LOCUS       sequence-434425-        4512 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 3 (OL3) 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 4512)
  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 4512)
  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-OL3del
FEATURES             Location/Qualifiers
     source          1..4512
                     /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             1022..1039
                     /codon_start=1
                     /product="thrombin recognition and cleavage site"
                     /label=thrombin site
                     /translation="LVPRGS"
     CDS             1046..1075
                     /codon_start=1
                     /product="10xHis affinity tag"
                     /label=10xHis
                     /translation="HHHHHHHHHH"
     CDS             1097..1141
                     /codon_start=1
                     /product="affinity and epitope tag derived from pancreatic 
                     ribonuclease A"
                     /label=S-Tag
                     /translation="KETAAAKFERQHMDS"
     primer_bind     complement(1179..1197)
                     /label=T7 Term
                     /note="T7 terminator, reverse primer"
     terminator      1193..1240
                     /label=T7 terminator
                     /note="transcription terminator for bacteriophage T7 RNA 
                     polymerase"
     CDS             complement(1411..2202)
                     /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(2203..2294)
                     /gene="bla"
                     /label=AmpR promoter
     rep_origin      complement(2342..3080)
                     /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(3290..4372)
                     /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     4334..4353
                     /label=LacI-R
                     /note="LacI, reverse primer"
     promoter        complement(4373..4450)
                     /gene="lacI"
                     /label=lacI promoter
     primer_bind     join(4496..4512,1..3)
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        join(4496..4512,1..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 ctggatggga atattaaaaa acaacggtgt gctgaataat tttctgctgt ggctgggggt
      661 tatcgatcaa ccgctgacca ttctgcatac caatctggcc gtttatatcg gcattgttta
      721 cgcttacgtg ccgtttatgg tactgccgat ttataccgcg ttgattcgta ttgattattc
      781 gctggtggaa gcagcgctgg atctcggtgc acgaccgctg aaaacgttct ttactgtgat
      841 cgtgccgctg actaaaggtg ggattattgc cggatcgatg ctggtgttta tcccggctgt
      901 gggcgagttt gtgatcccgc cggtggcctc ggcggtagcg atcatcatgt tgctgctgct
      961 aattgtgccg ataatgtggt tccacaaaca ccagcaaaaa agcgtgggag aacacggagg
     1021 gttggttccc cgtggtagtt ccgctcatca ccaccatcat caccatcacc accacggtgc
     1081 ataactcgag tctggtaaag aaaccgctgc tgcgaaattt gaacgccagc acatggactc
     1141 gtctactagc gcagcttaat taacctaggc tgctgccacc gctgagcaat aactagcata
     1201 accccttggg gcctctaaac gggtcttgag gggttttttg ctgaaacctc aggcatttga
     1261 gaagcacacg gtcacactgc ttccggtagt caataaaccg gtaaaccagc aatagacata
     1321 agcggctatt taacgaccct gccctgaacc gacgaccggg tcatcgtggc cggatcttgc
     1381 ggcccctcgg cttgaacgaa ttgttagaca ttatttgccg actaccttgg tgatctcgcc
     1441 tttcacgtag tggacaaatt cttccaactg atctgcgcgc gaggccaagc gatcttcttc
     1501 ttgtccaaga taagcctgtc tagcttcaag tatgacgggc tgatactggg ccggcaggcg
     1561 ctccattgcc cagtcggcag cgacatcctt cggcgcgatt ttgccggtta ctgcgctgta
     1621 ccaaatgcgg gacaacgtaa gcactacatt tcgctcatcg ccagcccagt cgggcggcga
     1681 gttccatagc gttaaggttt catttagcgc ctcaaataga tcctgttcag gaaccggatc
     1741 aaagagttcc tccgccgctg gacctaccaa ggcaacgcta tgttctcttg cttttgtcag
     1801 caagatagcc agatcaatgt cgatcgtggc tggctcgaag atacctgcaa gaatgtcatt
     1861 gcgctgccat tctccaaatt gcagttcgcg cttagctgga taacgccacg gaatgatgtc
     1921 gtcgtgcaca acaatggtga cttctacagc gcggagaatc tcgctctctc caggggaagc
     1981 cgaagtttcc aaaaggtcgt tgatcaaagc tcgccgcgtt gtttcatcaa gccttacggt
     2041 caccgtaacc agcaaatcaa tatcactgtg tggcttcagg ccgccatcca ctgcggagcc
     2101 gtacaaatgt acggccagca acgtcggttc gagatggcgc tcgatgacgc caactacctc
     2161 tgatagttga gtcgatactt cggcgatcac cgcttccctc atactcttcc tttttcaata
     2221 ttattgaagc atttatcagg gttattgtct catgagcgga tacatatttg aatgtattta
     2281 gaaaaataaa caaatagcta gctcactcgg tcgctacgct ccgggcgtga gactgcggcg
     2341 ggcgctgcgg acacatacaa agttacccac agattccgtg gataagcagg ggactaacat
     2401 gtgaggcaaa acagcagggc cgcgccggtg gcgtttttcc ataggctccg ccctcctgcc
     2461 agagttcaca taaacagacg cttttccggt gcatctgtgg gagccgtgag gctcaaccat
     2521 gaatctgaca gtacgggcga aacccgacag gacttaaaga tccccaccgt ttccggcggg
     2581 tcgctccctc ttgcgctctc ctgttccgac cctgccgttt accggatacc tgttccgcct
     2641 ttctccctta cgggaagtgt ggcgctttct catagctcac acactggtat ctcggctcgg
     2701 tgtaggtcgt tcgctccaag ctgggctgta agcaagaact ccccgttcag cccgactgct
     2761 gcgccttatc cggtaactgt tcacttgagt ccaacccgga aaagcacggt aaaacgccac
     2821 tggcagcagc cattggtaac tgggagttcg cagaggattt gtttagctaa acacgcggtt
     2881 gctcttgaag tgtgcgccaa agtccggcta cactggaagg acagatttgg ttgctgtgct
     2941 ctgcgaaagc cagttaccac ggttaagcag ttccccaact gacttaacct tcgatcaaac
     3001 cacctcccca ggtggttttt tcgtttacag ggcaaaagat tacgcgcaga aaaaaaggat
     3061 ctcaagaaga tcctttgatc ttttctactg aaccgctcta gatttcagtg caatttatct
     3121 cttcaaatgt agcacctgaa gtcagcccca tacgatataa gttgtaattc tcatgttagt
     3181 catgccccgc gcccaccgga aggagctgac tgggttgaag gctctcaagg gcatcggtcg
     3241 agatcccggt gcctaatgag tgagctaact tacattaatt gcgttgcgct cactgcccgc
     3301 tttccagtcg ggaaacctgt cgtgccagct gcattaatga atcggccaac gcgcggggag
     3361 aggcggtttg cgtattgggc gccagggtgg tttttctttt caccagtgag acgggcaaca
     3421 gctgattgcc cttcaccgcc tggccctgag agagttgcag caagcggtcc acgctggttt
     3481 gccccagcag gcgaaaatcc tgtttgatgg tggttaacgg cgggatataa catgagctgt
     3541 cttcggtatc gtcgtatccc actaccgaga tgtccgcacc aacgcgcagc ccggactcgg
     3601 taatggcgcg cattgcgccc agcgccatct gatcgttggc aaccagcatc gcagtgggaa
     3661 cgatgccctc attcagcatt tgcatggttt gttgaaaacc ggacatggca ctccagtcgc
     3721 cttcccgttc cgctatcggc tgaatttgat tgcgagtgag atatttatgc cagccagcca
     3781 gacgcagacg cgccgagaca gaacttaatg ggcccgctaa cagcgcgatt tgctggtgac
     3841 ccaatgcgac cagatgctcc acgcccagtc gcgtaccgtc ttcatgggag aaaataatac
     3901 tgttgatggg tgtctggtca gagacatcaa gaaataacgc cggaacatta gtgcaggcag
     3961 cttccacagc aatggcatcc tggtcatcca gcggatagtt aatgatcagc ccactgacgc
     4021 gttgcgcgag aagattgtgc accgccgctt tacaggcttc gacgccgctt cgttctacca
     4081 tcgacaccac cacgctggca cccagttgat cggcgcgaga tttaatcgcc gcgacaattt
     4141 gcgacggcgc gtgcagggcc agactggagg tggcaacgcc aatcagcaac gactgtttgc
     4201 ccgccagttg ttgtgccacg cggttgggaa tgtaattcag ctccgccatc gccgcttcca
     4261 ctttttcccg cgttttcgca gaaacgtggc tggcctggtt caccacgcgg gaaacggtct
     4321 gataagagac accggcatac tctgcgacat cgtataacgt tactggtttc acattcacca
     4381 ccctgaattg actctcttcc gggcgctatc atgccatacc gcgaaaggtt ttgcgccatt
     4441 cgatggtgtc cgggatctcg acgctctccc ttatgcgact cctgcattag gaaattaata
     4501 cgactcacta ta
//
