHis
                     /translation="HHHHHHHH"
     CDS             complement(6797..6814)
                     /codon_start=1
                     /product="thrombin recognition and cleavage site"
                     /label=thrombin site
                     /translation="LVPRGS"
ORIGIN
        1 aggtggcgac ctggtagcag agggtcgcca gcgagtaggc gacgttcagc ccccagagca
       61 ccgagaaagc catccagccc ttgctgcttt cccgggcgat cgcgcccatg gcggtcacgc
      121 agggcacgta gagcaggacg aagaccagat agctgtaggc cgccgcggcg aaggtggcga
      181 tgctgttcag gccgccgatc accaggctga ccaggatgat caccttgccg gcccgcacca
      241 cgaagccgcg caggcgcgac caggtctgca gcagcaggct cttaaggtgt ggcacatggt
      301 acagcggcag ttccatgacg aacggcgagg cctcgccgcg catcaaggtg tgcttgagca
      361 acagcccggt gaggatcgcc acgacgatgc cgagcaggta cagggagaag atcaccagcg
      421 cgccgccctg gccgaagaag gccccggcga atacggcgaa gatcgccagg cgcgcgccgc
      481 aggacatgaa cggcgccatc atgatggtaa tcagccgctc gcgctgcgcg tccagggtcc
      541 gcgcgcccat gatcgacggt acgttgcagc cgaaaccgac gatcagcggc acgaacgact
      601 tgcccggcag gcccagcgcc tgcatcaggc ggtccatgac gaaggccgcg cgtgccatgt
      661 agccggaatc ctcgagcagc gagaggaaca ggtacatcaa gccgatctgc ggcaccagcg
      721 gcaacaccgt gttgaccccg ccaccgatgc cctgggcgag gaacaccgtg agccaatcgg
      781 gcaggccgaa gcggataccc agccactgga tgccgtcgat gaagatcgcc gacgagccct
      841 tgtcgaagat cggctgtagc gccccgccga tgttgatggc gaagaagaac atcaggtaca
      901 tcaccagcag gaagatcggc agacccagcc agcgattcag gacgacccgg tcgagccatt
      961 gggtcagccg gtgcggctga gcctgctggt ggtcgcagac cgccgcacag atctcgccga
     1021 tcagccggta gcgcgcatcg acgatcgcca gctcgggctc ctcgccgcag ccccggcgcg
     1081 cctgttccag ggtggcgggc ggcaagccca gcgccgggcc gttgaagatg tcgccttcca
     1141 gcgcctgcaa cgccagccag cgcggctcga tggccgaggc cgccggagcg cgcgtttcca
     1201 gaaggtagcc gacctgcgcc tggatggccg gtgggtaatc caccgccagc gccgcctgcg
     1261 ggagctgtag ggagtcgatg gccgccttca gttcgtcgat gccatcggcg cgggtggata
     1321 ccagcggcac caccgggcag ccgagacgcc gcgccagacc gtcgatatcg atgcggatgc
     1381 gctggctacg ggcgatgtcg agcatgttca gcgcgacgat gcaggggatg cccatctccc
     1441 gcagttgcac cgtgaggtac aggttgcgct ccaggttcgc cgcatccacc acgttgacca
     1501 gcacgtccac ttcgccgctg gcgatgtagc ggcaggcgat ctgctcgtcc agcgaggcct
     1561 gggcggaaac gctggtcaac gaataggtgc cgggcaggtc gaccaggcgc accgcgtgac
     1621 ggacggtatg gaacgcaccc tccttgcgct ccacggtgac gccggcccag ttgccgaccc
     1681 gctggcgcga gccggtgagc tggttgaaca gggtggtctt gccggagttg ggattgccga
     1741 tcaggccgag ggtcaatgcg gtcatatgta tatctccttc ttaaagttaa acaaaattat
     1801 ttctagaggg gaattgttat ccgctcacaa ttcccctata gtgagtcgta ttaatttcgc
     1861 gggatcgaga tcgatctcga tcctctacgc cggacgcatc gtggccggca tcaccggcgc
     1921 cacaggtgcg gttgctggcg cctatatcgc cgacatcacc gatggggaag atcgggctcg
     1981 ccacttcggg ctcatgagcg cttgtttcgg cgtgggtatg gtggcaggcc ccgtggccgg
     2041 gggactgttg ggcgccatct ccttgcatgc accattcctt gcggcggcgg tgctcaacgg
     2101 cctcaaccta ctactgggct gcttcctaat gcaggagtcg cataagggag agcgtcgaga
     2161 tcccggacac catcgaatgg cgcaaaacct ttcgcggtat ggcatgatag cgcccggaag
     2221 agagtcaatt cagggtggtg aatgtgaaac cagtaacgtt atacgatgtc gcagagtatg
     2281 ccggtgtctc ttatcagacc gtttcccgcg tggtgaacca ggccagccac gtttctgcga
     2341 aaacgcggga aaaagtggaa gcggcgatgg cggagctgaa ttacattccc aaccgcgtgg
     2401 cacaacaact ggcgggcaaa cagtcgttgc tgattggcgt tgccacctcc agtctggccc
     2461 tgcacgcgcc gtcgcaaatt gtcgcggcga ttaaatctcg cgccgatcaa ctgggtgcca
     2521 gcgtggtggt gtcgatggta gaacgaagcg gcgtcgaagc ctgtaaagcg gcggtgcaca
     2581 atcttctcgc gcaacgcgtc agtgggctga tcattaacta tccgctggat gaccaggatg
     2641 ccattgctgt ggaagctgcc tgcactaatg ttccggcgtt atttcttgat gtctctgacc
     2701 agacacccat caacagtatt attttctccc atgaagacgg tacgcgactg ggcgtggagc
     2761 atctggtcgc attgggtcac cagcaaatcg cgctgttagc gggcccatta agttctgtct
     2821 cggcgcgtct gcgtctggct ggctggcata aatatctcac tcgcaatcaa attcagccga
     2881 tagcggaacg ggaaggcgac tggagtgcca tgtccggttt tcaacaaacc atgcaaatgc
     2941 tgaatgaggg catcgttccc actgcgatgc tggttgccaa cgatcagatg gcgctgggcg
     3001 caatgcgcgc cattaccgag tccgggctgc gcgttggtgc ggacatctcg gtagtgggat
     3061 acgacgatac cgaagacagc tcatgttata tcccgccgtt aaccaccatc aaacaggatt
     3121 ttcgcctgct ggggcaaacc agcgtggacc gcttgctgca actctctcag ggccaggcgg
     3181 tgaagggcaa tcagctgttg cccgtctcac tggtgaaaag aaaaaccacc ctggcgccca
     3241 atacgcaaac cgcctctccc cgcgcgttgg ccgattcatt aatgcagctg gcacgacagg
     3301 tttcccgact ggaaagcggg cagtgagcgc aacgcaatta atgtaagtta gctcactcat
     3361 taggcaccgg gatctcgacc gatgcccttg agagccttca acccagtcag ctccttccgg
     3421 tgggcgcggg gcatgactag catgatcgtg ctcctgtcgt tgaggacccg gctaggctgg
     3481 cggggttgcc ttactggtta gcagaatgaa tcaccgatac gcgagcgaac gtgaagcgac
     3541 tgctgctgca aaacgtctgc gacctgagca acaacatgaa tggtcttcgg tttccgtgtt
     3601 tcgtaaagtc tggaaacgcg gaagtcagcg ccctgcacca ttatgttccg gatctgcatc
     3661 gcaggatgct gctggctacc ctgtggaaca cctacatctg tattaacgaa gcgctggcat
     3721 tgaccctgag tgatttttct ctggtcccgc cgcatccata ccgccagttg tttaccctca
     3781 caacgttcca gtaaccgggc atgttcatca tcagtaaccc gtatcgtgag catcctctct
     3841 cgtttcatcg gtatcattac ccccatgaac agaaatcccc cttacacgga ggcatcagtg
     3901 accaaacagg aaaaaaccgc ccttaacatg gcccgcttta tcagaagcca gacattaacg
     3961 cttctggaga aactcaacga gctggacgcg gatgaacagg cagacatctg tgaatcgctt
     4021 cacgaccacg ctgatgagct ttaccgcagc tgcctcgcgc gtttcggtga tgacggtgaa
     4081 aacctctgac acatgcagct cccggagacg gtcacagctt gtctgtaagc ggatgccggg
     4141 agcagacaag cccgtcaggg cgcgtcagcg ggtgttggcg ggtgtcgggg cgcagccatg
     4201 acccagtcac gtagcgatag cggagtgtat actggcttaa ctatgcggca tcagagcaga
     4261 ttgtactgag agtgcaccat tgcggtgtga aataccgcac agatgcgtaa ggagaaaata
     4321 ccgcatcagg cgctcttccg cttcctcgct cactgactcg ctgcgctcgg tcgttcggct
     4381 gcggcgagcg gtatcagctc actcaaaggc ggtaatacgg ttatccacag aatcagggga
     4441 taacgcagga aagaacatgt gagcaaaagg ccagcaaaag gccaggaacc gtaaaaaggc
     4501 cgcgttgctg gcgtttttcc ataggctccg cccccctgac gagcatcaca aaaatcgacg
     4561 ctcaagtcag aggtggcgaa acccgacagg actataaaga taccaggcgt ttccccctgg
     4621 aagctccctc gtgcgctctc ctgttccgac cctgccgctt accggatacc tgtccgcctt
     4681 tctcccttcg ggaagcgtgg cgctttctca tagctcacgc tgtaggtatc tcagttcggt
     4741 gtaggtcgtt cgctccaagc tgggctgtgt gcacgaaccc cccgttcagc ccgaccgctg
     4801 cgccttatcc ggtaactatc gtcttgagtc caacccggta agacacgact tatcgccact
     4861 ggcagcagcc actggtaaca ggattagcag agcgaggtat gtaggcggtg ctacagagtt
     4921 cttgaagtgg tggcctaact acggctacac tagaaggaca gtatttggta tctgcgctct
     4981 gctgaagcca gttaccttcg gaaaaagagt tggtagctct tgatccggca aacaaaccac
     5041 cgctggtagc ggtggttttt ttgtttgcaa gcagcagatt acgcgcagaa aaaaaggatc
     5101 tcaagaagat cctttgatct tttctacggg gtctgacgct cagtggaacg aaaactcacg
     5161 ttaagggatt ttggtcatga acaataaaac tgtctgctta cataaacagt aatacaaggg
     5221 gtgttatgag ccatattcaa cgggaaacgt cttgctctag gccgcgatta aattccaaca
     5281 tggatgctga tttatatggg tataaatggg ctcgcgataa tgtcgggcaa tcaggtgcga
     5341 caatctatcg attgtatggg aagcccgatg cgccagagtt gtttctgaaa catggcaaag
     5401 gtagcgttgc caatgatgtt acagatgaga tggtcagact aaactggctg acggaattta
     5461 tgcctcttcc gaccatcaag cattttatcc gtactcctga tgatgcatgg ttactcacca
     5521 ctgcgatccc cgggaaaaca gcattccagg tattagaaga atatcctgat tcaggtgaaa
     5581 atattgttga tgcgctggca gtgttcctgc gccggttgca ttcgattcct gtttgtaatt
     5641 gtccttttaa cagcgatcgc gtatttcgtc tcgctcaggc gcaatcacga atgaataacg
     5701 gtttggttga tgcgagtgat tttgatgacg agcgtaatgg ctggcctgtt gaacaagtct
     5761 ggaaagaaat gcataaactt ttgccattct caccggattc agtcgtcact catggtgatt
     5821 tctcacttga taaccttatt tttgacgagg ggaaattaat aggttgtatt gatgttggac
     5881 gagtcggaat cgcagaccga taccaggatc ttgccatcct atggaactgc ctcggtgagt
     5941 tttctccttc attacagaaa cggctttttc aaaaatatgg tattgataat cctgatatga
     6001 ataaattgca gtttcatttg atgctcgatg agtttttcta agaattaatt catgagcgga
     6061 tacatatttg aatgtattta gaaaaataaa caaatagggg ttccgcgcac atttccccga
     6121 aaagtgccac ctaaattgta agcgttaata ttttgttaaa attcgcgtta aatttttgtt
     6181 aaatcagctc attttttaac caataggccg aaatcggcaa aatcccttat aaatcaaaag
     6241 aatagaccga gatagggttg agtgttgttc cagtttggaa caagagtcca ctattaaaga
     6301 acgtggactc caacgtcaaa gggcgaaaaa ccgtctatca gggcgatggc ccactacgtg
     6361 aaccatcacc ctaatcaagt tttttggggt cgaggtgccg taaagcacta aatcggaacc
     6421 ctaaagggag cccccgattt agagcttgac ggggaaagcc ggcgaacgtg gcgagaaagg
     6481 aagggaagaa agcgaaagga gcgggcgcta gggcgctggc aagtgtagcg gtcacgctgc
     6541 gcgtaaccac cacacccgcc gcgcttaatg cgccgctaca gggcgcgtcc cattcgccaa
     6601 tccggatata gttcctcctt tcagcaaaaa acccctcaag acccgtttag aggccccaag
     6661 gggttatgct agttattgct cagcggtggc agcagccaac tcagcttcct ttcgggcttt
     6721 gtttagcagc ctaggtatta atcaattagt ggtggtggtg gtggtggtgg tgctcgagtg
     6781 cggccgcaag cttcccacta ccgcgtggca ccaggtgaca tcctccctgc ccgctcgggg
     6841 catcggccat tcggcctggc agttgcagta cctgctcacg gccgaacaga cgcaggcagg
     6901 tcatcaggat caggttgaag agcagcacca cggcgatggt caggacgcta cgctcggggt
     6961 gcgcggcga
//
