LOCUS       sequence-434427-        4494 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 2 (OL2) 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 4494)
  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 4494)
  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-OL2del
FEATURES             Location/Qualifiers
     source          1..4494
                     /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             1004..1021
                     /codon_start=1
                     /product="thrombin recognition and cleavage site"
                     /label=thrombin site
                     /translation="LVPRGS"
     CDS             1028..1057
                     /codon_start=1
                     /product="10xHis affinity tag"
                     /label=10xHis
                     /translation="HHHHHHHHHH"
     CDS             1079..1123
                     /codon_start=1
                     /product="affinity and epitope tag derived from pancreatic 
                     ribonuclease A"
                     /label=S-Tag
                     /translation="KETAAAKFERQHMDS"
     primer_bind     complement(1161..1179)
                     /label=T7 Term
                     /note="T7 terminator, reverse primer"
     terminator      1175..1222
                     /label=T7 terminator
                     /note="transcription terminator for bacteriophage T7 RNA 
                     polymerase"
     CDS             complement(1393..2184)
                     /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(2185..2276)
                     /gene="bla"
                     /label=AmpR promoter
     rep_origin      complement(2324..3062)
                     /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(3272..4354)
                     /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     4316..4335
                     /label=LacI-R
                     /note="LacI, reverse primer"
     promoter        complement(4355..4432)
                     /gene="lacI"
                     /label=lacI promoter
     primer_bind     join(4478..4494,1..3)
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        join(4478..4494,1..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 ttgtctttaa
      301 aataagcctg gcggagatgg cgcgcgctat tccaccttat accgaactga tggagtgggc
      361 tgacgggcaa ctgtccatca ctcttaatct cggtaatttt ctgcaactga ccgacgatcc
      421 gctctatttc gatgcttatc tccagtcgct acaggtggct gcgatttcga cattttgctg
      481 tttactgatc ggctatccgc tggcgtgggc ggtggcgcac agtaagcctt cgacccgtaa
      541 tattttatta ctactggtga tcctgccgtc gtggacctcg tttctgatcc gcgtttatgc
      601 cctggccgtt tatatcggca ttgtttacgc ttacgtgccg tttatggtac tgccgattta
      661 taccgcgttg attcgtattg attattcgct ggtggaagca gcgctggatc tcggtgcacg
      721 accgctgaaa acgttcttta ctgtgatcgt gccgctgact aaaggtggga ttattgccgg
      781 atcgatgctg gtgtttatcc cggctgtggg cgagtttgtg atcccggaac tgctcggtgg
      841 cccggacagc atcatgatcg ggcgcgtgct atggcaagag tttttcaata accgcgactg
      901 gccggtggcc tcggcggtag cgatcatcat gttgctgctg ctaattgtgc cgataatgtg
      961 gttccacaaa caccagcaaa aaagcgtggg agaacacgga gggttggttc cccgtggtag
     1021 ttccgctcat caccaccatc atcaccatca ccaccacggt gcataactcg agtctggtaa
     1081 agaaaccgct gctgcgaaat ttgaacgcca gcacatggac tcgtctacta gcgcagctta
     1141 attaacctag gctgctgcca ccgctgagca ataactagca taaccccttg gggcctctaa
     1201 acgggtcttg aggggttttt tgctgaaacc tcaggcattt gagaagcaca cggtcacact
     1261 gcttccggta gtcaataaac cggtaaacca gcaatagaca taagcggcta tttaacgacc
     1321 ctgccctgaa ccgacgaccg ggtcatcgtg gccggatctt gcggcccctc ggcttgaacg
     1381 aattgttaga cattatttgc cgactacctt ggtgatctcg cctttcacgt agtggacaaa
     1441 ttcttccaac tgatctgcgc gcgaggccaa gcgatcttct tcttgtccaa gataagcctg
     1501 tctagcttca agtatgacgg gctgatactg ggccggcagg cgctccattg cccagtcggc
     1561 agcgacatcc ttcggcgcga ttttgccggt tactgcgctg taccaaatgc gggacaacgt
     1621 aagcactaca tttcgctcat cgccagccca gtcgggcggc gagttccata gcgttaaggt
     1681 ttcatttagc gcctcaaata gatcctgttc aggaaccgga tcaaagagtt cctccgccgc
     1741 tggacctacc aaggcaacgc tatgttctct tgcttttgtc agcaagatag ccagatcaat
     1801 gtcgatcgtg gctggctcga agatacctgc aagaatgtca ttgcgctgcc attctccaaa
     1861 ttgcagttcg cgcttagctg gataacgcca cggaatgatg tcgtcgtgca caacaatggt
     1921 gacttctaca gcgcggagaa tctcgctctc tccaggggaa gccgaagttt ccaaaaggtc
     1981 gttgatcaaa gctcgccgcg ttgtttcatc aagccttacg gtcaccgtaa ccagcaaatc
     2041 aatatcactg tgtggcttca ggccgccatc cactgcggag ccgtacaaat gtacggccag
     2101 caacgtcggt tcgagatggc gctcgatgac gccaactacc tctgatagtt gagtcgatac
     2161 ttcggcgatc accgcttccc tcatactctt cctttttcaa tattattgaa gcatttatca
     2221 gggttattgt ctcatgagcg gatacatatt tgaatgtatt tagaaaaata aacaaatagc
     2281 tagctcactc ggtcgctacg ctccgggcgt gagactgcgg cgggcgctgc ggacacatac
     2341 aaagttaccc acagattccg tggataagca ggggactaac atgtgaggca aaacagcagg
     2401 gccgcgccgg tggcgttttt ccataggctc cgccctcctg ccagagttca cataaacaga
     2461 cgcttttccg gtgcatctgt gggagccgtg aggctcaacc atgaatctga cagtacgggc
     2521 gaaacccgac aggacttaaa gatccccacc gtttccggcg ggtcgctccc tcttgcgctc
     2581 tcctgttccg accctgccgt ttaccggata cctgttccgc ctttctccct tacgggaagt
     2641 gtggcgcttt ctcatagctc acacactggt atctcggctc ggtgtaggtc gttcgctcca
     2701 agctgggctg taagcaagaa ctccccgttc agcccgactg ctgcgcctta tccggtaact
     2761 gttcacttga gtccaacccg gaaaagcacg gtaaaacgcc actggcagca gccattggta
     2821 actgggagtt cgcagaggat ttgtttagct aaacacgcgg ttgctcttga agtgtgcgcc
     2881 aaagtccggc tacactggaa ggacagattt ggttgctgtg ctctgcgaaa gccagttacc
     2941 acggttaagc agttccccaa ctgacttaac cttcgatcaa accacctccc caggtggttt
     3001 tttcgtttac agggcaaaag attacgcgca gaaaaaaagg atctcaagaa gatcctttga
     3061 tcttttctac tgaaccgctc tagatttcag tgcaatttat ctcttcaaat gtagcacctg
     3121 aagtcagccc catacgatat aagttgtaat tctcatgtta gtcatgcccc gcgcccaccg
     3181 gaaggagctg actgggttga aggctctcaa gggcatcggt cgagatcccg gtgcctaatg
     3241 agtgagctaa cttacattaa ttgcgttgcg ctcactgccc gctttccagt cgggaaacct
     3301 gtcgtgccag ctgcattaat gaatcggcca acgcgcgggg agaggcggtt tgcgtattgg
     3361 gcgccagggt ggtttttctt ttcaccagtg agacgggcaa cagctgattg cccttcaccg
     3421 cctggccctg agagagttgc agcaagcggt ccacgctggt ttgccccagc aggcgaaaat
     3481 cctgtttgat ggtggttaac ggcgggatat aacatgagct gtcttcggta tcgtcgtatc
     3541 ccactaccga gatgtccgca ccaacgcgca gcccggactc ggtaatggcg cgcattgcgc
     3601 ccagcgccat ctgatcgttg gcaaccagca tcgcagtggg aacgatgccc tcattcagca
     3661 tttgcatggt ttgttgaaaa ccggacatgg cactccagtc gccttcccgt tccgctatcg
     3721 gctgaatttg attgcgagtg agatatttat gccagccagc cagacgcaga cgcgccgaga
     3781 cagaacttaa tgggcccgct aacagcgcga tttgctggtg acccaatgcg accagatgct
     3841 ccacgcccag tcgcgtaccg tcttcatggg agaaaataat actgttgatg ggtgtctggt
     3901 cagagacatc aagaaataac gccggaacat tagtgcaggc agcttccaca gcaatggcat
     3961 cctggtcatc cagcggatag ttaatgatca gcccactgac gcgttgcgcg agaagattgt
     4021 gcaccgccgc tttacaggct tcgacgccgc ttcgttctac catcgacacc accacgctgg
     4081 cacccagttg atcggcgcga gatttaatcg ccgcgacaat ttgcgacggc gcgtgcaggg
     4141 ccagactgga ggtggcaacg ccaatcagca acgactgttt gcccgccagt tgttgtgcca
     4201 cgcggttggg aatgtaattc agctccgcca tcgccgcttc cactttttcc cgcgttttcg
     4261 cagaaacgtg gctggcctgg ttcaccacgc gggaaacggt ctgataagag acaccggcat
     4321 actctgcgac atcgtataac gttactggtt tcacattcac caccctgaat tgactctctt
     4381 ccgggcgcta tcatgccata ccgcgaaagg ttttgcgcca ttcgatggtg tccgggatct
     4441 cgacgctctc ccttatgcga ctcctgcatt aggaaattaa tacgactcac tata
//
