LOCUS       sequence-446359-        7290 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Study the interaction of FeoB in P. aeruginosa, specifically 
            focusing on the role of the outer loop 2 (OL2) by examining the 
            variant lacking OL2 in its interaction with exogenous peptides..
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 7290)
  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 7290)
  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-OL2del
FEATURES             Location/Qualifiers
     source          1..7290
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     CDS             955..1146
                     /codon_start=1
                     /gene="rop"
                     /product="Rop protein, which maintains plasmids at low copy
                     number"
                     /label=rop
                     /translation="MTKQEKTALNMARFIRSQTLTLLEKLNELDADEQADICESLHDHA
                     DELYRSCLARFGDDGENL"
     primer_bind     complement(1140..1162)
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     misc_feature    1248..1387
                     /label=bom
                     /note="basis of mobility region from pBR322"
     primer_bind     1303..1322
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     complement(1402..1419)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(1573..2161)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(1653..1672)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     complement(2272..2291)
                     /label=pENTR-R
                     /note="pENTR vectors, reverse primer"
     CDS             2283..3098
                     /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             2283..3098
                     /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(2356..2375)
                     /label=Kan-R
                     /note="Kanamycin resistance gene, reverse primer"
     rep_origin      complement(3190..3645)
                     /direction=LEFT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(3327..3348)
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     3539..3558
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     terminator      complement(3682..3729)
                     /label=T7 terminator
                     /note="transcription terminator for bacteriophage T7 RNA 
                     polymerase"
     primer_bind     3725..3743
                     /label=T7 Term
                     /note="T7 terminator, reverse primer"
     CDS             complement(3806..3829)
                     /codon_start=1
                     /product="8xHis affinity tag"
                     /label=8xHis
                     /translation="HHHHHHHH"
     CDS             complement(3854..3871)
                     /codon_start=1
                     /product="thrombin recognition and cleavage site"
                     /label=thrombin site
                     /translation="LVPRGS"
     RBS             6120..6142
                     /note="efficient ribosome binding site from bacteriophage 
                     T7 gene 10 (Olins and Rangwala, 1989)"
     protein_bind    6157..6181
                     /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(6181..6200)
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        complement(6182..6200)
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     primer_bind     complement(6288..6307)
                     /label=pBRrevBam
                     /note="pBR322 vectors, tet region, downstream of BamHI, 
                     reverse primer"
     promoter        6513..6590
                     /gene="lacI"
                     /label=lacI promoter
     CDS             join(6591..7290,1..383)
                     /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(6610..6629)
                     /label=LacI-R
                     /note="LacI, reverse primer"
ORIGIN
        1 atgagggcat cgttcccact gcgatgctgg ttgccaacga tcagatggcg ctgggcgcaa
       61 tgcgcgccat taccgagtcc gggctgcgcg ttggtgcgga catctcggta gtgggatacg
      121 acgataccga agacagctca tgttatatcc cgccgttaac caccatcaaa caggattttc
      181 gcctgctggg gcaaaccagc gtggaccgct tgctgcaact ctctcagggc caggcggtga
      241 agggcaatca gctgttgccc gtctcactgg tgaaaagaaa aaccaccctg gcgcccaata
      301 cgcaaaccgc ctctccccgc gcgttggccg attcattaat gcagctggca cgacaggttt
      361 cccgactgga aagcgggcag tgagcgcaac gcaattaatg taagttagct cactcattag
      421 gcaccgggat ctcgaccgat gcccttgaga gccttcaacc cagtcagctc cttccggtgg
      481 gcgcggggca tgactagcat gatcgtgctc ctgtcgttga ggacccggct aggctggcgg
      541 ggttgcctta ctggttagca gaatgaatca ccgatacgcg agcgaacgtg aagcgactgc
      601 tgctgcaaaa cgtctgcgac ctgagcaaca acatgaatgg tcttcggttt ccgtgtttcg
      661 taaagtctgg aaacgcggaa gtcagcgccc tgcaccatta tgttccggat ctgcatcgca
      721 ggatgctgct ggctaccctg tggaacacct acatctgtat taacgaagcg ctggcattga
      781 ccctgagtga tttttctctg gtcccgccgc atccataccg ccagttgttt accctcacaa
      841 cgttccagta accgggcatg ttcatcatca gtaacccgta tcgtgagcat cctctctcgt
      901 ttcatcggta tcattacccc catgaacaga aatccccctt acacggaggc atcagtgacc
      961 aaacaggaaa aaaccgccct taacatggcc cgctttatca gaagccagac attaacgctt
     1021 ctggagaaac tcaacgagct ggacgcggat gaacaggcag acatctgtga atcgcttcac
     1081 gaccacgctg atgagcttta ccgcagctgc ctcgcgcgtt tcggtgatga cggtgaaaac
     1141 ctctgacaca tgcagctccc ggagacggtc acagcttgtc tgtaagcgga tgccgggagc
     1201 agacaagccc gtcagggcgc gtcagcgggt gttggcgggt gtcggggcgc agccatgacc
     1261 cagtcacgta gcgatagcgg agtgtatact ggcttaacta tgcggcatca gagcagattg
     1321 tactgagagt gcaccattgc ggtgtgaaat accgcacaga tgcgtaagga gaaaataccg
     1381 catcaggcgc tcttccgctt cctcgctcac tgactcgctg cgctcggtcg ttcggctgcg
     1441 gcgagcggta tcagctcact caaaggcggt aatacggtta tccacagaat caggggataa
     1501 cgcaggaaag aacatgtgag caaaaggcca gcaaaaggcc aggaaccgta aaaaggccgc
     1561 gttgctggcg tttttccata ggctccgccc ccctgacgag catcacaaaa atcgacgctc
     1621 aagtcagagg tggcgaaacc cgacaggact ataaagatac caggcgtttc cccctggaag
     1681 ctccctcgtg cgctctcctg ttccgaccct gccgcttacc ggatacctgt ccgcctttct
     1741 cccttcggga agcgtggcgc tttctcatag ctcacgctgt aggtatctca gttcggtgta
     1801 ggtcgttcgc tccaagctgg gctgtgtgca cgaacccccc gttcagcccg accgctgcgc
     1861 cttatccggt aactatcgtc ttgagtccaa cccggtaaga cacgacttat cgccactggc
     1921 agcagccact ggtaacagga ttagcagagc gaggtatgta ggcggtgcta cagagttctt
     1981 gaagtggtgg cctaactacg gctacactag aaggacagta tttggtatct gcgctctgct
     2041 gaagccagtt accttcggaa aaagagttgg tagctcttga tccggcaaac aaaccaccgc
     2101 tggtagcggt ggtttttttg tttgcaagca gcagattacg cgcagaaaaa aaggatctca
     2161 agaagatcct ttgatctttt ctacggggtc tgacgctcag tggaacgaaa actcacgtta
     2221 agggattttg gtcatgaaca ataaaactgt ctgcttacat aaacagtaat acaaggggtg
     2281 ttatgagcca tattcaacgg gaaacgtctt gctctaggcc gcgattaaat tccaacatgg
     2341 atgctgattt atatgggtat aaatgggctc gcgataatgt cgggcaatca ggtgcgacaa
     2401 tctatcgatt gtatgggaag cccgatgcgc cagagttgtt tctgaaacat ggcaaaggta
     2461 gcgttgccaa tgatgttaca gatgagatgg tcagactaaa ctggctgacg gaatttatgc
     2521 ctcttccgac catcaagcat tttatccgta ctcctgatga tgcatggtta ctcaccactg
     2581 cgatccccgg gaaaacagca ttccaggtat tagaagaata tcctgattca ggtgaaaata
     2641 ttgttgatgc gctggcagtg ttcctgcgcc ggttgcattc gattcctgtt tgtaattgtc
     2701 cttttaacag cgatcgcgta tttcgtctcg ctcaggcgca atcacgaatg aataacggtt
     2761 tggttgatgc gagtgatttt gatgacgagc gtaatggctg gcctgttgaa caagtctgga
     2821 aagaaatgca taaacttttg ccattctcac cggattcagt cgtcactcat ggtgatttct
     2881 cacttgataa ccttattttt gacgagggga aattaatagg ttgtattgat gttggacgag
     2941 tcggaatcgc agaccgatac caggatcttg ccatcctatg gaactgcctc ggtgagtttt
     3001 ctccttcatt acagaaacgg ctttttcaaa aatatggtat tgataatcct gatatgaata
     3061 aattgcagtt tcatttgatg ctcgatgagt ttttctaaga attaattcat gagcggatac
     3121 atatttgaat gtatttagaa aaataaacaa ataggggttc cgcgcacatt tccccgaaaa
     3181 gtgccaccta aattgtaagc gttaatattt tgttaaaatt cgcgttaaat ttttgttaaa
     3241 tcagctcatt ttttaaccaa taggccgaaa tcggcaaaat cccttataaa tcaaaagaat
     3301 agaccgagat agggttgagt gttgttccag tttggaacaa gagtccacta ttaaagaacg
     3361 tggactccaa cgtcaaaggg cgaaaaaccg tctatcaggg cgatggccca ctacgtgaac
     3421 catcacccta atcaagtttt ttggggtcga ggtgccgtaa agcactaaat cggaacccta
     3481 aagggagccc ccgatttaga gcttgacggg gaaagccggc gaacgtggcg agaaaggaag
     3541 ggaagaaagc gaaaggagcg ggcgctaggg cgctggcaag tgtagcggtc acgctgcgcg
     3601 taaccaccac acccgccgcg cttaatgcgc cgctacaggg cgcgtcccat tcgccaatcc
     3661 ggatatagtt cctcctttca gcaaaaaacc cctcaagacc cgtttagagg ccccaagggg
     3721 ttatgctagt tattgctcag cggtggcagc agccaactca gcttcctttc gggctttgtt
     3781 tagcagccta ggtattaatc aattagtggt ggtggtggtg gtggtggtgc tcgagtgcgg
     3841 ccgcaagctt cccactaccg cgtggcacca ggtgacatcc tccctgcccg ctcggggcat
     3901 cggccattcg gcctggcagt tgcagtacct gctcacggcc gaacagacgc aggcaggtca
     3961 tcaggatcag gttgaagagc agcaccacgg cgatggtcag gacgctacgc tcggggtgcg
     4021 cggcgaaggt ggcgacctgg tagcagaggg tcgccagcga gtaggcgacg ttcagccccc
     4081 agagcaccga gaaagccatc cagcccttgc tgctttcccg ggcgatcgcg cccatggcgg
     4141 tcacgcaggg cacgtagagc aggacgaaga ccagatagct gtaggccgcg atggggctgc
     4201 cgaacttgct cgccatggtg cccatcgagc cggtctccat gtcgccgtcg gccatgctcg
     4261 cctccaccgg gttggccagc acgctgaggc tgaaggtatc gaccaggccg tcccaggtct
     4321 cgaccagggc ctcgcgcagt tgcccgggca ggtcgtagcc ttcgtagtcg aaggcctcgc
     4381 cctggatctg ctcggcggtg tagagggtgt tcagggtgcc gaccaccact tccttggcca
     4441 tcgccccggt gaccaggccg acggtggcct gccagttgtc cggctgcacc ccgagcggcg
     4501 ccagcagcgg agtgagacgc tggctgacgc tggccagcgc ggaatggccg atgtcgccct
     4561 gcaccggctt gccgtcgagg gtgatgctgt tcaggccgcc gatcaccagg ctgaccagga
     4621 tgatcacctt gccggcccgc accacgaagc cgcgcaggcg cgaccaggtc tgcagcagca
     4681 ggctcttaag gtgtggcaca tggtacagcg gcagttccat gacgaacggc gaggcctcgc
     4741 cgcgcatcaa ggtgtgcttg agcaacagcc cggtgaggat cgccacgacg atgccgagca
     4801 ggtacaggga gaagatcacc agcgcgccgc cctggccgaa gaaggccccg gcgaatacgg
     4861 cgaagatcgc caggcgcgcg ccgcaggaca tgaacggcgc catcatgatg gtaatcagcc
     4921 gctcgcgctg cgcgtccagg gtccgcgcgc ccatgatcga cggtacgttg cagccgaaac
     4981 cgacgatcag cggcacgaac gacttgcccg gcaggcccag cgccagcgag aggaacaggt
     5041 acatcaagcc gatctgcggc accagcggca acaccgtgtt gaccccgcca ccgatgccct
     5101 gggcgaggaa caccgtgagc caatcgggca ggccgaagcg gatacccagc cactggatgc
     5161 cgtcgatgaa gatcgccgac gagcccttgt cgaagatcgg ctgtagcgcc ccgccgatgt
     5221 tgatggcgaa gaagaacatc aggtacatca ccagcaggaa gatcggcaga cccagccagc
     5281 gattcaggac gacccggtcg agccattggg tcagccggtg cggctgagcc tgctggtggt
     5341 cgcagaccgc cgcacagatc tcgccgatca gccggtagcg cgcatcgacg atcgccagct
     5401 cgggctcctc gccgcagccc cggcgcgcct gttccagggt ggcgggcggc aagcccagcg
     5461 ccgggccgtt gaagatgtcg ccttccagcg cctgcaacgc cagccagcgc ggctcgatgg
     5521 ccgaggccgc cggagcgcgc gtttccagaa ggtagccgac ctgcgcctgg atggccggtg
     5581 ggtaatccac cgccagcgcc gcctgcggga gctgtaggga gtcgatggcc gccttcagtt
     5641 cgtcgatgcc atcggcgcgg gtggatacca gcggcaccac cgggcagccg agacgccgcg
     5701 ccagaccgtc gatatcgatg cggatgcgct ggctacgggc gatgtcgagc atgttcagcg
     5761 cgacgatgca ggggatgccc atctcccgca gttgcaccgt gaggtacagg ttgcgctcca
     5821 ggttcgccgc atccaccacg ttgaccagca cgtccacttc gccgctggcg atgtagcggc
     5881 aggcgatctg ctcgtccagc gaggcctggg cggaaacgct ggtcaacgaa taggtgccgg
     5941 gcaggtcgac caggcgcacc gcgtgacgga cggtatggaa cgcaccctcc ttgcgctcca
     6001 cggtgacgcc ggcccagttg ccgacccgct ggcgcgagcc ggtgagctgg ttgaacaggg
     6061 tggtcttgcc ggagttggga ttgccgatca ggccgagggt caatgcggtc atatgtatat
     6121 ctccttctta aagttaaaca aaattatttc tagaggggaa ttgttatccg ctcacaattc
     6181 ccctatagtg agtcgtatta atttcgcggg atcgagatcg atctcgatcc tctacgccgg
     6241 acgcatcgtg gccggcatca ccggcgccac aggtgcggtt gctggcgcct atatcgccga
     6301 catcaccgat ggggaagatc gggctcgcca cttcgggctc atgagcgctt gtttcggcgt
     6361 gggtatggtg gcaggccccg tggccggggg actgttgggc gccatctcct tgcatgcacc
     6421 attccttgcg gcggcggtgc tcaacggcct caacctacta ctgggctgct tcctaatgca
     6481 ggagtcgcat aagggagagc gtcgagatcc cggacaccat cgaatggcgc aaaacctttc
     6541 gcggtatggc atgatagcgc ccggaagaga gtcaattcag ggtggtgaat gtgaaaccag
     6601 taacgttata cgatgtcgca gagtatgccg gtgtctctta tcagaccgtt tcccgcgtgg
     6661 tgaaccaggc cagccacgtt tctgcgaaaa cgcgggaaaa agtggaagcg gcgatggcgg
     6721 agctgaatta cattcccaac cgcgtggcac aacaactggc gggcaaacag tcgttgctga
     6781 ttggcgttgc cacctccagt ctggccctgc acgcgccgtc gcaaattgtc gcggcgatta
     6841 aatctcgcgc cgatcaactg ggtgccagcg tggtggtgtc gatggtagaa cgaagcggcg
     6901 tcgaagcctg taaagcggcg gtgcacaatc ttctcgcgca acgcgtcagt gggctgatca
     6961 ttaactatcc gctggatgac caggatgcca ttgctgtgga agctgcctgc actaatgttc
     7021 cggcgttatt tcttgatgtc tctgaccaga cacccatcaa cagtattatt ttctcccatg
     7081 aagacggtac gcgactgggc gtggagcatc tggtcgcatt gggtcaccag caaatcgcgc
     7141 tgttagcggg cccattaagt tctgtctcgg cgcgtctgcg tctggctggc tggcataaat
     7201 atctcactcg caatcaaatt cagccgatag cggaacggga aggcgactgg agtgccatgt
     7261 ccggttttca acaaaccatg caaatgctga
//
