LOCUS       sequence-336757-        6973 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Encodes LacI-controlled expression of surface-displayed CCL20 and 
            PvirK-mNeonGreen reporter.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 6973)
  AUTHORS   Brink KR, Mu AM, Hoang KV, Groszman K, Gunn JS, Tabor JJ
  TITLE     High-throughput discovery of peptide activators of a bacterial 
            sensor kinase
  JOURNAL   bioRxiv
REFERENCE   2  (bases 1 to 6973)
  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: "bioRxiv"
COMMENT     Sequence Label: pKB235
FEATURES             Location/Qualifiers
     source          1..6973
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     terminator      496..582
                     /gene="Escherichia coli rrnB"
                     /label=rrnB T1 terminator
                     /note="transcription terminator T1 from the E. coli rrnB 
                     gene"
     promoter        join(755..760,761..776,777..783)
                     /label=tac promoter
                     /note="strong E. coli promoter; hybrid between the trp and 
                     lac UV5 promoters"
     protein_bind    791..807
                     /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     796..818
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     terminator      complement(2001..2028)
                     /label=T7Te terminator
                     /note="phage T7 early transcription terminator"
     terminator      complement(2044..2115)
                     /label=rrnB T1 terminator
                     /note="transcription terminator T1 from the E. coli rrnB 
                     gene"
     RBS             2841..2852
                     /note="strong bacterial ribosome binding site (Elowitz and 
                     Leibler, 2000)"
     primer_bind     complement(3050..3068)
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     promoter        3133..3237
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(3238..3306,3307..4098)
                     /codon_start=1
                     /gene="bla"
                     /product="beta-lactamase"
                     /label=AmpR
                     /note="confers resistance to ampicillin, carbenicillin, and
                     related antibiotics"
                     /translation="MSIQHFSVALIHFFAAFCLPVFAHPETLVKVKDAEDQLGARVGYI
                     ELDLNSGKILESFRPEERFPMMSTFKVLLCGAVLYRIDAGQEQLGRRIHYSQNDLVEYS
                     PVTEKHLTDGMTVRELCSAAITMSDNTAANLLLTTIGGPKELTAFLHNMGDHVTRLDRW
                     EPELNEAIPNDERDTTMPVAMATTLRKLLTGELLTLASRQQLIDWMEADKVAGPLLRSA
                     LPAGWFIADKSGAGERGSRGIIAALGPDGKPSRIVVIYTTGSQATMDERNRQIAEIGAS
                     LIKHW"
     primer_bind     complement(3456..3475)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     terminator      4128..4222
                     /label=lambda t0 terminator
                     /note="transcription terminator from phage lambda"
     rep_origin      4714..4936
                     /label=pSC101 ori
                     /note="low-copy replication origin that requires the Rep101
                     protein"
     CDS             4984..5934
                     /codon_start=1
                     /gene="rep101"
                     /product="RepA protein needed for replication with the 
                     pSC101 origin"
                     /label=Rep101
                     /translation="MSELVVFKANELAISRYDLTEHETKLILCCVALLNPTIENPTRKE
                     RTVSFTYNQYAQMMNISRENAYGVLAKATRELMTRTVEIMNPLVKGFEIFQWTNYAKFS
                     SEKLELVFSEEILPYLFQLKKFIKYNLEHVKSFENKYSMRIYEWLLKELTQKKTHKANI
                     EISLDEFKFMLMLENNYHEFKRLNQWVLKPISKDLNTYSNMKLVVDKRGRPTDTLIFQV
                     ELDRQMDLVTELENNQIKMNGDKIPTTITSDSYLHNGLRKTLHDALTAKIQLTSFEAKF
                     LSDMQSKYDLNGSFSWLTQKQRTTLENILAKYGRI"
     primer_bind     6196..6213
                     /label=L4440
                     /note="L4440 vector, forward primer"
     promoter        6303..6380
                     /gene="lacI (mutant)"
                     /label=lacIq promoter
                     /note="In the lacIq allele, a single base change in the 
                     promoter boosts expression of the lacI gene about 10-fold."
     CDS             join(6381..6973,1..490)
                     /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(6400..6419)
                     /label=LacI-R
                     /note="LacI, reverse primer"
ORIGIN
        1 ggctggctgg cataaatatc tcactcgcaa tcaaattcag ccgatagcgg aacgggaagg
       61 cgactggagt gccatgtccg gttttcaaca aaccatgcaa atgctgaatg agggcatcgt
      121 tcccactgcg atgctggttg ccaacgatca gatggcgctg ggcgcaatgc gcgccattac
      181 cgagtccggg ctgcgcgttg gtgcggatat ctcggtagtg ggatacgacg ataccgaaga
      241 cagctcatgt tatatcccgc cgttaaccac catcaaacag gattttcgcc tgctggggca
      301 aaccagcgtg gaccgcttgc tgcaactctc tcagggccag gcggtgaagg gcaatcagct
      361 gttgcccgtc tcactggtga aaagaaaaac caccctggcg cccaatacgc aaaccgcctc
      421 tccccgcgcg ttggccgatt cattaatgca gctggcacga caggtttccc gactggaaag
      481 cgggcagtga ggcatcaaat aaaacgaaag gctcagtcga aagactgggc ctttcgtttt
      541 atctgttgtt tgtcggtgaa cgctctcctg agtaggacaa atccgccgcc ctagacctag
      601 ggcgttcggc tgcggcgagc ggtatcagct cactcaaagg cggtaatacg taaatcactg
      661 cataattcgt gtcgctcaag gcgcactccc gttctggata atgttttttg cgccgacatc
      721 ataacggttc tggctaatat tctgaaatga gctgttgaca attaatcatc ggctcgtata
      781 atgtgtggaa ttgtgagcgg ataacaattt cacacagggc ccaagttcac ttaaaaagga
      841 gatcaacaat gaaagcaatt ttcgtactga aacatcttaa tcatgctatg gagtatttct
      901 gatgaaagct actaaactgg tactgggcgc ggtaatcctg ggttctactc tgctggcagg
      961 ttgctccagc aacgctaaaa tcgatcaggg aattaacccg tatgttggct ttgaaatggg
     1021 ttacgactgg ttaggtcgta tgccgtacaa aggcagcgtt gaaaacggtg catacaaagc
     1081 tcagggcgtt caactgaccg ctaaactggg ttacccaatc actgacgacc tggacatcta
     1141 cactcgtctg ggtggcatgg tatggcgtgc agacactaaa tccaacgttt atggtaaaaa
     1201 ccacgacacc ggcgtttctc cggtcttcgc tggcggtgtt gagtacgcga tcactcctga
     1261 aatcgctacc cgtctggaat accagtggac caacaacatc ggtgacgcac acaccatcgg
     1321 cactcgtccg gacaacggta ttggtaccag tcaagagcct gcagcgcccg ccgcagaggc
     1381 gactcctgct gctgaagctc cagctagcga agcgcctgca gcagaagctg ccccagcgga
     1441 tgctgccgaa gccccagccg ctggcatcag tcaggaacct gctgcaccag ctgcggaagc
     1501 tacaccagca gcggaggcac cagcgagtga agcaccggct gcggaagccg ctcctgcaga
     1561 tgccgctgag gctccagctg cgggtatcgg aggaacatcc aacttcgatt gttgcttggg
     1621 ctataccgac agaatccttc acccgaagtt catcgttggt ttcactcgtc aactggctaa
     1681 cgagggttgc gacatcaacg cgataatctt tcacacaaag aagaaactct cggtatgcgc
     1741 aaatccgaag caaacctggg tgaagtacat tgtccggttg ctgagcaaga aggtcaagaa
     1801 catgtaaaat taacgccgcg cagtttcaca ttataaatta aacaaccatc ttgcagagaa
     1861 gtatcttgcg ttacagttaa taccccgcca tcctcatagg tcgtcacact aatggagtac
     1921 gcagatgata cagctgcctg gcggcagtag cgcggtggtc ccacctgacc ccatgccgaa
     1981 ctcagaagtg aaactataaa cgcagaaagg cccacccgaa ggtgagccag tgtgactcta
     2041 gtagagagcg ttcaccgaca aacaacagat aaaacgaaag gcccagtctt tcgactgagc
     2101 ctttcgtttt atttgatgcc tggtcatttg tacagttcat ccatgcccat tacgtcggtg
     2161 aaggcttttt gccactcttt gaaattgagt tcagttttcg aatgtttcag ttccgttttg
     2221 cgaaacacat acatcggctg atttttcaga tagttcgcgg ccatgggttt cgcaaaggtg
     2281 tatgtggtgc gagccgtgct acggtagcgt ttaccgttac ccgtggtgta actccattta
     2341 aaggtagaaa taattgtttt atcattggga tacgtctttt tgctacgaca ccagtccgcc
     2401 gccgtcaggc tgttggtcat cactgggcca tccgccggga agccggtgcc tttaacttgt
     2461 gcttcacctt taatatgtga accttcgtaa gtgtaccgat aattcacggt caggctcgcc
     2521 ccatcctcga attgcatggt gcgatggacc tgatagccac tgccatccac catagccgcc
     2581 tgaaatgggc tcattccgtc tgggtagggc agatactgat gaaaaccata accaatatgc
     2641 ggtaccagaa tccacggact aaattgcaag tcgcccttgg tagatttcaa attcaattct
     2701 tcatagccgt cgttcgggtt gccggtgcct tggcccacca tgtcgaaatc aactccgttg
     2761 atgctgccga aaatatgcag ttcatgtgta gcaggcagcg acgccatgtt atcctcctca
     2821 cctttcgaaa ccatctagta tttctcctct ttactacggc tctactactc gatagactta
     2881 taaccgagac ggtagcctga ttgagttaaa cgtaaaggca acgaagaagg tctgtacaga
     2941 cgatgttgtt aaacagttta tcaatggcgg tatccgacgt cggccgtagg cgtcggagat
     3001 tccggagcgt aatagggagt agacggaacg cggaagcgat aaaaattctg cctcgtgata
     3061 cgcctatttt tataggttaa tgtcatgata ataatggttt attagacgtc aggtggcacc
     3121 atgggaaatg tgcgcggaac ccctatttgt ttatttttct aaatacatta aaatatgtat
     3181 ccgctcatga gacaataacc ctgataaatg cttcaataat attgaaaaag gaagagtatg
     3241 agtattcaac atttcagtgt cgcccttatt cacttttttg cggcattttg ccttcccgtt
     3301 tttgctcacc cagaaacgct ggtgaaagta aaagatgctg aagatcagtt gggtgcacga
     3361 gtgggttaca tcgaactgga tctcaacagc ggtaagatcc ttgagagttt tcgccccgaa
     3421 gaacgttttc caatgatgag cacttttaaa gttctgctat gtggcgcggt attataccgt
     3481 attgacgccg ggcaagagca actcggtcgc cgcatacact attctcagaa tgacttggtt
     3541 gagtactcac cagtaacaga aaagcatctt acggatggca tgacagtaag agaattatgc
     3601 agtgctgcca taaccatgag tgataacact gcggccaact tacttctgac aacgatcgga
     3661 ggaccgaagg agctaaccgc ttttttgcac aacatggggg atcatgtaac tcgccttgat
     3721 cgttgggaac cggagctgaa tgaagccata ccaaacgacg agcgtgacac cacgatgcct
     3781 gtagcaatgg caacaacgtt gcgcaaacta ttaactggcg aactacttac tctagcttcc
     3841 cggcaacaat taatagactg gatggaggcg gataaagttg caggaccact tctgcgctcg
     3901 gcccttccgg ctggctggtt tattgctgat aaatctggag ccggtgagcg tggatcacgc
     3961 ggtatcattg cagcactggg gccagatggt aagccctccc gtatcgtagt tatctacacg
     4021 acggggagtc aggcaactat ggatgaacga aatagacaga tcgctgagat aggtgcctca
     4081 ctgattaagc attggtaact gtcagaccaa gtttacgagc tcgcttggac tcctgttgat
     4141 agatccagta atgacatcag aactccatct ggatttgttc agaacgctcg gttgccgccg
     4201 ggcgtttttt attggtgaga atccaagcac tagggacagt aagacgggta agcctgttga
     4261 tgataccgct gccttactgg gtgcattagc cagtctgaat gacctgtcac gggataatcc
     4321 gaagtggtca gactggaaaa tcagagggca ggaactgctg aacagcaaaa agtcagatag
     4381 caccacatag cagaccagcc ataaaacgcc ctgagaagcc cgtgacgggc ttttcttgta
     4441 ttatgggtag tttccttgca tgaatccata aaaggcgcct gtagtgccat ttacccacat
     4501 tcactgccag agccgtgagc gcagcgaact gaatgtcacg aaaaaggcag cgactcaggt
     4561 gcctgatggt cggagacaaa aggaatatta agcgatttgc acgagcttgc gagggtgcta
     4621 cttaagcctt tagggtttta aggtctgttt tgtagaggag caaacagcgt ttgcgacatc
     4681 cttttgtaat actgcggaac tgactaaagt agtgagttat acacagggct gggatctatt
     4741 ctttttatct ttttttattc tttctttatt ctataaatta taaccacttg aatataaaca
     4801 aaaaaaacac acaaaggtct agcggaattt acagagggtc tagcagaatt tacaagtttt
     4861 ccagcaaagg tctagcagaa tttacagata cccacaactc aaaggaaaag gacatgtaat
     4921 tatcattgac tagcccatct caattggtat agtgattaaa atcacctaga ccaattgaga
     4981 tgtatgtctg aattagttgt tttcaaagca aatgaactag cgattagtcg ctatgactta
     5041 acggagcatg aaaccaagct aattttatgc tgtgtggcac tactcaaccc cactattgaa
     5101 aaccctacaa ggaaagaacg gacggtatcg ttcacttata accaatacgc tcagatgatg
     5161 aacatcagta gggaaaatgc ttatggtgta ttagctaaag caaccagaga gctgatgacg
     5221 agaactgtgg aaatcatgaa tcctttggtt aaaggctttg agattttcca gtggacaaac
     5281 tatgccaagt tctcaagcga aaaattagaa ttagttttta gtgaagagat attgccttat
     5341 cttttccagt taaaaaaatt cataaaatat aatctggaac atgttaagtc ttttgaaaac
     5401 aaatactcta tgaggattta tgagtggtta ttaaaagaac taacacaaaa gaaaactcac
     5461 aaggcaaata tagagattag ccttgatgaa tttaagttca tgttaatgct tgaaaataac
     5521 taccatgagt ttaaaaggct taaccaatgg gttttgaaac caataagtaa agatttaaac
     5581 acttacagca atatgaaatt ggtggttgat aagcgaggcc gcccgactga tacgttgatt
     5641 ttccaagttg aactagatag acaaatggat ctcgtaaccg aacttgagaa caaccagata
     5701 aaaatgaatg gtgacaaaat accaacaacc attacatcag attcctacct acataacgga
     5761 ctaagaaaaa cactacacga tgctttaact gcaaaaattc agctcaccag ttttgaggca
     5821 aaatttttga gtgacatgca aagtaagtat gatctcaatg gttcgttctc atggctcacg
     5881 caaaaacaac gaaccacact agagaacata ctggctaaat acggaaggat ctgaggttct
     5941 tatggctctt gtatctatca gtgaagcatc aagactaaca aacaaaagta gaacaactgt
     6001 tcaccgttac atatcaaagg gaaaactgtc catatgcaca gatgaaaacg gtgtaaaaaa
     6061 gatagataca tcagagcttt tacgagtttt tggtgcattc aaagctgttc accatgaaca
     6121 gatcgacaat gtaacctcga gattaccgcc tttgagtgag ctgataccgc tcgccgcagc
     6181 cgaacgaccg agcgcagcga gtcagtgagc gaggaagcct gcataacgcg aagtaatctt
     6241 ttcggtttta aagaaaaagg gcagggtggt gacaccttgc cgaagcggca tgcatttacg
     6301 ttgacaccat cgaatggtgc aaaacctttc gcggtatggc atgatagcgc ccggaagaga
     6361 gtcaattcag ggtggtgaat gtgaaaccag taacgttata cgatgtcgca gagtatgccg
     6421 gtgtctctta tcagaccgtt tcccgcgtgg tgaaccaggc cagccacgtt tctgcgaaaa
     6481 cgcgggaaaa agtggaagcg gcgatggcgg agctgaatta cattcccaac cgcgtggcac
     6541 aacaactggc gggcaaacag tcgttgctga ttggcgttgc cacctccagt ctggccctgc
     6601 acgcgccgtc gcaaattgtc gcggcgatta aatctcgcgc cgatcaactg ggtgccagcg
     6661 tggtggtgtc gatggtagaa cgaagcggcg tcgaagcctg taaagcggcg gtgcacaatc
     6721 ttctcgcgca acgcgtcagt gggctgatca ttaactatcc gctggatgac caggatgcca
     6781 ttgctgtgga agctgcctgc actaatgttc cggcgttatt tcttgatgtc tctgaccaga
     6841 cacccatcaa cagtattatt ttctcccatg aagacggtac gcgactgggc gtggagcatc
     6901 tggtcgcatt gggtcaccag caaatcgcgc tgttagcggg cccattaagt tctgtctcgg
     6961 cgcgtctgcg tct
//
