LOCUS       sequence-335297-        6973 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Encodes LacI-controlled expression of surface-displayed TCP 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: pKB236
FEATURES             Location/Qualifiers
     source          1..6973
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     terminator      complement(313..340)
                     /label=T7Te terminator
                     /note="phage T7 early transcription terminator"
     terminator      complement(356..427)
                     /label=rrnB T1 terminator
                     /note="transcription terminator T1 from the E. coli rrnB 
                     gene"
     RBS             1153..1164
                     /note="strong bacterial ribosome binding site (Elowitz and 
                     Leibler, 2000)"
     primer_bind     complement(1362..1380)
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     promoter        1445..1549
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(1550..1618,1619..2410)
                     /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(1768..1787)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     terminator      2440..2534
                     /label=lambda t0 terminator
                     /note="transcription terminator from phage lambda"
     rep_origin      3026..3248
                     /label=pSC101 ori
                     /note="low-copy replication origin that requires the Rep101
                     protein"
     CDS             3296..4246
                     /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     4508..4525
                     /label=L4440
                     /note="L4440 vector, forward primer"
     promoter        4615..4692
                     /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             4693..5775
                     /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(4712..4731)
                     /label=LacI-R
                     /note="LacI, reverse primer"
     terminator      5781..5867
                     /gene="Escherichia coli rrnB"
                     /label=rrnB T1 terminator
                     /note="transcription terminator T1 from the E. coli rrnB 
                     gene"
     promoter        join(6040..6045,6046..6061,6062..6068)
                     /label=tac promoter
                     /note="strong E. coli promoter; hybrid between the trp and 
                     lac UV5 promoters"
     protein_bind    6076..6092
                     /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     6081..6103
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
ORIGIN
        1 agggtaaacg aggtgatgct tgtgagggtg atagtggcgg tccatttgtt atgaagagtc
       61 cgttcaacaa cagatggtat cagatgggta ttgtatcttg gggtgagggt tgtgatcgtg
      121 atggtaagta tggattctat actcatgtat tccgtttgaa gaagtggatt cagaaggtta
      181 ttgatcagtt tggtgagtaa catcctcata ggtcgtcaca ctaatggagt acgcagatga
      241 tacagctgcc tggcggcagt agcgcggtgg tcccacctga ccccatgccg aactcagaag
      301 tgaaactata aacgcagaaa ggcccacccg aaggtgagcc agtgtgactc tagtagagag
      361 cgttcaccga caaacaacag ataaaacgaa aggcccagtc tttcgactga gcctttcgtt
      421 ttatttgatg cctggtcatt tgtacagttc atccatgccc attacgtcgg tgaaggcttt
      481 ttgccactct ttgaaattga gttcagtttt cgaatgtttc agttccgttt tgcgaaacac
      541 atacatcggc tgatttttca gatagttcgc ggccatgggt ttcgcaaagg tgtatgtggt
      601 gcgagccgtg ctacggtagc gtttaccgtt acccgtggtg taactccatt taaaggtaga
      661 aataattgtt ttatcattgg gatacgtctt tttgctacga caccagtccg ccgccgtcag
      721 gctgttggtc atcactgggc catccgccgg gaagccggtg cctttaactt gtgcttcacc
      781 tttaatatgt gaaccttcgt aagtgtaccg ataattcacg gtcaggctcg ccccatcctc
      841 gaattgcatg gtgcgatgga cctgatagcc actgccatcc accatagccg cctgaaatgg
      901 gctcattccg tctgggtagg gcagatactg atgaaaacca taaccaatat gcggtaccag
      961 aatccacgga ctaaattgca agtcgccctt ggtagatttc aaattcaatt cttcatagcc
     1021 gtcgttcggg ttgccggtgc cttggcccac catgtcgaaa tcaactccgt tgatgctgcc
     1081 gaaaatatgc agttcatgtg tagcaggcag cgacgccatg ttatcctcct cacctttcga
     1141 aaccatctag tatttctcct ctttactacg gctctactac tcgatagact tataaccgag
     1201 acggtagcct gattgagtta aacgtaaagg caacgaagaa ggtctgtaca gacgatgttg
     1261 ttaaacagtt tatcaatggc ggtatccgac gtcggccgta ggcgtcggag attccggagc
     1321 gtaataggga gtagacggaa cgcggaagcg ataaaaattc tgcctcgtga tacgcctatt
     1381 tttataggtt aatgtcatga taataatggt ttattagacg tcaggtggca ccatgggaaa
     1441 tgtgcgcgga acccctattt gtttattttt ctaaatacat taaaatatgt atccgctcat
     1501 gagacaataa ccctgataaa tgcttcaata atattgaaaa aggaagagta tgagtattca
     1561 acatttcagt gtcgccctta ttcacttttt tgcggcattt tgccttcccg tttttgctca
     1621 cccagaaacg ctggtgaaag taaaagatgc tgaagatcag ttgggtgcac gagtgggtta
     1681 catcgaactg gatctcaaca gcggtaagat ccttgagagt tttcgccccg aagaacgttt
     1741 tccaatgatg agcactttta aagttctgct atgtggcgcg gtattatacc gtattgacgc
     1801 cgggcaagag caactcggtc gccgcataca ctattctcag aatgacttgg ttgagtactc
     1861 accagtaaca gaaaagcatc ttacggatgg catgacagta agagaattat gcagtgctgc
     1921 cataaccatg agtgataaca ctgcggccaa cttacttctg acaacgatcg gaggaccgaa
     1981 ggagctaacc gcttttttgc acaacatggg ggatcatgta actcgccttg atcgttggga
     2041 accggagctg aatgaagcca taccaaacga cgagcgtgac accacgatgc ctgtagcaat
     2101 ggcaacaacg ttgcgcaaac tattaactgg cgaactactt actctagctt cccggcaaca
     2161 attaatagac tggatggagg cggataaagt tgcaggacca cttctgcgct cggcccttcc
     2221 ggctggctgg tttattgctg ataaatctgg agccggtgag cgtggatcac gcggtatcat
     2281 tgcagcactg gggccagatg gtaagccctc ccgtatcgta gttatctaca cgacggggag
     2341 tcaggcaact atggatgaac gaaatagaca gatcgctgag ataggtgcct cactgattaa
     2401 gcattggtaa ctgtcagacc aagtttacga gctcgcttgg actcctgttg atagatccag
     2461 taatgacatc agaactccat ctggatttgt tcagaacgct cggttgccgc cgggcgtttt
     2521 ttattggtga gaatccaagc actagggaca gtaagacggg taagcctgtt gatgataccg
     2581 ctgccttact gggtgcatta gccagtctga atgacctgtc acgggataat ccgaagtggt
     2641 cagactggaa aatcagaggg caggaactgc tgaacagcaa aaagtcagat agcaccacat
     2701 agcagaccag ccataaaacg ccctgagaag cccgtgacgg gcttttcttg tattatgggt
     2761 agtttccttg catgaatcca taaaaggcgc ctgtagtgcc atttacccac attcactgcc
     2821 agagccgtga gcgcagcgaa ctgaatgtca cgaaaaaggc agcgactcag gtgcctgatg
     2881 gtcggagaca aaaggaatat taagcgattt gcacgagctt gcgagggtgc tacttaagcc
     2941 tttagggttt taaggtctgt tttgtagagg agcaaacagc gtttgcgaca tccttttgta
     3001 atactgcgga actgactaaa gtagtgagtt atacacaggg ctgggatcta ttctttttat
     3061 ctttttttat tctttcttta ttctataaat tataaccact tgaatataaa caaaaaaaac
     3121 acacaaaggt ctagcggaat ttacagaggg tctagcagaa tttacaagtt ttccagcaaa
     3181 ggtctagcag aatttacaga tacccacaac tcaaaggaaa aggacatgta attatcattg
     3241 actagcccat ctcaattggt atagtgatta aaatcaccta gaccaattga gatgtatgtc
     3301 tgaattagtt gttttcaaag caaatgaact agcgattagt cgctatgact taacggagca
     3361 tgaaaccaag ctaattttat gctgtgtggc actactcaac cccactattg aaaaccctac
     3421 aaggaaagaa cggacggtat cgttcactta taaccaatac gctcagatga tgaacatcag
     3481 tagggaaaat gcttatggtg tattagctaa agcaaccaga gagctgatga cgagaactgt
     3541 ggaaatcatg aatcctttgg ttaaaggctt tgagattttc cagtggacaa actatgccaa
     3601 gttctcaagc gaaaaattag aattagtttt tagtgaagag atattgcctt atcttttcca
     3661 gttaaaaaaa ttcataaaat ataatctgga acatgttaag tcttttgaaa acaaatactc
     3721 tatgaggatt tatgagtggt tattaaaaga actaacacaa aagaaaactc acaaggcaaa
     3781 tatagagatt agccttgatg aatttaagtt catgttaatg cttgaaaata actaccatga
     3841 gtttaaaagg cttaaccaat gggttttgaa accaataagt aaagatttaa acacttacag
     3901 caatatgaaa ttggtggttg ataagcgagg ccgcccgact gatacgttga ttttccaagt
     3961 tgaactagat agacaaatgg atctcgtaac cgaacttgag aacaaccaga taaaaatgaa
     4021 tggtgacaaa ataccaacaa ccattacatc agattcctac ctacataacg gactaagaaa
     4081 aacactacac gatgctttaa ctgcaaaaat tcagctcacc agttttgagg caaaattttt
     4141 gagtgacatg caaagtaagt atgatctcaa tggttcgttc tcatggctca cgcaaaaaca
     4201 acgaaccaca ctagagaaca tactggctaa atacggaagg atctgaggtt cttatggctc
     4261 ttgtatctat cagtgaagca tcaagactaa caaacaaaag tagaacaact gttcaccgtt
     4321 acatatcaaa gggaaaactg tccatatgca cagatgaaaa cggtgtaaaa aagatagata
     4381 catcagagct tttacgagtt tttggtgcat tcaaagctgt tcaccatgaa cagatcgaca
     4441 atgtaacctc gagattaccg cctttgagtg agctgatacc gctcgccgca gccgaacgac
     4501 cgagcgcagc gagtcagtga gcgaggaagc ctgcataacg cgaagtaatc ttttcggttt
     4561 taaagaaaaa gggcagggtg gtgacacctt gccgaagcgg catgcattta cgttgacacc
     4621 atcgaatggt gcaaaacctt tcgcggtatg gcatgatagc gcccggaaga gagtcaattc
     4681 agggtggtga atgtgaaacc agtaacgtta tacgatgtcg cagagtatgc cggtgtctct
     4741 tatcagaccg tttcccgcgt ggtgaaccag gccagccacg tttctgcgaa aacgcgggaa
     4801 aaagtggaag cggcgatggc ggagctgaat tacattccca accgcgtggc acaacaactg
     4861 gcgggcaaac agtcgttgct gattggcgtt gccacctcca gtctggccct gcacgcgccg
     4921 tcgcaaattg tcgcggcgat taaatctcgc gccgatcaac tgggtgccag cgtggtggtg
     4981 tcgatggtag aacgaagcgg cgtcgaagcc tgtaaagcgg cggtgcacaa tcttctcgcg
     5041 caacgcgtca gtgggctgat cattaactat ccgctggatg accaggatgc cattgctgtg
     5101 gaagctgcct gcactaatgt tccggcgtta tttcttgatg tctctgacca gacacccatc
     5161 aacagtatta ttttctccca tgaagacggt acgcgactgg gcgtggagca tctggtcgca
     5221 ttgggtcacc agcaaatcgc gctgttagcg ggcccattaa gttctgtctc ggcgcgtctg
     5281 cgtctggctg gctggcataa atatctcact cgcaatcaaa ttcagccgat agcggaacgg
     5341 gaaggcgact ggagtgccat gtccggtttt caacaaacca tgcaaatgct gaatgagggc
     5401 atcgttccca ctgcgatgct ggttgccaac gatcagatgg cgctgggcgc aatgcgcgcc
     5461 attaccgagt ccgggctgcg cgttggtgcg gatatctcgg tagtgggata cgacgatacc
     5521 gaagacagct catgttatat cccgccgtta accaccatca aacaggattt tcgcctgctg
     5581 gggcaaacca gcgtggaccg cttgctgcaa ctctctcagg gccaggcggt gaagggcaat
     5641 cagctgttgc ccgtctcact ggtgaaaaga aaaaccaccc tggcgcccaa tacgcaaacc
     5701 gcctctcccc gcgcgttggc cgattcatta atgcagctgg cacgacaggt ttcccgactg
     5761 gaaagcgggc agtgaggcat caaataaaac gaaaggctca gtcgaaagac tgggcctttc
     5821 gttttatctg ttgtttgtcg gtgaacgctc tcctgagtag gacaaatccg ccgccctaga
     5881 cctagggcgt tcggctgcgg cgagcggtat cagctcactc aaaggcggta atacgtaaat
     5941 cactgcataa ttcgtgtcgc tcaaggcgca ctcccgttct ggataatgtt ttttgcgccg
     6001 acatcataac ggttctggct aatattctga aatgagctgt tgacaattaa tcatcggctc
     6061 gtataatgtg tggaattgtg agcggataac aatttcacac agggcccaag ttcacttaaa
     6121 aaggagatca acaatgaaag caattttcgt actgaaacat cttaatcatg ctatggagta
     6181 tttctgatga aagctactaa actggtactg ggcgcggtaa tcctgggttc tactctgctg
     6241 gcaggttgct ccagcaacgc taaaatcgat cagggaatta acccgtatgt tggctttgaa
     6301 atgggttacg actggttagg tcgtatgccg tacaaaggca gcgttgaaaa cggtgcatac
     6361 aaagctcagg gcgttcaact gaccgctaaa ctgggttacc caatcactga cgacctggac
     6421 atctacactc gtctgggtgg catggtatgg cgtgcagaca ctaaatccaa cgtttatggt
     6481 aaaaaccacg acaccggcgt ttctccggtc ttcgctggcg gtgttgagta cgcgatcact
     6541 cctgaaatcg ctacccgtct ggaataccag tggaccaaca acatcggtga cgcacacacc
     6601 atcggcactc gtccggacaa cggtattggt accagtcaag agcctgcagc gcccgccgca
     6661 gaggcgactc ctgctgctga agctccagct agcgaagcgc ctgcagcaga agctgcccca
     6721 gcggatgctg ccgaagcccc agccgctggc atcagtcagg aacctgctgc accagctgcg
     6781 gaagctacac cagcagcgga ggcaccagcg agtgaagcac cggctgcgga agccgctcct
     6841 gcagatgccg ctgaggctcc agctgcgggt atcggaggaa caaatttacc aattgtggaa
     6901 cgtccagttt gcaaggattc tacccgtatc cgtattactg acaacatgtt ctgtgctggt
     6961 tataagcccg atg
//
