LOCUS       sequence-434424-        4569 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Study the interaction of PotH in E. coli, specifically focusing on 
            the variant in which outer loop 2 (OL2) is substituted with OL3 in 
            its interaction with exogenous peptides..
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 4569)
  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 4569)
  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-OL3/2sub
FEATURES             Location/Qualifiers
     source          1..4569
                     /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             1079..1096
                     /codon_start=1
                     /product="thrombin recognition and cleavage site"
                     /label=thrombin site
                     /translation="LVPRGS"
     CDS             1103..1132
                     /codon_start=1
                     /product="10xHis affinity tag"
                     /label=10xHis
                     /translation="HHHHHHHHHH"
     CDS             1154..1198
                     /codon_start=1
                     /product="affinity and epitope tag derived from pancreatic 
                     ribonuclease A"
                     /label=S-Tag
                     /translation="KETAAAKFERQHMDS"
     primer_bind     complement(1236..1254)
                     /label=T7 Term
                     /note="T7 terminator, reverse primer"
     terminator      1250..1297
                     /label=T7 terminator
                     /note="transcription terminator for bacteriophage T7 RNA 
                     polymerase"
     CDS             complement(1468..2259)
                     /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(2260..2351)
                     /gene="bla"
                     /label=AmpR promoter
     rep_origin      complement(2399..3137)
                     /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(3347..4429)
                     /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     4391..4410
                     /label=LacI-R
                     /note="LacI, reverse primer"
     promoter        complement(4430..4507)
                     /gene="lacI"
                     /label=lacI promoter
     primer_bind     join(4553..4569,1..3)
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        join(4553..4569,1..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 cgagctgctg ggcggccccg acagcatcat gatcggcaga gtgctgtggc aggagttctt
      661 caacaacaga gactggctgg ccgtttatat cggcattgtt tacgcttacg tgccgtttat
      721 ggtactgccg atttataccg cgttgattcg tattgattat tcgctggtgg aagcagcgct
      781 ggatctcggt gcacgaccgc tgaaaacgtt ctttactgtg atcgtgccgc tgactaaagg
      841 tgggattatt gccggatcga tgctggtgtt tatcccggct gtgggcgagt ttgtgatccc
      901 ggaactgctc ggtggcccgg acagcatcat gatcgggcgc gtgctatggc aagagttttt
      961 caataaccgc gactggccgg tggcctcggc ggtagcgatc atcatgttgc tgctgctaat
     1021 tgtgccgata atgtggttcc acaaacacca gcaaaaaagc gtgggagaac acggagggtt
     1081 ggttccccgt ggtagttccg ctcatcacca ccatcatcac catcaccacc acggtgcata
     1141 actcgagtct ggtaaagaaa ccgctgctgc gaaatttgaa cgccagcaca tggactcgtc
     1201 tactagcgca gcttaattaa cctaggctgc tgccaccgct gagcaataac tagcataacc
     1261 ccttggggcc tctaaacggg tcttgagggg ttttttgctg aaacctcagg catttgagaa
     1321 gcacacggtc acactgcttc cggtagtcaa taaaccggta aaccagcaat agacataagc
     1381 ggctatttaa cgaccctgcc ctgaaccgac gaccgggtca tcgtggccgg atcttgcggc
     1441 ccctcggctt gaacgaattg ttagacatta tttgccgact accttggtga tctcgccttt
     1501 cacgtagtgg acaaattctt ccaactgatc tgcgcgcgag gccaagcgat cttcttcttg
     1561 tccaagataa gcctgtctag cttcaagtat gacgggctga tactgggccg gcaggcgctc
     1621 cattgcccag tcggcagcga catccttcgg cgcgattttg ccggttactg cgctgtacca
     1681 aatgcgggac aacgtaagca ctacatttcg ctcatcgcca gcccagtcgg gcggcgagtt
     1741 ccatagcgtt aaggtttcat ttagcgcctc aaatagatcc tgttcaggaa ccggatcaaa
     1801 gagttcctcc gccgctggac ctaccaaggc aacgctatgt tctcttgctt ttgtcagcaa
     1861 gatagccaga tcaatgtcga tcgtggctgg ctcgaagata cctgcaagaa tgtcattgcg
     1921 ctgccattct ccaaattgca gttcgcgctt agctggataa cgccacggaa tgatgtcgtc
     1981 gtgcacaaca atggtgactt ctacagcgcg gagaatctcg ctctctccag gggaagccga
     2041 agtttccaaa aggtcgttga tcaaagctcg ccgcgttgtt tcatcaagcc ttacggtcac
     2101 cgtaaccagc aaatcaatat cactgtgtgg cttcaggccg ccatccactg cggagccgta
     2161 caaatgtacg gccagcaacg tcggttcgag atggcgctcg atgacgccaa ctacctctga
     2221 tagttgagtc gatacttcgg cgatcaccgc ttccctcata ctcttccttt ttcaatatta
     2281 ttgaagcatt tatcagggtt attgtctcat gagcggatac atatttgaat gtatttagaa
     2341 aaataaacaa atagctagct cactcggtcg ctacgctccg ggcgtgagac tgcggcgggc
     2401 gctgcggaca catacaaagt tacccacaga ttccgtggat aagcagggga ctaacatgtg
     2461 aggcaaaaca gcagggccgc gccggtggcg tttttccata ggctccgccc tcctgccaga
     2521 gttcacataa acagacgctt ttccggtgca tctgtgggag ccgtgaggct caaccatgaa
     2581 tctgacagta cgggcgaaac ccgacaggac ttaaagatcc ccaccgtttc cggcgggtcg
     2641 ctccctcttg cgctctcctg ttccgaccct gccgtttacc ggatacctgt tccgcctttc
     2701 tcccttacgg gaagtgtggc gctttctcat agctcacaca ctggtatctc ggctcggtgt
     2761 aggtcgttcg ctccaagctg ggctgtaagc aagaactccc cgttcagccc gactgctgcg
     2821 ccttatccgg taactgttca cttgagtcca acccggaaaa gcacggtaaa acgccactgg
     2881 cagcagccat tggtaactgg gagttcgcag aggatttgtt tagctaaaca cgcggttgct
     2941 cttgaagtgt gcgccaaagt ccggctacac tggaaggaca gatttggttg ctgtgctctg
     3001 cgaaagccag ttaccacggt taagcagttc cccaactgac ttaaccttcg atcaaaccac
     3061 ctccccaggt ggttttttcg tttacagggc aaaagattac gcgcagaaaa aaaggatctc
     3121 aagaagatcc tttgatcttt tctactgaac cgctctagat ttcagtgcaa tttatctctt
     3181 caaatgtagc acctgaagtc agccccatac gatataagtt gtaattctca tgttagtcat
     3241 gccccgcgcc caccggaagg agctgactgg gttgaaggct ctcaagggca tcggtcgaga
     3301 tcccggtgcc taatgagtga gctaacttac attaattgcg ttgcgctcac tgcccgcttt
     3361 ccagtcggga aacctgtcgt gccagctgca ttaatgaatc ggccaacgcg cggggagagg
     3421 cggtttgcgt attgggcgcc agggtggttt ttcttttcac cagtgagacg ggcaacagct
     3481 gattgccctt caccgcctgg ccctgagaga gttgcagcaa gcggtccacg ctggtttgcc
     3541 ccagcaggcg aaaatcctgt ttgatggtgg ttaacggcgg gatataacat gagctgtctt
     3601 cggtatcgtc gtatcccact accgagatgt ccgcaccaac gcgcagcccg gactcggtaa
     3661 tggcgcgcat tgcgcccagc gccatctgat cgttggcaac cagcatcgca gtgggaacga
     3721 tgccctcatt cagcatttgc atggtttgtt gaaaaccgga catggcactc cagtcgcctt
     3781 cccgttccgc tatcggctga atttgattgc gagtgagata tttatgccag ccagccagac
     3841 gcagacgcgc cgagacagaa cttaatgggc ccgctaacag cgcgatttgc tggtgaccca
     3901 atgcgaccag atgctccacg cccagtcgcg taccgtcttc atgggagaaa ataatactgt
     3961 tgatgggtgt ctggtcagag acatcaagaa ataacgccgg aacattagtg caggcagctt
     4021 ccacagcaat ggcatcctgg tcatccagcg gatagttaat gatcagccca ctgacgcgtt
     4081 gcgcgagaag attgtgcacc gccgctttac aggcttcgac gccgcttcgt tctaccatcg
     4141 acaccaccac gctggcaccc agttgatcgg cgcgagattt aatcgccgcg acaatttgcg
     4201 acggcgcgtg cagggccaga ctggaggtgg caacgccaat cagcaacgac tgtttgcccg
     4261 ccagttgttg tgccacgcgg ttgggaatgt aattcagctc cgccatcgcc gcttccactt
     4321 tttcccgcgt tttcgcagaa acgtggctgg cctggttcac cacgcgggaa acggtctgat
     4381 aagagacacc ggcatactct gcgacatcgt ataacgttac tggtttcaca ttcaccaccc
     4441 tgaattgact ctcttccggg cgctatcatg ccataccgcg aaaggttttg cgccattcga
     4501 tggtgtccgg gatctcgacg ctctccctta tgcgactcct gcattaggaa attaatacga
     4561 ctcactata
//
