LOCUS       sequence-434430-        4428 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Study the interaction of PotH in E. coli, specifically focusing on 
            the role of the outer loop 1 (OL1) by examining the variant lacking 
            OL1 in its interaction with exogenous peptides..
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 4428)
  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 4428)
  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: PCDF Bravo-PotH-OL1del
FEATURES             Location/Qualifiers
     source          1..4428
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     protein_bind    3..27
                     /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)."
     CDS             80..103
                     /codon_start=1
                     /product="FLAG(R) epitope tag, followed by an enterokinase 
                     cleavage site"
                     /label=FLAG
                     /translation="DYKDDDDK"
     CDS             110..133
                     /codon_start=1
                     /product="recognition and cleavage site for human 
                     rhinovirus 3C and PreScission proteases"
                     /label=HRV 3C site
                     /translation="LEVLFQGP"
     CDS             938..955
                     /codon_start=1
                     /product="thrombin recognition and cleavage site"
                     /label=thrombin site
                     /translation="LVPRGS"
     CDS             962..991
                     /codon_start=1
                     /product="10xHis affinity tag"
                     /label=10xHis
                     /translation="HHHHHHHHHH"
     CDS             1013..1057
                     /codon_start=1
                     /product="affinity and epitope tag derived from pancreatic 
                     ribonuclease A"
                     /label=S-Tag
                     /translation="KETAAAKFERQHMDS"
     primer_bind     complement(1095..1113)
                     /label=T7 Term
                     /note="T7 terminator, reverse primer"
     terminator      1109..1156
                     /label=T7 terminator
                     /note="transcription terminator for bacteriophage T7 RNA 
                     polymerase"
     CDS             complement(1327..2118)
                     /codon_start=1
                     /gene="aadA"
                     /product="aminoglycoside adenylyltransferase (Murphy, 
                     1985)"
                     /label=SmR
                     /note="confers resistance to spectinomycin and 
                     streptomycin"
                     /translation="MREAVIAEVSTQLSEVVGVIERHLEPTLLAVHLYGSAVDGGLKPH
                     SDIDLLVTVTVRLDETTRRALINDLLETSASPGESEILRAVEVTIVVHDDIIPWRYPAK
                     RELQFGEWQRNDILAGIFEPATIDIDLAILLTKAREHSVALVGPAAEELFDPVPEQDLF
                     EALNETLTLWNSPPDWAGDERNVVLTLSRIWYSAVTGKIAPKDVAADWAMERLPAQYQP
                     VILEARQAYLGQEEDRLASRADQLEEFVHYVKGEITKVVGK"
     promoter        complement(2119..2210)
                     /gene="bla"
                     /label=AmpR promoter
     rep_origin      complement(2258..2996)
                     /direction=LEFT
                     /label=CloDF13 ori
                     /note="Plasmids containing the CloDF13 (CDF) origin of 
                     replication can be propagated in E. coli cells that contain
                     additional plasmids with compatible origins."
     CDS             complement(3206..4288)
                     /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     4250..4269
                     /label=LacI-R
                     /note="LacI, reverse primer"
     promoter        complement(4289..4366)
                     /gene="lacI"
                     /label=lacI promoter
     primer_bind     join(4412..4428,1..3)
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        join(4412..4428,1..2)
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
ORIGIN
        1 ggggaattgt gagcggataa caattcccct gtagaaataa ttttgtttaa ctttaataag
       61 gagatatacc atggcaagcg actacaagga tgacgacgac aagggtgcac ttgaagtcct
      121 ctttcaagga ccgagcagcc cctcaagtac acttgaacct gctgcccagt cgaaaccgcc
      181 gggcggattt aagctgtggt tgtcgcagct gcaaatgaag catgggcgca aactggtcat
      241 tgcgttgcca tatatctggt tgatcttgct gtttctgctg ccatttctga ttgtcgtggc
      301 tgcgatttcg acattttgct gtttactgat cggctatccg ctggcgtggg cggtggcgca
      361 cagtaagcct tcgacccgta atattttatt actactggtg atcctgccgt cgtggacctc
      421 gtttctgatc cgcgtttatg cctggatggg aatattaaaa aacaacggtg tgctgaataa
      481 ttttctgctg tggctggggg ttatcgatca accgctgacc attctgcata ccaatctggc
      541 cgtttatatc ggcattgttt acgcttacgt gccgtttatg gtactgccga tttataccgc
      601 gttgattcgt attgattatt cgctggtgga agcagcgctg gatctcggtg cacgaccgct
      661 gaaaacgttc tttactgtga tcgtgccgct gactaaaggt gggattattg ccggatcgat
      721 gctggtgttt atcccggctg tgggcgagtt tgtgatcccg gaactgctcg gtggcccgga
      781 cagcatcatg atcgggcgcg tgctatggca agagtttttc aataaccgcg actggccggt
      841 ggcctcggcg gtagcgatca tcatgttgct gctgctaatt gtgccgataa tgtggttcca
      901 caaacaccag caaaaaagcg tgggagaaca cggagggttg gttccccgtg gtagttccgc
      961 tcatcaccac catcatcacc atcaccacca cggtgcataa ctcgagtctg gtaaagaaac
     1021 cgctgctgcg aaatttgaac gccagcacat ggactcgtct actagcgcag cttaattaac
     1081 ctaggctgct gccaccgctg agcaataact agcataaccc cttggggcct ctaaacgggt
     1141 cttgaggggt tttttgctga aacctcaggc atttgagaag cacacggtca cactgcttcc
     1201 ggtagtcaat aaaccggtaa accagcaata gacataagcg gctatttaac gaccctgccc
     1261 tgaaccgacg accgggtcat cgtggccgga tcttgcggcc cctcggcttg aacgaattgt
     1321 tagacattat ttgccgacta ccttggtgat ctcgcctttc acgtagtgga caaattcttc
     1381 caactgatct gcgcgcgagg ccaagcgatc ttcttcttgt ccaagataag cctgtctagc
     1441 ttcaagtatg acgggctgat actgggccgg caggcgctcc attgcccagt cggcagcgac
     1501 atccttcggc gcgattttgc cggttactgc gctgtaccaa atgcgggaca acgtaagcac
     1561 tacatttcgc tcatcgccag cccagtcggg cggcgagttc catagcgtta aggtttcatt
     1621 tagcgcctca aatagatcct gttcaggaac cggatcaaag agttcctccg ccgctggacc
     1681 taccaaggca acgctatgtt ctcttgcttt tgtcagcaag atagccagat caatgtcgat
     1741 cgtggctggc tcgaagatac ctgcaagaat gtcattgcgc tgccattctc caaattgcag
     1801 ttcgcgctta gctggataac gccacggaat gatgtcgtcg tgcacaacaa tggtgacttc
     1861 tacagcgcgg agaatctcgc tctctccagg ggaagccgaa gtttccaaaa ggtcgttgat
     1921 caaagctcgc cgcgttgttt catcaagcct tacggtcacc gtaaccagca aatcaatatc
     1981 actgtgtggc ttcaggccgc catccactgc ggagccgtac aaatgtacgg ccagcaacgt
     2041 cggttcgaga tggcgctcga tgacgccaac tacctctgat agttgagtcg atacttcggc
     2101 gatcaccgct tccctcatac tcttcctttt tcaatattat tgaagcattt atcagggtta
     2161 ttgtctcatg agcggataca tatttgaatg tatttagaaa aataaacaaa tagctagctc
     2221 actcggtcgc tacgctccgg gcgtgagact gcggcgggcg ctgcggacac atacaaagtt
     2281 acccacagat tccgtggata agcaggggac taacatgtga ggcaaaacag cagggccgcg
     2341 ccggtggcgt ttttccatag gctccgccct cctgccagag ttcacataaa cagacgcttt
     2401 tccggtgcat ctgtgggagc cgtgaggctc aaccatgaat ctgacagtac gggcgaaacc
     2461 cgacaggact taaagatccc caccgtttcc ggcgggtcgc tccctcttgc gctctcctgt
     2521 tccgaccctg ccgtttaccg gatacctgtt ccgcctttct cccttacggg aagtgtggcg
     2581 ctttctcata gctcacacac tggtatctcg gctcggtgta ggtcgttcgc tccaagctgg
     2641 gctgtaagca agaactcccc gttcagcccg actgctgcgc cttatccggt aactgttcac
     2701 ttgagtccaa cccggaaaag cacggtaaaa cgccactggc agcagccatt ggtaactggg
     2761 agttcgcaga ggatttgttt agctaaacac gcggttgctc ttgaagtgtg cgccaaagtc
     2821 cggctacact ggaaggacag atttggttgc tgtgctctgc gaaagccagt taccacggtt
     2881 aagcagttcc ccaactgact taaccttcga tcaaaccacc tccccaggtg gttttttcgt
     2941 ttacagggca aaagattacg cgcagaaaaa aaggatctca agaagatcct ttgatctttt
     3001 ctactgaacc gctctagatt tcagtgcaat ttatctcttc aaatgtagca cctgaagtca
     3061 gccccatacg atataagttg taattctcat gttagtcatg ccccgcgccc accggaagga
     3121 gctgactggg ttgaaggctc tcaagggcat cggtcgagat cccggtgcct aatgagtgag
     3181 ctaacttaca ttaattgcgt tgcgctcact gcccgctttc cagtcgggaa acctgtcgtg
     3241 ccagctgcat taatgaatcg gccaacgcgc ggggagaggc ggtttgcgta ttgggcgcca
     3301 gggtggtttt tcttttcacc agtgagacgg gcaacagctg attgcccttc accgcctggc
     3361 cctgagagag ttgcagcaag cggtccacgc tggtttgccc cagcaggcga aaatcctgtt
     3421 tgatggtggt taacggcggg atataacatg agctgtcttc ggtatcgtcg tatcccacta
     3481 ccgagatgtc cgcaccaacg cgcagcccgg actcggtaat ggcgcgcatt gcgcccagcg
     3541 ccatctgatc gttggcaacc agcatcgcag tgggaacgat gccctcattc agcatttgca
     3601 tggtttgttg aaaaccggac atggcactcc agtcgccttc ccgttccgct atcggctgaa
     3661 tttgattgcg agtgagatat ttatgccagc cagccagacg cagacgcgcc gagacagaac
     3721 ttaatgggcc cgctaacagc gcgatttgct ggtgacccaa tgcgaccaga tgctccacgc
     3781 ccagtcgcgt accgtcttca tgggagaaaa taatactgtt gatgggtgtc tggtcagaga
     3841 catcaagaaa taacgccgga acattagtgc aggcagcttc cacagcaatg gcatcctggt
     3901 catccagcgg atagttaatg atcagcccac tgacgcgttg cgcgagaaga ttgtgcaccg
     3961 ccgctttaca ggcttcgacg ccgcttcgtt ctaccatcga caccaccacg ctggcaccca
     4021 gttgatcggc gcgagattta atcgccgcga caatttgcga cggcgcgtgc agggccagac
     4081 tggaggtggc aacgccaatc agcaacgact gtttgcccgc cagttgttgt gccacgcggt
     4141 tgggaatgta attcagctcc gccatcgccg cttccacttt ttcccgcgtt ttcgcagaaa
     4201 cgtggctggc ctggttcacc acgcgggaaa cggtctgata agagacaccg gcatactctg
     4261 cgacatcgta taacgttact ggtttcacat tcaccaccct gaattgactc tcttccgggc
     4321 gctatcatgc cataccgcga aaggttttgc gccattcgat ggtgtccggg atctcgacgc
     4381 tctcccttat gcgactcctg cattaggaaa ttaatacgac tcactata
//
