LOCUS       sequence-434435-        7011 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 OL2 in its interaction with exogenous peptides..
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 7011)
  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 7011)
  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-OL2/4sub
FEATURES             Location/Qualifiers
     source          1..7011
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     RBS             824..846
                     /note="efficient ribosome binding site from bacteriophage 
                     T7 gene 10 (Olins and Rangwala, 1989)"
     protein_bind    861..885
                     /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(885..904)
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        complement(886..904)
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     primer_bind     complement(992..1011)
                     /label=pBRrevBam
                     /note="pBR322 vectors, tet region, downstream of BamHI, 
                     reverse primer"
     promoter        1217..1294
                     /gene="lacI"
                     /label=lacI promoter
     CDS             1295..2377
                     /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(1314..1333)
                     /label=LacI-R
                     /note="LacI, reverse primer"
     CDS             2949..3140
                     /codon_start=1
                     /gene="rop"
                     /product="Rop protein, which maintains plasmids at low copy
                     number"
                     /label=rop
                     /translation="MTKQEKTALNMARFIRSQTLTLLEKLNELDADEQADICESLHDHA
                     DELYRSCLARFGDDGENL"
     primer_bind     complement(3134..3156)
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     misc_feature    3242..3381
                     /label=bom
                     /note="basis of mobility region from pBR322"
     primer_bind     3297..3316
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     complement(3396..3413)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(3567..4155)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(3647..3666)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     complement(4266..4285)
                     /label=pENTR-R
                     /note="pENTR vectors, reverse primer"
     CDS             4277..5092
                     /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             4277..5092
                     /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(4350..4369)
                     /label=Kan-R
                     /note="Kanamycin resistance gene, reverse primer"
     rep_origin      complement(5184..5639)
                     /direction=LEFT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(5321..5342)
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     5533..5552
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     terminator      complement(5676..5723)
                     /label=T7 terminator
                     /note="transcription terminator for bacteriophage T7 RNA 
                     polymerase"
     primer_bind     5719..5737
                     /label=T7 Term
                     /note="T7 terminator, reverse primer"
     CDS             complement(5800..5823)
                     /codon_start=1
                     /product="8xHis affinity tag"
                     /label=8xHis
                     /translation="HHHHHHHH"
     CDS             complement(5848..5865)
                     /codon_start=1
                     /product="thrombin recognition and cleavage site"
                     /label=thrombin site
                     /translation="LVPRGS"
ORIGIN
        1 gtcgagccat tgggtcagcc ggtgcggctg agcctgctgg tggtcgcaga ccgccgcaca
       61 gatctcgccg atcagccggt agcgcgcatc gacgatcgcc agctcgggct cctcgccgca
      121 gccccggcgc gcctgttcca gggtggcggg cggcaagccc agcgccgggc cgttgaagat
      181 gtcgccttcc agcgcctgca acgccagcca gcgcggctcg atggccgagg ccgccggagc
      241 gcgcgtttcc agaaggtagc cgacctgcgc ctggatggcc ggtgggtaat ccaccgccag
      301 cgccgcctgc gggagctgta gggagtcgat ggccgccttc agttcgtcga tgccatcggc
      361 gcgggtggat accagcggca ccaccgggca gccgagacgc cgcgccagac cgtcgatatc
      421 gatgcggatg cgctggctac gggcgatgtc gagcatgttc agcgcgacga tgcaggggat
      481 gcccatctcc cgcagttgca ccgtgaggta caggttgcgc tccaggttcg ccgcatccac
      541 cacgttgacc agcacgtcca cttcgccgct ggcgatgtag cggcaggcga tctgctcgtc
      601 cagcgaggcc tgggcggaaa cgctggtcaa cgaataggtg ccgggcaggt cgaccaggcg
      661 caccgcgtga cggacggtat ggaacgcacc ctccttgcgc tccacggtga cgccggccca
      721 gttgccgacc cgctggcgcg agccggtgag ctggttgaac agggtggtct tgccggagtt
      781 gggattgccg atcaggccga gggtcaatgc ggtcatatgt atatctcctt cttaaagtta
      841 aacaaaatta tttctagagg ggaattgtta tccgctcaca attcccctat agtgagtcgt
      901 attaatttcg cgggatcgag atcgatctcg atcctctacg ccggacgcat cgtggccggc
      961 atcaccggcg ccacaggtgc ggttgctggc gcctatatcg ccgacatcac cgatggggaa
     1021 gatcgggctc gccacttcgg gctcatgagc gcttgtttcg gcgtgggtat ggtggcaggc
     1081 cccgtggccg ggggactgtt gggcgccatc tccttgcatg caccattcct tgcggcggcg
     1141 gtgctcaacg gcctcaacct actactgggc tgcttcctaa tgcaggagtc gcataaggga
     1201 gagcgtcgag atcccggaca ccatcgaatg gcgcaaaacc tttcgcggta tggcatgata
     1261 gcgcccggaa gagagtcaat tcagggtggt gaatgtgaaa ccagtaacgt tatacgatgt
     1321 cgcagagtat gccggtgtct cttatcagac cgtttcccgc gtggtgaacc aggccagcca
     1381 cgtttctgcg aaaacgcggg aaaaagtgga agcggcgatg gcggagctga attacattcc
     1441 caaccgcgtg gcacaacaac tggcgggcaa acagtcgttg ctgattggcg ttgccacctc
     1501 cagtctggcc ctgcacgcgc cgtcgcaaat tgtcgcggcg attaaatctc gcgccgatca
     1561 actgggtgcc agcgtggtgg tgtcgatggt agaacgaagc ggcgtcgaag cctgtaaagc
     1621 ggcggtgcac aatcttctcg cgcaacgcgt cagtgggctg atcattaact atccgctgga
     1681 tgaccaggat gccattgctg tggaagctgc ctgcactaat gttccggcgt tatttcttga
     1741 tgtctctgac cagacaccca tcaacagtat tattttctcc catgaagacg gtacgcgact
     1801 gggcgtggag catctggtcg cattgggtca ccagcaaatc gcgctgttag cgggcccatt
     1861 aagttctgtc tcggcgcgtc tgcgtctggc tggctggcat aaatatctca ctcgcaatca
     1921 aattcagccg atagcggaac gggaaggcga ctggagtgcc atgtccggtt ttcaacaaac
     1981 catgcaaatg ctgaatgagg gcatcgttcc cactgcgatg ctggttgcca acgatcagat
     2041 ggcgctgggc gcaatgcgcg ccattaccga gtccgggctg cgcgttggtg cggacatctc
     2101 ggtagtggga tacgacgata ccgaagacag ctcatgttat atcccgccgt taaccaccat
     2161 caaacaggat tttcgcctgc tggggcaaac cagcgtggac cgcttgctgc aactctctca
     2221 gggccaggcg gtgaagggca atcagctgtt gcccgtctca ctggtgaaaa gaaaaaccac
     2281 cctggcgccc aatacgcaaa ccgcctctcc ccgcgcgttg gccgattcat taatgcagct
     2341 ggcacgacag gtttcccgac tggaaagcgg gcagtgagcg caacgcaatt aatgtaagtt
     2401 agctcactca ttaggcaccg ggatctcgac cgatgccctt gagagccttc aacccagtca
     2461 gctccttccg gtgggcgcgg ggcatgacta gcatgatcgt gctcctgtcg ttgaggaccc
     2521 ggctaggctg gcggggttgc cttactggtt agcagaatga atcaccgata cgcgagcgaa
     2581 cgtgaagcga ctgctgctgc aaaacgtctg cgacctgagc aacaacatga atggtcttcg
     2641 gtttccgtgt ttcgtaaagt ctggaaacgc ggaagtcagc gccctgcacc attatgttcc
     2701 ggatctgcat cgcaggatgc tgctggctac cctgtggaac acctacatct gtattaacga
     2761 agcgctggca ttgaccctga gtgatttttc tctggtcccg ccgcatccat accgccagtt
     2821 gtttaccctc acaacgttcc agtaaccggg catgttcatc atcagtaacc cgtatcgtga
     2881 gcatcctctc tcgtttcatc ggtatcatta cccccatgaa cagaaatccc ccttacacgg
     2941 aggcatcagt gaccaaacag gaaaaaaccg cccttaacat ggcccgcttt atcagaagcc
     3001 agacattaac gcttctggag aaactcaacg agctggacgc ggatgaacag gcagacatct
     3061 gtgaatcgct tcacgaccac gctgatgagc tttaccgcag ctgcctcgcg cgtttcggtg
     3121 atgacggtga aaacctctga cacatgcagc tcccggagac ggtcacagct tgtctgtaag
     3181 cggatgccgg gagcagacaa gcccgtcagg gcgcgtcagc gggtgttggc gggtgtcggg
     3241 gcgcagccat gacccagtca cgtagcgata gcggagtgta tactggctta actatgcggc
     3301 atcagagcag attgtactga gagtgcacca ttgcggtgtg aaataccgca cagatgcgta
     3361 aggagaaaat accgcatcag gcgctcttcc gcttcctcgc tcactgactc gctgcgctcg
     3421 gtcgttcggc tgcggcgagc ggtatcagct cactcaaagg cggtaatacg gttatccaca
     3481 gaatcagggg ataacgcagg aaagaacatg tgagcaaaag gccagcaaaa ggccaggaac
     3541 cgtaaaaagg ccgcgttgct ggcgtttttc cataggctcc gcccccctga cgagcatcac
     3601 aaaaatcgac gctcaagtca gaggtggcga aacccgacag gactataaag ataccaggcg
     3661 tttccccctg gaagctccct cgtgcgctct cctgttccga ccctgccgct taccggatac
     3721 ctgtccgcct ttctcccttc gggaagcgtg gcgctttctc atagctcacg ctgtaggtat
     3781 ctcagttcgg tgtaggtcgt tcgctccaag ctgggctgtg tgcacgaacc ccccgttcag
     3841 cccgaccgct gcgccttatc cggtaactat cgtcttgagt ccaacccggt aagacacgac
     3901 ttatcgccac tggcagcagc cactggtaac aggattagca gagcgaggta tgtaggcggt
     3961 gctacagagt tcttgaagtg gtggcctaac tacggctaca ctagaaggac agtatttggt
     4021 atctgcgctc tgctgaagcc agttaccttc ggaaaaagag ttggtagctc ttgatccggc
     4081 aaacaaacca ccgctggtag cggtggtttt tttgtttgca agcagcagat tacgcgcaga
     4141 aaaaaaggat ctcaagaaga tcctttgatc ttttctacgg ggtctgacgc tcagtggaac
     4201 gaaaactcac gttaagggat tttggtcatg aacaataaaa ctgtctgctt acataaacag
     4261 taatacaagg ggtgttatga gccatattca acgggaaacg tcttgctcta ggccgcgatt
     4321 aaattccaac atggatgctg atttatatgg gtataaatgg gctcgcgata atgtcgggca
     4381 atcaggtgcg acaatctatc gattgtatgg gaagcccgat gcgccagagt tgtttctgaa
     4441 acatggcaaa ggtagcgttg ccaatgatgt tacagatgag atggtcagac taaactggct
     4501 gacggaattt atgcctcttc cgaccatcaa gcattttatc cgtactcctg atgatgcatg
     4561 gttactcacc actgcgatcc ccgggaaaac agcattccag gtattagaag aatatcctga
     4621 ttcaggtgaa aatattgttg atgcgctggc agtgttcctg cgccggttgc attcgattcc
     4681 tgtttgtaat tgtcctttta acagcgatcg cgtatttcgt ctcgctcagg cgcaatcacg
     4741 aatgaataac ggtttggttg atgcgagtga ttttgatgac gagcgtaatg gctggcctgt
     4801 tgaacaagtc tggaaagaaa tgcataaact tttgccattc tcaccggatt cagtcgtcac
     4861 tcatggtgat ttctcacttg ataaccttat ttttgacgag gggaaattaa taggttgtat
     4921 tgatgttgga cgagtcggaa tcgcagaccg ataccaggat cttgccatcc tatggaactg
     4981 cctcggtgag ttttctcctt cattacagaa acggcttttt caaaaatatg gtattgataa
     5041 tcctgatatg aataaattgc agtttcattt gatgctcgat gagtttttct aagaattaat
     5101 tcatgagcgg atacatattt gaatgtattt agaaaaataa acaaataggg gttccgcgca
     5161 catttccccg aaaagtgcca cctaaattgt aagcgttaat attttgttaa aattcgcgtt
     5221 aaatttttgt taaatcagct cattttttaa ccaataggcc gaaatcggca aaatccctta
     5281 taaatcaaaa gaatagaccg agatagggtt gagtgttgtt ccagtttgga acaagagtcc
     5341 actattaaag aacgtggact ccaacgtcaa agggcgaaaa accgtctatc agggcgatgg
     5401 cccactacgt gaaccatcac cctaatcaag ttttttgggg tcgaggtgcc gtaaagcact
     5461 aaatcggaac cctaaaggga gcccccgatt tagagcttga cggggaaagc cggcgaacgt
     5521 ggcgagaaag gaagggaaga aagcgaaagg agcgggcgct agggcgctgg caagtgtagc
     5581 ggtcacgctg cgcgtaacca ccacacccgc cgcgcttaat gcgccgctac agggcgcgtc
     5641 ccattcgcca atccggatat agttcctcct ttcagcaaaa aacccctcaa gacccgttta
     5701 gaggccccaa ggggttatgc tagttattgc tcagcggtgg cagcagccaa ctcagcttcc
     5761 tttcgggctt tgtttagcag cctaggtatt aatcaattag tggtggtggt ggtggtggtg
     5821 gtgctcgagt gcggccgcaa gcttcccact accgcgtggc accaggtgac atcctccctg
     5881 cccgctcggg gcatcggcca ttcggcctgg cagttgcagt acctgctcac ggccgaacag
     5941 acgcaggcag gtcatcagga tcaggttgaa gagcagcacc acggcgatgg tcaggacgct
     6001 acgctcgggg tgcgcggcga aggtggcgac ctggtagcag agggtcgcca gcgagtaggc
     6061 gacgttcagc ccccagagca ccgagaaagc catccagccc ttgctgcttt cccgggcgat
     6121 cgcgcccatg gcggtcacgc agggcacgta gagcaggacg aagaccagat agctgtaggc
     6181 cgcctgcatc aggcggtcca tgacgaaggc cgcgcgtgcc atgtagccgg aatcctcgag
     6241 gatgctgttc aggccgccga tcaccaggct gaccaggatg atcaccttgc cggcccgcac
     6301 cacgaagccg cgcaggcgcg accaggtctg cagcagcagg ctcttaaggt gtggcacatg
     6361 gtacagcggc agttccatga cgaacggcga ggcctcgccg cgcatcaagg tgtgcttgag
     6421 caacagcccg gtgaggatcg ccacgacgat gccgagcagg tacagggaga agatcaccag
     6481 cgcgccgccc tggccgaaga aggccccggc gaatacggcg aagatcgcca ggcgcgcgcc
     6541 gcaggacatg aacggcgcca tcatgatggt aatcagccgc tcgcgctgcg cgtccagggt
     6601 ccgcgcgccc atgatcgacg gtacgttgca gccgaaaccg acgatcagcg gcacgaacga
     6661 cttgcccggc aggcccagcg cctgcatcag gcggtccatg acgaaggccg cgcgtgccat
     6721 gtagccggaa tcctcgagca gcgagaggaa caggtacatc aagccgatct gcggcaccag
     6781 cggcaacacc gtgttgaccc cgccaccgat gccctgggcg aggaacaccg tgagccaatc
     6841 gggcaggccg aagcggatac ccagccactg gatgccgtcg atgaagatcg ccgacgagcc
     6901 cttgtcgaag atcggctgta gcgccccgcc gatgttgatg gcgaagaaga acatcaggta
     6961 catcaccagc aggaagatcg gcagacccag ccagcgattc aggacgaccc g
//
