LOCUS       sequence-335958-        6973 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Encodes LacI-controlled expression of surface-displayed TCP fragment
            lacking C-terminal alpha-helix sequence 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: pKB236.1
FEATURES             Location/Qualifiers
     source          1..6973
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     terminator      488..574
                     /gene="Escherichia coli rrnB"
                     /label=rrnB T1 terminator
                     /note="transcription terminator T1 from the E. coli rrnB 
                     gene"
     promoter        join(747..752,753..768,769..775)
                     /label=tac promoter
                     /note="strong E. coli promoter; hybrid between the trp and 
                     lac UV5 promoters"
     protein_bind    783..799
                     /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     788..810
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     terminator      complement(1993..2020)
                     /label=T7Te terminator
                     /note="phage T7 early transcription terminator"
     terminator      complement(2036..2107)
                     /label=rrnB T1 terminator
                     /note="transcription terminator T1 from the E. coli rrnB 
                     gene"
     RBS             2833..2844
                     /note="strong bacterial ribosome binding site (Elowitz and 
                     Leibler, 2000)"
     primer_bind     complement(3042..3060)
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     promoter        3125..3229
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(3230..3298,3299..4090)
                     /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(3448..3467)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     terminator      4120..4214
                     /label=lambda t0 terminator
                     /note="transcription terminator from phage lambda"
     rep_origin      4706..4928
                     /label=pSC101 ori
                     /note="low-copy replication origin that requires the Rep101
                     protein"
     CDS             4976..5926
                     /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     6188..6205
                     /label=L4440
                     /note="L4440 vector, forward primer"
     promoter        6295..6372
                     /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(6373..6973,1..482)
                     /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(6392..6411)
                     /label=LacI-R
                     /note="LacI, reverse primer"
ORIGIN
        1 ggcataaata tctcactcgc aatcaaattc agccgatagc ggaacgggaa ggcgactgga
       61 gtgccatgtc cggttttcaa caaaccatgc aaatgctgaa tgagggcatc gttcccactg
      121 cgatgctggt tgccaacgat cagatggcgc tgggcgcaat gcgcgccatt accgagtccg
      181 ggctgcgcgt tggtgcggat atctcggtag tgggatacga cgataccgaa gacagctcat
      241 gttatatccc gccgttaacc accatcaaac aggattttcg cctgctgggg caaaccagcg
      301 tggaccgctt gctgcaactc tctcagggcc aggcggtgaa gggcaatcag ctgttgcccg
      361 tctcactggt gaaaagaaaa accaccctgg cgcccaatac gcaaaccgcc tctccccgcg
      421 cgttggccga ttcattaatg cagctggcac gacaggtttc ccgactggaa agcgggcagt
      481 gaggcatcaa ataaaacgaa aggctcagtc gaaagactgg gcctttcgtt ttatctgttg
      541 tttgtcggtg aacgctctcc tgagtaggac aaatccgccg ccctagacct agggcgttcg
      601 gctgcggcga gcggtatcag ctcactcaaa ggcggtaata cgtaaatcac tgcataattc
      661 gtgtcgctca aggcgcactc ccgttctgga taatgttttt tgcgccgaca tcataacggt
      721 tctggctaat attctgaaat gagctgttga caattaatca tcggctcgta taatgtgtgg
      781 aattgtgagc ggataacaat ttcacacagg gcccaagttc acttaaaaag gagatcaaca
      841 atgaaagcaa ttttcgtact gaaacatctt aatcatgcta tggagtattt ctgatgaaag
      901 ctactaaact ggtactgggc gcggtaatcc tgggttctac tctgctggca ggttgctcca
      961 gcaacgctaa aatcgatcag ggaattaacc cgtatgttgg ctttgaaatg ggttacgact
     1021 ggttaggtcg tatgccgtac aaaggcagcg ttgaaaacgg tgcatacaaa gctcagggcg
     1081 ttcaactgac cgctaaactg ggttacccaa tcactgacga cctggacatc tacactcgtc
     1141 tgggtggcat ggtatggcgt gcagacacta aatccaacgt ttatggtaaa aaccacgaca
     1201 ccggcgtttc tccggtcttc gctggcggtg ttgagtacgc gatcactcct gaaatcgcta
     1261 cccgtctgga ataccagtgg accaacaaca tcggtgacgc acacaccatc ggcactcgtc
     1321 cggacaacgg tattggtacc agtcaagagc ctgcagcgcc cgccgcagag gcgactcctg
     1381 ctgctgaagc tccagctagc gaagcgcctg cagcagaagc tgccccagcg gatgctgccg
     1441 aagccccagc cgctggcatc agtcaggaac ctgctgcacc agctgcggaa gctacaccag
     1501 cagcggaggc accagcgagt gaagcaccgg ctgcggaagc cgctcctgca gatgccgctg
     1561 aggctccagc tgcgggtatc ggaggaacaa atttaccaat tgtggaacgt ccagtttgca
     1621 aggattctac ccgtatccgt attactgaca acatgttctg tgctggttat aagcccgatg
     1681 agggtaaacg aggtgatgct tgtgagggtg atagtggcgg tccatttgtt atgaagagtc
     1741 cgttcaacaa cagatggtat cagatgggta ttgtatcttg gggtgagggt tgtgatcgtg
     1801 atggtaagta tggattctat actcatgtat tccgttagca tcttgcagag aagtatcttg
     1861 cgttacagtt aataccccgc catcctcata ggtcgtcaca ctaatggagt acgcagatga
     1921 tacagctgcc tggcggcagt agcgcggtgg tcccacctga ccccatgccg aactcagaag
     1981 tgaaactata aacgcagaaa ggcccacccg aaggtgagcc agtgtgactc tagtagagag
     2041 cgttcaccga caaacaacag ataaaacgaa aggcccagtc tttcgactga gcctttcgtt
     2101 ttatttgatg cctggtcatt tgtacagttc atccatgccc attacgtcgg tgaaggcttt
     2161 ttgccactct ttgaaattga gttcagtttt cgaatgtttc agttccgttt tgcgaaacac
     2221 atacatcggc tgatttttca gatagttcgc ggccatgggt ttcgcaaagg tgtatgtggt
     2281 gcgagccgtg ctacggtagc gtttaccgtt acccgtggtg taactccatt taaaggtaga
     2341 aataattgtt ttatcattgg gatacgtctt tttgctacga caccagtccg ccgccgtcag
     2401 gctgttggtc atcactgggc catccgccgg gaagccggtg cctttaactt gtgcttcacc
     2461 tttaatatgt gaaccttcgt aagtgtaccg ataattcacg gtcaggctcg ccccatcctc
     2521 gaattgcatg gtgcgatgga cctgatagcc actgccatcc accatagccg cctgaaatgg
     2581 gctcattccg tctgggtagg gcagatactg atgaaaacca taaccaatat gcggtaccag
     2641 aatccacgga ctaaattgca agtcgccctt ggtagatttc aaattcaatt cttcatagcc
     2701 gtcgttcggg ttgccggtgc cttggcccac catgtcgaaa tcaactccgt tgatgctgcc
     2761 gaaaatatgc agttcatgtg tagcaggcag cgacgccatg ttatcctcct cacctttcga
     2821 aaccatctag tatttctcct ctttactacg gctctactac tcgatagact tataaccgag
     2881 acggtagcct gattgagtta aacgtaaagg caacgaagaa ggtctgtaca gacgatgttg
     2941 ttaaacagtt tatcaatggc ggtatccgac gtcggccgta ggcgtcggag attccggagc
     3001 gtaataggga gtagacggaa cgcggaagcg ataaaaattc tgcctcgtga tacgcctatt
     3061 tttataggtt aatgtcatga taataatggt ttattagacg tcaggtggca ccatgggaaa
     3121 tgtgcgcgga acccctattt gtttattttt ctaaatacat taaaatatgt atccgctcat
     3181 gagacaataa ccctgataaa tgcttcaata atattgaaaa aggaagagta tgagtattca
     3241 acatttcagt gtcgccctta ttcacttttt tgcggcattt tgccttcccg tttttgctca
     3301 cccagaaacg ctggtgaaag taaaagatgc tgaagatcag ttgggtgcac gagtgggtta
     3361 catcgaactg gatctcaaca gcggtaagat ccttgagagt tttcgccccg aagaacgttt
     3421 tccaatgatg agcactttta aagttctgct atgtggcgcg gtattatacc gtattgacgc
     3481 cgggcaagag caactcggtc gccgcataca ctattctcag aatgacttgg ttgagtactc
     3541 accagtaaca gaaaagcatc ttacggatgg catgacagta agagaattat gcagtgctgc
     3601 cataaccatg agtgataaca ctgcggccaa cttacttctg acaacgatcg gaggaccgaa
     3661 ggagctaacc gcttttttgc acaacatggg ggatcatgta actcgccttg atcgttggga
     3721 accggagctg aatgaagcca taccaaacga cgagcgtgac accacgatgc ctgtagcaat
     3781 ggcaacaacg ttgcgcaaac tattaactgg cgaactactt actctagctt cccggcaaca
     3841 attaatagac tggatggagg cggataaagt tgcaggacca cttctgcgct cggcccttcc
     3901 ggctggctgg tttattgctg ataaatctgg agccggtgag cgtggatcac gcggtatcat
     3961 tgcagcactg gggccagatg gtaagccctc ccgtatcgta gttatctaca cgacggggag
     4021 tcaggcaact atggatgaac gaaatagaca gatcgctgag ataggtgcct cactgattaa
     4081 gcattggtaa ctgtcagacc aagtttacga gctcgcttgg actcctgttg atagatccag
     4141 taatgacatc agaactccat ctggatttgt tcagaacgct cggttgccgc cgggcgtttt
     4201 ttattggtga gaatccaagc actagggaca gtaagacggg taagcctgtt gatgataccg
     4261 ctgccttact gggtgcatta gccagtctga atgacctgtc acgggataat ccgaagtggt
     4321 cagactggaa aatcagaggg caggaactgc tgaacagcaa aaagtcagat agcaccacat
     4381 agcagaccag ccataaaacg ccctgagaag cccgtgacgg gcttttcttg tattatgggt
     4441 agtttccttg catgaatcca taaaaggcgc ctgtagtgcc atttacccac attcactgcc
     4501 agagccgtga gcgcagcgaa ctgaatgtca cgaaaaaggc agcgactcag gtgcctgatg
     4561 gtcggagaca aaaggaatat taagcgattt gcacgagctt gcgagggtgc tacttaagcc
     4621 tttagggttt taaggtctgt tttgtagagg agcaaacagc gtttgcgaca tccttttgta
     4681 atactgcgga actgactaaa gtagtgagtt atacacaggg ctgggatcta ttctttttat
     4741 ctttttttat tctttcttta ttctataaat tataaccact tgaatataaa caaaaaaaac
     4801 acacaaaggt ctagcggaat ttacagaggg tctagcagaa tttacaagtt ttccagcaaa
     4861 ggtctagcag aatttacaga tacccacaac tcaaaggaaa aggacatgta attatcattg
     4921 actagcccat ctcaattggt atagtgatta aaatcaccta gaccaattga gatgtatgtc
     4981 tgaattagtt gttttcaaag caaatgaact agcgattagt cgctatgact taacggagca
     5041 tgaaaccaag ctaattttat gctgtgtggc actactcaac cccactattg aaaaccctac
     5101 aaggaaagaa cggacggtat cgttcactta taaccaatac gctcagatga tgaacatcag
     5161 tagggaaaat gcttatggtg tattagctaa agcaaccaga gagctgatga cgagaactgt
     5221 ggaaatcatg aatcctttgg ttaaaggctt tgagattttc cagtggacaa actatgccaa
     5281 gttctcaagc gaaaaattag aattagtttt tagtgaagag atattgcctt atcttttcca
     5341 gttaaaaaaa ttcataaaat ataatctgga acatgttaag tcttttgaaa acaaatactc
     5401 tatgaggatt tatgagtggt tattaaaaga actaacacaa aagaaaactc acaaggcaaa
     5461 tatagagatt agccttgatg aatttaagtt catgttaatg cttgaaaata actaccatga
     5521 gtttaaaagg cttaaccaat gggttttgaa accaataagt aaagatttaa acacttacag
     5581 caatatgaaa ttggtggttg ataagcgagg ccgcccgact gatacgttga ttttccaagt
     5641 tgaactagat agacaaatgg atctcgtaac cgaacttgag aacaaccaga taaaaatgaa
     5701 tggtgacaaa ataccaacaa ccattacatc agattcctac ctacataacg gactaagaaa
     5761 aacactacac gatgctttaa ctgcaaaaat tcagctcacc agttttgagg caaaattttt
     5821 gagtgacatg caaagtaagt atgatctcaa tggttcgttc tcatggctca cgcaaaaaca
     5881 acgaaccaca ctagagaaca tactggctaa atacggaagg atctgaggtt cttatggctc
     5941 ttgtatctat cagtgaagca tcaagactaa caaacaaaag tagaacaact gttcaccgtt
     6001 acatatcaaa gggaaaactg tccatatgca cagatgaaaa cggtgtaaaa aagatagata
     6061 catcagagct tttacgagtt tttggtgcat tcaaagctgt tcaccatgaa cagatcgaca
     6121 atgtaacctc gagattaccg cctttgagtg agctgatacc gctcgccgca gccgaacgac
     6181 cgagcgcagc gagtcagtga gcgaggaagc ctgcataacg cgaagtaatc ttttcggttt
     6241 taaagaaaaa gggcagggtg gtgacacctt gccgaagcgg catgcattta cgttgacacc
     6301 atcgaatggt gcaaaacctt tcgcggtatg gcatgatagc gcccggaaga gagtcaattc
     6361 agggtggtga atgtgaaacc agtaacgtta tacgatgtcg cagagtatgc cggtgtctct
     6421 tatcagaccg tttcccgcgt ggtgaaccag gccagccacg tttctgcgaa aacgcgggaa
     6481 aaagtggaag cggcgatggc ggagctgaat tacattccca accgcgtggc acaacaactg
     6541 gcgggcaaac agtcgttgct gattggcgtt gccacctcca gtctggccct gcacgcgccg
     6601 tcgcaaattg tcgcggcgat taaatctcgc gccgatcaac tgggtgccag cgtggtggtg
     6661 tcgatggtag aacgaagcgg cgtcgaagcc tgtaaagcgg cggtgcacaa tcttctcgcg
     6721 caacgcgtca gtgggctgat cattaactat ccgctggatg accaggatgc cattgctgtg
     6781 gaagctgcct gcactaatgt tccggcgtta tttcttgatg tctctgacca gacacccatc
     6841 aacagtatta ttttctccca tgaagacggt acgcgactgg gcgtggagca tctggtcgca
     6901 ttgggtcacc agcaaatcgc gctgttagcg ggcccattaa gttctgtctc ggcgcgtctg
     6961 cgtctggctg gct
//
