LOCUS       sequence-335964-        6973 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Encodes LacI-controlled expression of surface-displayed hBD3 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: pKB234
FEATURES             Location/Qualifiers
     source          1..6973
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     932..951
                     /label=LacI-R
                     /note="LacI, reverse primer"
     promoter        complement(971..1048)
                     /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."
     primer_bind     complement(1138..1155)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     CDS             complement(1417..2367)
                     /codon_start=1
                     /gene="rep101"
                     /product="RepA protein needed for replication with the 
                     pSC101 origin"
                     /label=Rep101
                     /translation="MSELVVFKANELAISRYDLTEHETKLILCCVALLNPTIENPTRKE
                     RTVSFTYNQYAQMMNISRENAYGVLAKATRELMTRTVEIMNPLVKGFEIFQWTNYAKFS
                     SEKLELVFSEEILPYLFQLKKFIKYNLEHVKSFENKYSMRIYEWLLKELTQKKTHKANI
                     EISLDEFKFMLMLENNYHEFKRLNQWVLKPISKDLNTYSNMKLVVDKRGRPTDTLIFQV
                     ELDRQMDLVTELENNQIKMNGDKIPTTITSDSYLHNGLRKTLHDALTAKIQLTSFEAKF
                     LSDMQSKYDLNGSFSWLTQKQRTTLENILAKYGRI"
     rep_origin      2415..2637
                     /label=pSC101 ori
                     /note="low-copy replication origin that requires the Rep101
                     protein"
     terminator      complement(3129..3223)
                     /label=lambda t0 terminator
                     /note="transcription terminator from phage lambda"
     CDS             complement(join(3253..4044,4045..4113))
                     /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     3876..3895
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(4114..4218)
                     /gene="bla"
                     /label=AmpR promoter
     primer_bind     4283..4301
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     RBS             4499..4510
                     /note="strong bacterial ribosome binding site (Elowitz and 
                     Leibler, 2000)"
     terminator      5236..5307
                     /label=rrnB T1 terminator
                     /note="transcription terminator T1 from the E. coli rrnB 
                     gene"
     terminator      5323..5350
                     /label=T7Te terminator
                     /note="phage T7 early transcription terminator"
     primer_bind     complement(6533..6555)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    6544..6560
                     /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)."
     promoter        complement(join(6568..6574,6575..6590,6591..6596))
                     /label=tac promoter
                     /note="strong E. coli promoter; hybrid between the trp and 
                     lac UV5 promoters"
     terminator      complement(6769..6855)
                     /gene="Escherichia coli rrnB"
                     /label=rrnB T1 terminator
                     /note="transcription terminator T1 from the E. coli rrnB 
                     gene"
     CDS             complement(join(6861..6973,1..970))
                     /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"
ORIGIN
        1 ccagtgagac gggcaacagc tgattgccct tcaccgcctg gccctgagag agttgcagca
       61 agcggtccac gctggtttgc cccagcaggc gaaaatcctg tttgatggtg gttaacggcg
      121 ggatataaca tgagctgtct tcggtatcgt cgtatcccac taccgagata tccgcaccaa
      181 cgcgcagccc ggactcggta atggcgcgca ttgcgcccag cgccatctga tcgttggcaa
      241 ccagcatcgc agtgggaacg atgccctcat tcagcatttg catggtttgt tgaaaaccgg
      301 acatggcact ccagtcgcct tcccgttccg ctatcggctg aatttgattg cgagtgagat
      361 atttatgcca gccagccaga cgcagacgcg ccgagacaga acttaatggg cccgctaaca
      421 gcgcgatttg ctggtgaccc aatgcgacca gatgctccac gcccagtcgc gtaccgtctt
      481 catgggagaa aataatactg ttgatgggtg tctggtcaga gacatcaaga aataacgccg
      541 gaacattagt gcaggcagct tccacagcaa tggcatcctg gtcatccagc ggatagttaa
      601 tgatcagccc actgacgcgt tgcgcgagaa gattgtgcac cgccgcttta caggcttcga
      661 cgccgcttcg ttctaccatc gacaccacca cgctggcacc cagttgatcg gcgcgagatt
      721 taatcgccgc gacaatttgc gacggcgcgt gcagggccag actggaggtg gcaacgccaa
      781 tcagcaacga ctgtttgccc gccagttgtt gtgccacgcg gttgggaatg taattcagct
      841 ccgccatcgc cgcttccact ttttcccgcg ttttcgcaga aacgtggctg gcctggttca
      901 ccacgcggga aacggtctga taagagacac cggcatactc tgcgacatcg tataacgtta
      961 ctggtttcac attcaccacc ctgaattgac tctcttccgg gcgctatcat gccataccgc
     1021 gaaaggtttt gcaccattcg atggtgtcaa cgtaaatgca tgccgcttcg gcaaggtgtc
     1081 accaccctgc cctttttctt taaaaccgaa aagattactt cgcgttatgc aggcttcctc
     1141 gctcactgac tcgctgcgct cggtcgttcg gctgcggcga gcggtatcag ctcactcaaa
     1201 ggcggtaatc tcgaggttac attgtcgatc tgttcatggt gaacagcttt gaatgcacca
     1261 aaaactcgta aaagctctga tgtatctatc ttttttacac cgttttcatc tgtgcatatg
     1321 gacagttttc cctttgatat gtaacggtga acagttgttc tacttttgtt tgttagtctt
     1381 gatgcttcac tgatagatac aagagccata agaacctcag atccttccgt atttagccag
     1441 tatgttctct agtgtggttc gttgtttttg cgtgagccat gagaacgaac cattgagatc
     1501 atacttactt tgcatgtcac tcaaaaattt tgcctcaaaa ctggtgagct gaatttttgc
     1561 agttaaagca tcgtgtagtg tttttcttag tccgttatgt aggtaggaat ctgatgtaat
     1621 ggttgttggt attttgtcac cattcatttt tatctggttg ttctcaagtt cggttacgag
     1681 atccatttgt ctatctagtt caacttggaa aatcaacgta tcagtcgggc ggcctcgctt
     1741 atcaaccacc aatttcatat tgctgtaagt gtttaaatct ttacttattg gtttcaaaac
     1801 ccattggtta agccttttaa actcatggta gttattttca agcattaaca tgaacttaaa
     1861 ttcatcaagg ctaatctcta tatttgcctt gtgagttttc ttttgtgtta gttcttttaa
     1921 taaccactca taaatcctca tagagtattt gttttcaaaa gacttaacat gttccagatt
     1981 atattttatg aattttttta actggaaaag ataaggcaat atctcttcac taaaaactaa
     2041 ttctaatttt tcgcttgaga acttggcata gtttgtccac tggaaaatct caaagccttt
     2101 aaccaaagga ttcatgattt ccacagttct cgtcatcagc tctctggttg ctttagctaa
     2161 tacaccataa gcattttccc tactgatgtt catcatctga gcgtattggt tataagtgaa
     2221 cgataccgtc cgttctttcc ttgtagggtt ttcaatagtg gggttgagta gtgccacaca
     2281 gcataaaatt agcttggttt catgctccgt taagtcatag cgactaatcg ctagttcatt
     2341 tgctttgaaa acaactaatt cagacataca tctcaattgg tctaggtgat tttaatcact
     2401 ataccaattg agatgggcta gtcaatgata attacatgtc cttttccttt gagttgtggg
     2461 tatctgtaaa ttctgctaga cctttgctgg aaaacttgta aattctgcta gaccctctgt
     2521 aaattccgct agacctttgt gtgttttttt tgtttatatt caagtggtta taatttatag
     2581 aataaagaaa gaataaaaaa agataaaaag aatagatccc agccctgtgt ataactcact
     2641 actttagtca gttccgcagt attacaaaag gatgtcgcaa acgctgtttg ctcctctaca
     2701 aaacagacct taaaacccta aaggcttaag tagcaccctc gcaagctcgt gcaaatcgct
     2761 taatattcct tttgtctccg accatcaggc acctgagtcg ctgccttttt cgtgacattc
     2821 agttcgctgc gctcacggct ctggcagtga atgtgggtaa atggcactac aggcgccttt
     2881 tatggattca tgcaaggaaa ctacccataa tacaagaaaa gcccgtcacg ggcttctcag
     2941 ggcgttttat ggctggtctg ctatgtggtg ctatctgact ttttgctgtt cagcagttcc
     3001 tgccctctga ttttccagtc tgaccacttc ggattatccc gtgacaggtc attcagactg
     3061 gctaatgcac ccagtaaggc agcggtatca tcaacaggct tacccgtctt actgtcccta
     3121 gtgcttggat tctcaccaat aaaaaacgcc cggcggcaac cgagcgttct gaacaaatcc
     3181 agatggagtt ctgatgtcat tactggatct atcaacagga gtccaagcga gctcgtaaac
     3241 ttggtctgac agttaccaat gcttaatcag tgaggcacct atctcagcga tctgtctatt
     3301 tcgttcatcc atagttgcct gactccccgt cgtgtagata actacgatac gggagggctt
     3361 accatctggc cccagtgctg caatgatacc gcgtgatcca cgctcaccgg ctccagattt
     3421 atcagcaata aaccagccag ccggaagggc cgagcgcaga agtggtcctg caactttatc
     3481 cgcctccatc cagtctatta attgttgccg ggaagctaga gtaagtagtt cgccagttaa
     3541 tagtttgcgc aacgttgttg ccattgctac aggcatcgtg gtgtcacgct cgtcgtttgg
     3601 tatggcttca ttcagctccg gttcccaacg atcaaggcga gttacatgat cccccatgtt
     3661 gtgcaaaaaa gcggttagct ccttcggtcc tccgatcgtt gtcagaagta agttggccgc
     3721 agtgttatca ctcatggtta tggcagcact gcataattct cttactgtca tgccatccgt
     3781 aagatgcttt tctgttactg gtgagtactc aaccaagtca ttctgagaat agtgtatgcg
     3841 gcgaccgagt tgctcttgcc cggcgtcaat acggtataat accgcgccac atagcagaac
     3901 tttaaaagtg ctcatcattg gaaaacgttc ttcggggcga aaactctcaa ggatcttacc
     3961 gctgttgaga tccagttcga tgtaacccac tcgtgcaccc aactgatctt cagcatcttt
     4021 tactttcacc agcgtttctg ggtgagcaaa aacgggaagg caaaatgccg caaaaaagtg
     4081 aataagggcg acactgaaat gttgaatact catactcttc ctttttcaat attattgaag
     4141 catttatcag ggttattgtc tcatgagcgg atacatattt taatgtattt agaaaaataa
     4201 acaaataggg gttccgcgca catttcccat ggtgccacct gacgtctaat aaaccattat
     4261 tatcatgaca ttaacctata aaaataggcg tatcacgagg cagaattttt atcgcttccg
     4321 cgttccgtct actccctatt acgctccgga atctccgacg cctacggccg acgtcggata
     4381 ccgccattga taaactgttt aacaacatcg tctgtacaga ccttcttcgt tgcctttacg
     4441 tttaactcaa tcaggctacc gtctcggtta taagtctatc gagtagtaga gccgtagtaa
     4501 agaggagaaa tactagatgg tttcgaaagg tgaggaggat aacatggcgt cgctgcctgc
     4561 tacacatgaa ctgcatattt tcggcagcat caacggagtt gatttcgaca tggtgggcca
     4621 aggcaccggc aacccgaacg acggctatga agaattgaat ttgaaatcta ccaagggcga
     4681 cttgcaattt agtccgtgga ttctggtacc gcatattggt tatggttttc atcagtatct
     4741 gccctaccca gacggaatga gcccatttca ggcggctatg gtggatggca gtggctatca
     4801 ggtccatcgc accatgcaat tcgaggatgg ggcgagcctg accgtgaatt atcggtacac
     4861 ttacgaaggt tcacatatta aaggtgaagc acaagttaaa ggcaccggct tcccggcgga
     4921 tggcccagtg atgaccaaca gcctgacggc ggcggactgg tgtcgtagca aaaagacgta
     4981 tcccaatgat aaaacaatta tttctacctt taaatggagt tacaccacgg gtaacggtaa
     5041 acgctaccgt agcacggctc gcaccacata cacctttgcg aaacccatgg ccgcgaacta
     5101 tctgaaaaat cagccgatgt atgtgtttcg caaaacggaa ctgaaacatt cgaaaactga
     5161 actcaatttc aaagagtggc aaaaagcctt caccgacgta atgggcatgg atgaactgta
     5221 caaatgacca ggcatcaaat aaaacgaaag gctcagtcga aagactgggc ctttcgtttt
     5281 atctgttgtt tgtcggtgaa cgctctctac tagagtcaca ctggctcacc ttcgggtggg
     5341 cctttctgcg tttatagttt cacttctgag ttcggcatgg ggtcaggtgg gaccaccgcg
     5401 ctactgccgc caggcagctg tatcatctgc gtactccatt agtgtgacga cctatgagga
     5461 tggcggggta ttaactgtaa cgcaagatac ttctctgcaa gatggttgtt taatttataa
     5521 tgtgaaactg cgcggcgtta attttccctc aaatggtccc gtaatgcaaa agaagacctt
     5581 agggtgggaa gcttctacgg aaacgttgta tcctgttact tcttacgacg gcaacacttt
     5641 ctaccacgag tagaacactt accaatttgc tcctccttag gcaaacaaga caggacagca
     5701 caacgaccac ctctaacgcg acagtagtac ttctggaggg tgtttatgat tcctgttcct
     5761 ccgatacccg cagctggagc ctcagcggca tctgcaggag cggcttccgc agccggtgct
     5821 tcactcgctg gtgcctccgc tgctggtgta gcttccgcag ctggtgcagc aggttcctga
     5881 ctgatgccag cggctggggc ttcggcagca tccgctgggg cagcttctgc tgcaggcgct
     5941 tcgctagctg gagcttcagc agcaggagtc gcctctgcgg cgggcgctgc aggctcttga
     6001 ctggtaccaa taccgttgtc cggacgagtg ccgatggtgt gtgcgtcacc gatgttgttg
     6061 gtccactggt attccagacg ggtagcgatt tcaggagtga tcgcgtactc aacaccgcca
     6121 gcgaagaccg gagaaacgcc ggtgtcgtgg tttttaccat aaacgttgga tttagtgtct
     6181 gcacgccata ccatgccacc cagacgagtg tagatgtcca ggtcgtcagt gattgggtaa
     6241 cccagtttag cggtcagttg aacgccctga gctttgtatg caccgttttc aacgctgcct
     6301 ttgtacggca tacgacctaa ccagtcgtaa cccatttcaa agccaacata cgggttaatt
     6361 ccctgatcga ttttagcgtt gctggagcaa cctgccagca gagtagaacc caggattacc
     6421 gcgcccagta ccagtttagt agctttcatc agaaatactc catagcatga ttaagatgtt
     6481 tcagtacgaa aattgctttc attgttgatc tcctttttaa gtgaacttgg gccctgtgtg
     6541 aaattgttat ccgctcacaa ttccacacat tatacgagcc gatgattaat tgtcaacagc
     6601 tcatttcaga atattagcca gaaccgttat gatgtcggcg caaaaaacat tatccagaac
     6661 gggagtgcgc cttgagcgac acgaattatg cagtgattta cgtattaccg cctttgagtg
     6721 agctgatacc gctcgccgca gccgaacgcc ctaggtctag ggcggcggat ttgtcctact
     6781 caggagagcg ttcaccgaca aacaacagat aaaacgaaag gcccagtctt tcgactgagc
     6841 ctttcgtttt atttgatgcc tcactgcccg ctttccagtc gggaaacctg tcgtgccagc
     6901 tgcattaatg aatcggccaa cgcgcgggga gaggcggttt gcgtattggg cgccagggtg
     6961 gtttttcttt tca
//
