LOCUS       sequence-270545-        5445 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  TetR, lacI-Plac-ssdsbA-mscarC, Integrative plasmid for the 
            expression of fully-secreted prey. C-terminal mScarlet-I fusion..
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 5445)
  AUTHORS   Lim HC, Bernhardt TG
  TITLE     A PopZ-Linked Apical Recruitment Assay for Studying Protein-Protein 
            Interactions in the Bacterial Cell Envelope.
  JOURNAL   Mol Microbiol. 2019 Sep 24. doi: 10.1111/mmi.14391.
  PUBMED    31550057
REFERENCE   2  (bases 1 to 5445)
  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: "Mol 
            Microbiol. 2019 Sep 24. doi: 10.1111/mmi.14391."
COMMENT     Sequence Label: pHCL147
FEATURES             Location/Qualifiers
     source          1..5445
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     1..20
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        1..19
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     protein_bind    20..44
                     /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)."
     RBS             59..81
                     /note="efficient ribosome binding site from bacteriophage 
                     T7 gene 10 (Olins and Rangwala, 1989)"
     protein_bind    1172..1398
                     /gene="attP"
                     /label=HK022 attP
                     /bound_moiety="HK022 integrase"
                     /note="attachment site of phage HK022"
     rep_origin      1432..1820
                     /label=R6K gamma ori
                     /note="gamma replication origin from E. coli plasmid R6K; 
                     requires the R6K initiator protein pi for replication"
     CDS             complement(2027..3232)
                     /codon_start=1
                     /gene="tetA from transposon Tn10"
                     /product="tetracycline efflux protein"
                     /label=TcR
                     /note="confers resistance to tetracycline"
                     /translation="MNSSTKIALVITLLDAMGIGLIMPVLPTLLREFIASEDIANHFGV
                     LLALYALMQVIFAPWLGKMSDRFGRRPVLLLSLIGASLDYLLLAFSSALWMLYLGRLLS
                     GITGATGAVAASVIADTTSASQRVKWFGWLGASFGLGLIAGPIIGGFAGEISPHSPFFI
                     AALLNIVTFLVVMFWFRETKNTRDNTDTEVGVETQSNSVYITLFKTMPILLIIYFSAQL
                     IGQIPATVWVLFTENRFGWNSMMVGFSLAGLGLLHSVFQAFVAGRIATKWGEKTAVLLG
                     FIADSSAFAFLAFISEGWLVFPVLILLAGGGIALPALQGVMSIQTKSHQQGALQGLLVS
                     LTNATGVIGPLLFAVIYNHSLPIWDGWIWIIGLAFYCIIILLSMTFMLTPQAQGSKQET
                     SA"
     promoter        3240..3295
                     /gene="tetR"
                     /label=tetR/tetA promoters
                     /note="overlapping promoters for bacterial tetR and tetA"
     protein_bind    3241..3259
                     /gene="tetO"
                     /label=tet operator
                     /bound_moiety="tetracycline repressor TetR"
                     /note="bacterial operator O2 for the tetR and tetA genes"
     protein_bind    3271..3289
                     /label=tet operator
                     /bound_moiety="tetracycline repressor TetR"
                     /note="bacterial operator O1 for the tetR and tetA genes"
     CDS             3311..3937
                     /codon_start=1
                     /gene="tetR"
                     /product="tetracycline repressor TetR"
                     /label=TetR
                     /note="TetR binds to the tetracycline operator tetO to 
                     inhibit transcription. This inhibition can be relieved by 
                     adding tetracycline or doxycycline."
                     /translation="MMSRLDKSKVINSALELLNEVGIEGLTTRKLAQKLGVEQPTLYWH
                     VKNKRALLDALAIEMLDRHHTHFCPLEGESWQDFLRNNAKSFRCALLSHRDGAKVHLGT
                     RPTEKQYETLENQLAFLCQQGFSLENALYALSAVGHFTLGCVLEDQEHQVAKEERETPT
                     TDSMPPLLRQAIELFDHQGAEPAFLFGLELIICGLEKQLKCESGS"
     primer_bind     complement(3387..3406)
                     /label=Tet-R
                     /note="Tetracycline resistance gene, reverse primer"
     primer_bind     4081..4099
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     promoter        4119..4196
                     /gene="lacI"
                     /label=lacI promoter
     CDS             4197..5279
                     /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(4216..4235)
                     /label=LacI-R
                     /note="LacI, reverse primer"
     protein_bind    5292..5313
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     promoter        join(5328..5333,5334..5351,5352..5358)
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    5366..5382
                     /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     5371..5393
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     primer_bind     5390..5406
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     5390..5406
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
ORIGIN
        1 taatacgact cactataggg gaattgtgag cggataacaa ttcccctcta gaaataattt
       61 tgtttaactt taagaaggag atatacatat gaaaaagatt tggctggcgc tggctggttt
      121 agttttagcg tttagcgcat cggcggcgca gtatgaagga tcctctggtc tcgagggtcc
      181 ggctggtctg gtttctaaag gtgaagcagt tatcaaggag ttcatgcgct ttaaggttca
      241 catggaaggg agcatgaacg gacacgagtt cgagatcgaa ggggaggggg aaggccgccc
      301 ttatgagggt actcaaactg ccaagctgaa ggtcacaaag gggggtccct tgccgttttc
      361 ttgggacatt ctgtcacctc agttcatgta cgggtcccgc gcgtttatta agcatccggc
      421 tgacatccca gattactata agcagtcatt cccggaaggg ttcaaatggg aacgcgtgat
      481 gaacttcgaa gatgggggag ccgtcactgt tacccaggac actagcctgg aggatggcac
      541 gttgatttat aaggtcaagc tgcgtggaac aaattttccc ccagatgggc cagttatgca
      601 gaagaaaaca atggggtggg aagcgagtac ggagcgctta taccccgaag atggcgtcct
      661 gaaaggggac atcaaaatgg ccctgcgcct gaaggacggg ggacgttatt tagcggactt
      721 caagacaacc tataaagcta agaaacccgt gcaaatgccc ggtgcttaca acgtggaccg
      781 caagctggat atcacaagcc ataatgaaga ttacaccgtg gtagagcagt acgagcgcag
      841 tgagggccgc cactcaaccg gaggtatgga tgaactgtat aagtaagctt atcgatctca
      901 cgataatatc cgggtaggcg caatcacttt cgtctctact ccgttacaaa gcgaggctgg
      961 gtatttcccg gcctttctgt tatccgaaat ccactgaaag cacagcggct ggctgaggag
     1021 ataaataata aacgaggggc tgtatgcaca aagcatcttc tgttgagtta agaacgagta
     1081 tcgagatggc acatagcctt gctcaaattg gaatcaggtt tgtgccaata ccagtagaaa
     1141 cagacgaaga agctagctaa tgctctgtct caggtcacta atactatcta agtagttgat
     1201 tcatagtgac tggatatgtt gcgttttgtc gcattatgta gtctatcatt taaccacaga
     1261 ttagtgtaat gcgatgattt ttaagtgatt aatgttattt tgtcatcctt taggtgaata
     1321 agttgtatat ttaaaatctc tttaattatc agtaaattaa tgtaagtagg tcattattag
     1381 tcaaaataaa atcatttgtc gatttcaatt ttgtcccatg gctaattccc atgtcagccg
     1441 ttaagtgttc ctgtgtcact caaaattgct ttgagaggct ctaagggctt ctcagtgcgt
     1501 tacatccctg gcttgttgtc cacaaccgtt aaaccttaaa acctttaaaa gccttatata
     1561 ttcttttttt tcttataaaa cttaaaacct tagaggctat ttaagttgct gatttatatt
     1621 aattttattg ttcaaacatg agagcttagt acgtgaaaca tgagagctta gtacgttagc
     1681 catgagagct tagtacgtta gccatgaggg tttagttcgt taaacatgag agcttagtac
     1741 gttaaacatg agagcttagt acgtgaaaca tgagagctta gtacgtacta tcaacaggtt
     1801 gaactgctga tcttcagatc ctctacgccg gacgcatcgt ggccggatct tgcggccgca
     1861 ggccaattta ttgctattta ccgcggcttt ttattgagct tgaaagataa ataaaataga
     1921 taggttttat ttgaagctaa atcttcttta tcgtaaaaaa tgccctcttg ggttatcaag
     1981 agggtcatta tatttcgcgg aataacatca tttggtgacg aaataactaa gcacttgtct
     2041 cctgtttact cccctgagct tgaggggtta acatgaaggt catcgatagc aggataataa
     2101 tacagtaaaa cgctaaacca ataatccaaa tccagccatc ccaaattggt agtgaatgat
     2161 tataaataac agcaaacagt aatgggccaa taacaccggt tgcattggta aggctcacca
     2221 ataatccctg taaagcacct tgctgatgac tctttgtttg gatagacatc actccctgta
     2281 atgcaggtaa agcgatccca ccaccagcca ataaaattaa aacagggaaa actaaccaac
     2341 cttcagatat aaacgctaaa aaggcaaatg cactactatc tgcaataaat ccgagcagta
     2401 ctgccgtttt ttcgccccat ttagtggcta ttcttcctgc cacaaaggct tggaatactg
     2461 agtgtaaaag accaagaccc gctaatgaaa agccaaccat catgctattc catccaaaac
     2521 gattttcggt aaatagcacc cacaccgttg cgggaatttg gcctatcaat tgcgctgaaa
     2581 aataaataat caacaaaatg ggcatcgttt taaataaagt gatgtatacc gagttcgatt
     2641 gcgtctcaac ccctacttcg gtatctgtat tatcacgtgt atttttggtt tcacggaacc
     2701 aaaacataac cacaaggaaa gtgacaatat ttagcaacgc agcgataaaa aagggactat
     2761 gcggtgaaat ctctcctgca aaaccaccaa taataggccc cgctattaaa ccaagcccaa
     2821 aacttgcccc taaccaaccg aaccacttca cgcgttgaga agctgaggtg gtatcggcaa
     2881 tgaccgatgc cgcgacagcc ccagtagctc ctgtgatccc tgaaagcaaa cggcctaaat
     2941 acagcatcca aagcgcactt gaaaaagcca gcaataagta atccagcgat gcgcctatta
     3001 atgacaacaa cagcactggg cgccgaccaa atcggtcaga catttttcca agccaaggag
     3061 caaagataac ctgcattaac gcataaagtg caagcaatac gccaaagtgg ttagcgatat
     3121 cttccgaagc aataaattca cgtaataacg ttggcaagac tggcatgata aggccaatcc
     3181 ccatggcatc gagtaacgta attaccaatg cgatctttgt cgaactattc atttcacttt
     3241 tctctatcac tgatagggag tggtaaaata actctatcaa tgatagagtg tcaacaaaaa
     3301 ttaggaatta atgatgtcca gattagataa aagtaaagtg attaacagcg cattagagct
     3361 gcttaatgag gtcggaatcg aaggtttaac aacccgtaaa ctcgcccaga agctaggtgt
     3421 agagcagcct acattgtatt ggcatgtaaa aaataagcgg gctttgctcg acgccttagc
     3481 cattgagatg ttagataggc accatactca cttttgccct ttagaagggg aaagctggca
     3541 agatttttta cgtaataacg ctaaaagttt tagatgtgct ttactaagtc atcgcgatgg
     3601 agcaaaagta catttaggta cacggcctac agaaaaacag tatgaaactc tcgaaaatca
     3661 attagccttt ttatgccaac aaggtttttc actagagaat gcattatatg cactcagcgc
     3721 tgtggggcat tttactttag gttgcgtatt ggaagatcaa gagcatcaag tcgctaaaga
     3781 agaaagggaa acacctacta ctgatagtat gccgccatta ttacgacaag ctatcgaatt
     3841 atttgatcac caaggtgcag agccagcctt cttattcggc cttgaattga tcatctgcgg
     3901 attagaaaaa caacttaaat gtgaaagtgg gtcttaaaag cagcataacc tttttccgtg
     3961 atggtaactt cacggtaacc aagatgtcga gttaaccacc ctttagagat ctgcacattt
     4021 ccccgaaaag tgccacctga cgtctaagaa accattatta tcatgacatt aacctataaa
     4081 aataggcgta tcacgaggcc ctttcgtctt caaattccga caccatcgaa tggcgcaaaa
     4141 cctttcgcgg tatggcatga tagcgcccgg aagagagtca attcagggtg gtgaatgtga
     4201 aaccagtaac gttatacgat gtcgcagagt atgccggtgt ctcttatcag accgtttccc
     4261 gcgtggtgaa ccaggccagc cacgtttctg cgaaaacgcg ggaaaaagtg gaagcggcga
     4321 tggcggagct gaattacatt cccaaccgcg tggcacaaca actggcgggc aaacagtcgt
     4381 tgctgattgg cgttgccacc tccagtctgg ccctgcacgc gccgtcgcaa attgtcgcgg
     4441 cgattaaatc tcgcgccgat caactgggtg ccagcgtggt ggtgtcgatg gtagaacgaa
     4501 gcggcgtcga agcctgtaaa gcggcggtgc acaatcttct cgcgcaacgc gtcagtgggc
     4561 tgatcattaa ctatccgctg gatgaccagg atgccattgc tgtggaagct gcctgcacta
     4621 atgttccggc gttatttctt gatgtctctg accagacacc catcaacagt attattttct
     4681 cccatgaaga cggtacgcga ctgggcgtgg agcatctggt cgcattgggt caccagcaaa
     4741 tcgcgctgtt agcgggccca ttaagttctg tctcggcgcg tctgcgtctg gctggctggc
     4801 ataaatatct cactcgcaat caaattcagc cgatagcgga acgggaaggc gactggagtg
     4861 ccatgtccgg ttttcaacaa accatgcaaa tgctgaatga gggcatcgtt cccactgcga
     4921 tgctggttgc caacgatcag atggcgctgg gcgcaatgcg cgccattacc gagtccgggc
     4981 tgcgcgttgg tgcggatatc tcggtagtgg gatacgacga taccgaagac agctcatgtt
     5041 atatcccgcc gttaaccacc atcaaacagg attttcgcct gctggggcaa accagcgtgg
     5101 accgcttgct gcaactctct cagggccagg cggtgaaggg caatcagctg ttgcccgtct
     5161 cactggtgaa aagaaaaacc accctggcgc ccaatacgca aaccgcctct ccccgcgcgt
     5221 tggccgattc attaatgcag ctggcacgac aggtttcccg actggaaagc gggcagtgag
     5281 cgcaacgcaa ttaatgtgag ttagctcact cattaggcac cccaggcttt acactttatg
     5341 cttccggctc gtatgttgtg tggaattgtg agcggataac aatttcacac aggaaacagc
     5401 tatgaccatg attacgaatt cccggggatc tcgatcccgc gaaat
//
