LOCUS       sequence-335296-        6973 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Encodes LacI-controlled expression of surface-displayed TCP 
            C-terminal alpha-helix fragment 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: pKB236.2
FEATURES             Location/Qualifiers
     source          1..6973
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     terminator      447..533
                     /gene="Escherichia coli rrnB"
                     /label=rrnB T1 terminator
                     /note="transcription terminator T1 from the E. coli rrnB 
                     gene"
     promoter        join(706..711,712..727,728..734)
                     /label=tac promoter
                     /note="strong E. coli promoter; hybrid between the trp and 
                     lac UV5 promoters"
     protein_bind    742..758
                     /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     747..769
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     terminator      complement(1952..1979)
                     /label=T7Te terminator
                     /note="phage T7 early transcription terminator"
     terminator      complement(1995..2066)
                     /label=rrnB T1 terminator
                     /note="transcription terminator T1 from the E. coli rrnB 
                     gene"
     RBS             2792..2803
                     /note="strong bacterial ribosome binding site (Elowitz and 
                     Leibler, 2000)"
     primer_bind     complement(3001..3019)
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     promoter        3084..3188
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(3189..3257,3258..4049)
                     /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(3407..3426)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     terminator      4079..4173
                     /label=lambda t0 terminator
                     /note="transcription terminator from phage lambda"
     rep_origin      4665..4887
                     /label=pSC101 ori
                     /note="low-copy replication origin that requires the Rep101
                     protein"
     CDS             4935..5885
                     /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     6147..6164
                     /label=L4440
                     /note="L4440 vector, forward primer"
     promoter        6254..6331
                     /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(6332..6973,1..441)
                     /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(6351..6370)
                     /label=LacI-R
                     /note="LacI, reverse primer"
ORIGIN
        1 gaacgggaag gcgactggag tgccatgtcc ggttttcaac aaaccatgca aatgctgaat
       61 gagggcatcg ttcccactgc gatgctggtt gccaacgatc agatggcgct gggcgcaatg
      121 cgcgccatta ccgagtccgg gctgcgcgtt ggtgcggata tctcggtagt gggatacgac
      181 gataccgaag acagctcatg ttatatcccg ccgttaacca ccatcaaaca ggattttcgc
      241 ctgctggggc aaaccagcgt ggaccgcttg ctgcaactct ctcagggcca ggcggtgaag
      301 ggcaatcagc tgttgcccgt ctcactggtg aaaagaaaaa ccaccctggc gcccaatacg
      361 caaaccgcct ctccccgcgc gttggccgat tcattaatgc agctggcacg acaggtttcc
      421 cgactggaaa gcgggcagtg aggcatcaaa taaaacgaaa ggctcagtcg aaagactggg
      481 cctttcgttt tatctgttgt ttgtcggtga acgctctcct gagtaggaca aatccgccgc
      541 cctagaccta gggcgttcgg ctgcggcgag cggtatcagc tcactcaaag gcggtaatac
      601 gtaaatcact gcataattcg tgtcgctcaa ggcgcactcc cgttctggat aatgtttttt
      661 gcgccgacat cataacggtt ctggctaata ttctgaaatg agctgttgac aattaatcat
      721 cggctcgtat aatgtgtgga attgtgagcg gataacaatt tcacacaggg cccaagttca
      781 cttaaaaagg agatcaacaa tgaaagcaat tttcgtactg aaacatctta atcatgctat
      841 ggagtatttc tgatgaaagc tactaaactg gtactgggcg cggtaatcct gggttctact
      901 ctgctggcag gttgctccag caacgctaaa atcgatcagg gaattaaccc gtatgttggc
      961 tttgaaatgg gttacgactg gttaggtcgt atgccgtaca aaggcagcgt tgaaaacggt
     1021 gcatacaaag ctcagggcgt tcaactgacc gctaaactgg gttacccaat cactgacgac
     1081 ctggacatct acactcgtct gggtggcatg gtatggcgtg cagacactaa atccaacgtt
     1141 tatggtaaaa accacgacac cggcgtttct ccggtcttcg ctggcggtgt tgagtacgcg
     1201 atcactcctg aaatcgctac ccgtctggaa taccagtgga ccaacaacat cggtgacgca
     1261 cacaccatcg gcactcgtcc ggacaacggt attggtacca gtcaagagcc tgcagcgccc
     1321 gccgcagagg cgactcctgc tgctgaagct ccagctagcg aagcgcctgc agcagaagct
     1381 gccccagcgg atgctgccga agccccagcc gctggcatca gtcaggaacc tgctgcacca
     1441 gctgcggaag ctacaccagc agcggaggca ccagcgagtg aagcaccggc tgcggaagcc
     1501 gctcctgcag atgccgctga ggctccagct gcgggtatcg gaggaacagt attccgtttg
     1561 aagaagtgga ttcagaaggt tattgatcag tttggtgagt aaaagtcgtt ttcagattac
     1621 aatgcaaatg tccaccgccg accagtttca acgccatatc acaacgaccc tcaagaccgc
     1681 catctgcagg atacaacgtt tccgtagaag cttcccaccc taaggtcttc ttttgcatta
     1741 cgggaccatt tgagggaaaa ttaacgccgc gcagtttcac attataaatt aaacaaccat
     1801 cttgcagaga agtatcttgc gttacagtta ataccccgcc atcctcatag gtcgtcacac
     1861 taatggagta cgcagatgat acagctgcct ggcggcagta gcgcggtggt cccacctgac
     1921 cccatgccga actcagaagt gaaactataa acgcagaaag gcccacccga aggtgagcca
     1981 gtgtgactct agtagagagc gttcaccgac aaacaacaga taaaacgaaa ggcccagtct
     2041 ttcgactgag cctttcgttt tatttgatgc ctggtcattt gtacagttca tccatgccca
     2101 ttacgtcggt gaaggctttt tgccactctt tgaaattgag ttcagttttc gaatgtttca
     2161 gttccgtttt gcgaaacaca tacatcggct gatttttcag atagttcgcg gccatgggtt
     2221 tcgcaaaggt gtatgtggtg cgagccgtgc tacggtagcg tttaccgtta cccgtggtgt
     2281 aactccattt aaaggtagaa ataattgttt tatcattggg atacgtcttt ttgctacgac
     2341 accagtccgc cgccgtcagg ctgttggtca tcactgggcc atccgccggg aagccggtgc
     2401 ctttaacttg tgcttcacct ttaatatgtg aaccttcgta agtgtaccga taattcacgg
     2461 tcaggctcgc cccatcctcg aattgcatgg tgcgatggac ctgatagcca ctgccatcca
     2521 ccatagccgc ctgaaatggg ctcattccgt ctgggtaggg cagatactga tgaaaaccat
     2581 aaccaatatg cggtaccaga atccacggac taaattgcaa gtcgcccttg gtagatttca
     2641 aattcaattc ttcatagccg tcgttcgggt tgccggtgcc ttggcccacc atgtcgaaat
     2701 caactccgtt gatgctgccg aaaatatgca gttcatgtgt agcaggcagc gacgccatgt
     2761 tatcctcctc acctttcgaa accatctagt atttctcctc tttactacgg ctctactact
     2821 cgatagactt ataaccgaga cggtagcctg attgagttaa acgtaaaggc aacgaagaag
     2881 gtctgtacag acgatgttgt taaacagttt atcaatggcg gtatccgacg tcggccgtag
     2941 gcgtcggaga ttccggagcg taatagggag tagacggaac gcggaagcga taaaaattct
     3001 gcctcgtgat acgcctattt ttataggtta atgtcatgat aataatggtt tattagacgt
     3061 caggtggcac catgggaaat gtgcgcggaa cccctatttg tttatttttc taaatacatt
     3121 aaaatatgta tccgctcatg agacaataac cctgataaat gcttcaataa tattgaaaaa
     3181 ggaagagtat gagtattcaa catttcagtg tcgcccttat tcactttttt gcggcatttt
     3241 gccttcccgt ttttgctcac ccagaaacgc tggtgaaagt aaaagatgct gaagatcagt
     3301 tgggtgcacg agtgggttac atcgaactgg atctcaacag cggtaagatc cttgagagtt
     3361 ttcgccccga agaacgtttt ccaatgatga gcacttttaa agttctgcta tgtggcgcgg
     3421 tattataccg tattgacgcc gggcaagagc aactcggtcg ccgcatacac tattctcaga
     3481 atgacttggt tgagtactca ccagtaacag aaaagcatct tacggatggc atgacagtaa
     3541 gagaattatg cagtgctgcc ataaccatga gtgataacac tgcggccaac ttacttctga
     3601 caacgatcgg aggaccgaag gagctaaccg cttttttgca caacatgggg gatcatgtaa
     3661 ctcgccttga tcgttgggaa ccggagctga atgaagccat accaaacgac gagcgtgaca
     3721 ccacgatgcc tgtagcaatg gcaacaacgt tgcgcaaact attaactggc gaactactta
     3781 ctctagcttc ccggcaacaa ttaatagact ggatggaggc ggataaagtt gcaggaccac
     3841 ttctgcgctc ggcccttccg gctggctggt ttattgctga taaatctgga gccggtgagc
     3901 gtggatcacg cggtatcatt gcagcactgg ggccagatgg taagccctcc cgtatcgtag
     3961 ttatctacac gacggggagt caggcaacta tggatgaacg aaatagacag atcgctgaga
     4021 taggtgcctc actgattaag cattggtaac tgtcagacca agtttacgag ctcgcttgga
     4081 ctcctgttga tagatccagt aatgacatca gaactccatc tggatttgtt cagaacgctc
     4141 ggttgccgcc gggcgttttt tattggtgag aatccaagca ctagggacag taagacgggt
     4201 aagcctgttg atgataccgc tgccttactg ggtgcattag ccagtctgaa tgacctgtca
     4261 cgggataatc cgaagtggtc agactggaaa atcagagggc aggaactgct gaacagcaaa
     4321 aagtcagata gcaccacata gcagaccagc cataaaacgc cctgagaagc ccgtgacggg
     4381 cttttcttgt attatgggta gtttccttgc atgaatccat aaaaggcgcc tgtagtgcca
     4441 tttacccaca ttcactgcca gagccgtgag cgcagcgaac tgaatgtcac gaaaaaggca
     4501 gcgactcagg tgcctgatgg tcggagacaa aaggaatatt aagcgatttg cacgagcttg
     4561 cgagggtgct acttaagcct ttagggtttt aaggtctgtt ttgtagagga gcaaacagcg
     4621 tttgcgacat ccttttgtaa tactgcggaa ctgactaaag tagtgagtta tacacagggc
     4681 tgggatctat tctttttatc tttttttatt ctttctttat tctataaatt ataaccactt
     4741 gaatataaac aaaaaaaaca cacaaaggtc tagcggaatt tacagagggt ctagcagaat
     4801 ttacaagttt tccagcaaag gtctagcaga atttacagat acccacaact caaaggaaaa
     4861 ggacatgtaa ttatcattga ctagcccatc tcaattggta tagtgattaa aatcacctag
     4921 accaattgag atgtatgtct gaattagttg ttttcaaagc aaatgaacta gcgattagtc
     4981 gctatgactt aacggagcat gaaaccaagc taattttatg ctgtgtggca ctactcaacc
     5041 ccactattga aaaccctaca aggaaagaac ggacggtatc gttcacttat aaccaatacg
     5101 ctcagatgat gaacatcagt agggaaaatg cttatggtgt attagctaaa gcaaccagag
     5161 agctgatgac gagaactgtg gaaatcatga atcctttggt taaaggcttt gagattttcc
     5221 agtggacaaa ctatgccaag ttctcaagcg aaaaattaga attagttttt agtgaagaga
     5281 tattgcctta tcttttccag ttaaaaaaat tcataaaata taatctggaa catgttaagt
     5341 cttttgaaaa caaatactct atgaggattt atgagtggtt attaaaagaa ctaacacaaa
     5401 agaaaactca caaggcaaat atagagatta gccttgatga atttaagttc atgttaatgc
     5461 ttgaaaataa ctaccatgag tttaaaaggc ttaaccaatg ggttttgaaa ccaataagta
     5521 aagatttaaa cacttacagc aatatgaaat tggtggttga taagcgaggc cgcccgactg
     5581 atacgttgat tttccaagtt gaactagata gacaaatgga tctcgtaacc gaacttgaga
     5641 acaaccagat aaaaatgaat ggtgacaaaa taccaacaac cattacatca gattcctacc
     5701 tacataacgg actaagaaaa acactacacg atgctttaac tgcaaaaatt cagctcacca
     5761 gttttgaggc aaaatttttg agtgacatgc aaagtaagta tgatctcaat ggttcgttct
     5821 catggctcac gcaaaaacaa cgaaccacac tagagaacat actggctaaa tacggaagga
     5881 tctgaggttc ttatggctct tgtatctatc agtgaagcat caagactaac aaacaaaagt
     5941 agaacaactg ttcaccgtta catatcaaag ggaaaactgt ccatatgcac agatgaaaac
     6001 ggtgtaaaaa agatagatac atcagagctt ttacgagttt ttggtgcatt caaagctgtt
     6061 caccatgaac agatcgacaa tgtaacctcg agattaccgc ctttgagtga gctgataccg
     6121 ctcgccgcag ccgaacgacc gagcgcagcg agtcagtgag cgaggaagcc tgcataacgc
     6181 gaagtaatct tttcggtttt aaagaaaaag ggcagggtgg tgacaccttg ccgaagcggc
     6241 atgcatttac gttgacacca tcgaatggtg caaaaccttt cgcggtatgg catgatagcg
     6301 cccggaagag agtcaattca gggtggtgaa tgtgaaacca gtaacgttat acgatgtcgc
     6361 agagtatgcc ggtgtctctt atcagaccgt ttcccgcgtg gtgaaccagg ccagccacgt
     6421 ttctgcgaaa acgcgggaaa aagtggaagc ggcgatggcg gagctgaatt acattcccaa
     6481 ccgcgtggca caacaactgg cgggcaaaca gtcgttgctg attggcgttg ccacctccag
     6541 tctggccctg cacgcgccgt cgcaaattgt cgcggcgatt aaatctcgcg ccgatcaact
     6601 gggtgccagc gtggtggtgt cgatggtaga acgaagcggc gtcgaagcct gtaaagcggc
     6661 ggtgcacaat cttctcgcgc aacgcgtcag tgggctgatc attaactatc cgctggatga
     6721 ccaggatgcc attgctgtgg aagctgcctg cactaatgtt ccggcgttat ttcttgatgt
     6781 ctctgaccag acacccatca acagtattat tttctcccat gaagacggta cgcgactggg
     6841 cgtggagcat ctggtcgcat tgggtcacca gcaaatcgcg ctgttagcgg gcccattaag
     6901 ttctgtctcg gcgcgtctgc gtctggctgg ctggcataaa tatctcactc gcaatcaaat
     6961 tcagccgata gcg
//
