LOCUS       sequence-270547-        5478 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  TetR, lacI-Plac-ssdsbA-mscarN, Integrative plasmid for the 
            expression of fully-screted prey. N-terminal mScarlet-I fusion..
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 5478)
  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 5478)
  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: pHCL151
FEATURES             Location/Qualifiers
     source          1..5478
                     /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    1205..1431
                     /gene="attP"
                     /label=HK022 attP
                     /bound_moiety="HK022 integrase"
                     /note="attachment site of phage HK022"
     rep_origin      1465..1853
                     /label=R6K gamma ori
                     /note="gamma replication origin from E. coli plasmid R6K; 
                     requires the R6K initiator protein pi for replication"
     CDS             complement(2060..3265)
                     /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        3273..3328
                     /gene="tetR"
                     /label=tetR/tetA promoters
                     /note="overlapping promoters for bacterial tetR and tetA"
     protein_bind    3274..3292
                     /gene="tetO"
                     /label=tet operator
                     /bound_moiety="tetracycline repressor TetR"
                     /note="bacterial operator O2 for the tetR and tetA genes"
     protein_bind    3304..3322
                     /label=tet operator
                     /bound_moiety="tetracycline repressor TetR"
                     /note="bacterial operator O1 for the tetR and tetA genes"
     CDS             3344..3970
                     /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(3420..3439)
                     /label=Tet-R
                     /note="Tetracycline resistance gene, reverse primer"
     primer_bind     4114..4132
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     promoter        4152..4229
                     /gene="lacI"
                     /label=lacI promoter
     CDS             4230..5312
                     /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(4249..4268)
                     /label=LacI-R
                     /note="LacI, reverse primer"
     protein_bind    5325..5346
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     promoter        join(5361..5366,5367..5384,5385..5391)
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    5399..5415
                     /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     5404..5426
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     primer_bind     5423..5439
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     5423..5439
                     /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 tctatggttt ctaaaggtga
      181 agcagttatc aaggagttca tgcgctttaa ggttcacatg gaagggagcg ttaacggaca
      241 cgagttcgag atcgaagggg agggggaagg ccgcccttat gagggtactc aaactgccaa
      301 gctgaaggtc acaaaggggg gtcccttgcc gttttcttgg gacattctgt cacctcagtt
      361 catgtacggg tcccgcgcgt ttattaagca tccggctgac atcccagatt actataagca
      421 gtcattcccg gaagggttca aatgggaacg cgtgatgaac ttcgaagatg ggggagccgt
      481 cactgttacc caggacacta gcctggagga tggcacgttg atttataagg tcaagctgcg
      541 tggaacaaat tttcccccag atgggccagt tatgcagaag aaaacaatgg ggtgggaagc
      601 gagtacggag cgcttatacc ccgaagatgg cgtcctgaaa ggggacatca aaatggccct
      661 gcgcctgaag gacgggggac gttatttagc ggacttcaag acaacctata aagctaagaa
      721 acccgtgcaa atgcccggtg cttacaacgt ggaccgcaag ctggatatca caagccataa
      781 tgaagattac accgtggtag agcagtacga gcgcagtgag ggccgccact caaccggagg
      841 tatggatgaa ctgtataagt ctggcggtgg ttctccggct ggtctgggcg gtggatccgg
      901 tatctcatcc ctcgagtaag cttatcgatc tcacgataat atccgggtag gcgcaatcac
      961 tttcgtctct actccgttac aaagcgaggc tgggtatttc ccggcctttc tgttatccga
     1021 aatccactga aagcacagcg gctggctgag gagataaata ataaacgagg ggctgtatgc
     1081 acaaagcatc ttctgttgag ttaagaacga gtatcgagat ggcacatagc cttgctcaaa
     1141 ttggaatcag gtttgtgcca ataccagtag aaacagacga agaagctagc taatgctctg
     1201 tctcaggtca ctaatactat ctaagtagtt gattcatagt gactggatat gttgcgtttt
     1261 gtcgcattat gtagtctatc atttaaccac agattagtgt aatgcgatga tttttaagtg
     1321 attaatgtta ttttgtcatc ctttaggtga ataagttgta tatttaaaat ctctttaatt
     1381 atcagtaaat taatgtaagt aggtcattat tagtcaaaat aaaatcattt gtcgatttca
     1441 attttgtccc atggctaatt cccatgtcag ccgttaagtg ttcctgtgtc actcaaaatt
     1501 gctttgagag gctctaaggg cttctcagtg cgttacatcc ctggcttgtt gtccacaacc
     1561 gttaaacctt aaaaccttta aaagccttat atattctttt ttttcttata aaacttaaaa
     1621 ccttagaggc tatttaagtt gctgatttat attaatttta ttgttcaaac atgagagctt
     1681 agtacgtgaa acatgagagc ttagtacgtt agccatgaga gcttagtacg ttagccatga
     1741 gggtttagtt cgttaaacat gagagcttag tacgttaaac atgagagctt agtacgtgaa
     1801 acatgagagc ttagtacgta ctatcaacag gttgaactgc tgatcttcag atcctctacg
     1861 ccggacgcat cgtggccgga tcttgcggcc gcaggccaat ttattgctat ttaccgcggc
     1921 tttttattga gcttgaaaga taaataaaat agataggttt tatttgaagc taaatcttct
     1981 ttatcgtaaa aaatgccctc ttgggttatc aagagggtca ttatatttcg cggaataaca
     2041 tcatttggtg acgaaataac taagcacttg tctcctgttt actcccctga gcttgagggg
     2101 ttaacatgaa ggtcatcgat agcaggataa taatacagta aaacgctaaa ccaataatcc
     2161 aaatccagcc atcccaaatt ggtagtgaat gattataaat aacagcaaac agtaatgggc
     2221 caataacacc ggttgcattg gtaaggctca ccaataatcc ctgtaaagca ccttgctgat
     2281 gactctttgt ttggatagac atcactccct gtaatgcagg taaagcgatc ccaccaccag
     2341 ccaataaaat taaaacaggg aaaactaacc aaccttcaga tataaacgct aaaaaggcaa
     2401 atgcactact atctgcaata aatccgagca gtactgccgt tttttcgccc catttagtgg
     2461 ctattcttcc tgccacaaag gcttggaata ctgagtgtaa aagaccaaga cccgctaatg
     2521 aaaagccaac catcatgcta ttccatccaa aacgattttc ggtaaatagc acccacaccg
     2581 ttgcgggaat ttggcctatc aattgcgctg aaaaataaat aatcaacaaa atgggcatcg
     2641 ttttaaataa agtgatgtat accgagttcg attgcgtctc aacccctact tcggtatctg
     2701 tattatcacg tgtatttttg gtttcacgga accaaaacat aaccacaagg aaagtgacaa
     2761 tatttagcaa cgcagcgata aaaaagggac tatgcggtga aatctctcct gcaaaaccac
     2821 caataatagg ccccgctatt aaaccaagcc caaaacttgc ccctaaccaa ccgaaccact
     2881 tcacgcgttg agaagctgag gtggtatcgg caatgaccga tgccgcgaca gccccagtag
     2941 ctcctgtgat ccctgaaagc aaacggccta aatacagcat ccaaagcgca cttgaaaaag
     3001 ccagcaataa gtaatccagc gatgcgccta ttaatgacaa caacagcact gggcgccgac
     3061 caaatcggtc agacattttt ccaagccaag gagcaaagat aacctgcatt aacgcataaa
     3121 gtgcaagcaa tacgccaaag tggttagcga tatcttccga agcaataaat tcacgtaata
     3181 acgttggcaa gactggcatg ataaggccaa tccccatggc atcgagtaac gtaattacca
     3241 atgcgatctt tgtcgaacta ttcatttcac ttttctctat cactgatagg gagtggtaaa
     3301 ataactctat caatgataga gtgtcaacaa aaattaggaa ttaatgatgt ccagattaga
     3361 taaaagtaaa gtgattaaca gcgcattaga gctgcttaat gaggtcggaa tcgaaggttt
     3421 aacaacccgt aaactcgccc agaagctagg tgtagagcag cctacattgt attggcatgt
     3481 aaaaaataag cgggctttgc tcgacgcctt agccattgag atgttagata ggcaccatac
     3541 tcacttttgc cctttagaag gggaaagctg gcaagatttt ttacgtaata acgctaaaag
     3601 ttttagatgt gctttactaa gtcatcgcga tggagcaaaa gtacatttag gtacacggcc
     3661 tacagaaaaa cagtatgaaa ctctcgaaaa tcaattagcc tttttatgcc aacaaggttt
     3721 ttcactagag aatgcattat atgcactcag cgctgtgggg cattttactt taggttgcgt
     3781 attggaagat caagagcatc aagtcgctaa agaagaaagg gaaacaccta ctactgatag
     3841 tatgccgcca ttattacgac aagctatcga attatttgat caccaaggtg cagagccagc
     3901 cttcttattc ggccttgaat tgatcatctg cggattagaa aaacaactta aatgtgaaag
     3961 tgggtcttaa aagcagcata acctttttcc gtgatggtaa cttcacggta accaagatgt
     4021 cgagttaacc accctttaga gatctgcaca tttccccgaa aagtgccacc tgacgtctaa
     4081 gaaaccatta ttatcatgac attaacctat aaaaataggc gtatcacgag gccctttcgt
     4141 cttcaaattc cgacaccatc gaatggcgca aaacctttcg cggtatggca tgatagcgcc
     4201 cggaagagag tcaattcagg gtggtgaatg tgaaaccagt aacgttatac gatgtcgcag
     4261 agtatgccgg tgtctcttat cagaccgttt cccgcgtggt gaaccaggcc agccacgttt
     4321 ctgcgaaaac gcgggaaaaa gtggaagcgg cgatggcgga gctgaattac attcccaacc
     4381 gcgtggcaca acaactggcg ggcaaacagt cgttgctgat tggcgttgcc acctccagtc
     4441 tggccctgca cgcgccgtcg caaattgtcg cggcgattaa atctcgcgcc gatcaactgg
     4501 gtgccagcgt ggtggtgtcg atggtagaac gaagcggcgt cgaagcctgt aaagcggcgg
     4561 tgcacaatct tctcgcgcaa cgcgtcagtg ggctgatcat taactatccg ctggatgacc
     4621 aggatgccat tgctgtggaa gctgcctgca ctaatgttcc ggcgttattt cttgatgtct
     4681 ctgaccagac acccatcaac agtattattt tctcccatga agacggtacg cgactgggcg
     4741 tggagcatct ggtcgcattg ggtcaccagc aaatcgcgct gttagcgggc ccattaagtt
     4801 ctgtctcggc gcgtctgcgt ctggctggct ggcataaata tctcactcgc aatcaaattc
     4861 agccgatagc ggaacgggaa ggcgactgga gtgccatgtc cggttttcaa caaaccatgc
     4921 aaatgctgaa tgagggcatc gttcccactg cgatgctggt tgccaacgat cagatggcgc
     4981 tgggcgcaat gcgcgccatt accgagtccg ggctgcgcgt tggtgcggat atctcggtag
     5041 tgggatacga cgataccgaa gacagctcat gttatatccc gccgttaacc accatcaaac
     5101 aggattttcg cctgctgggg caaaccagcg tggaccgctt gctgcaactc tctcagggcc
     5161 aggcggtgaa gggcaatcag ctgttgcccg tctcactggt gaaaagaaaa accaccctgg
     5221 cgcccaatac gcaaaccgcc tctccccgcg cgttggccga ttcattaatg cagctggcac
     5281 gacaggtttc ccgactggaa agcgggcagt gagcgcaacg caattaatgt gagttagctc
     5341 actcattagg caccccaggc tttacacttt atgcttccgg ctcgtatgtt gtgtggaatt
     5401 gtgagcggat aacaatttca cacaggaaac agctatgacc atgattacga attcccgggg
     5461 atctcgatcc cgcgaaat
//
