LOCUS       sequence-334667-        6973 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Encodes LacI-controlled expression of surface-displayed hBD3 
            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: pKB234.1
FEATURES             Location/Qualifiers
     source          1..6973
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     911..930
                     /label=LacI-R
                     /note="LacI, reverse primer"
     promoter        complement(950..1027)
                     /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(1117..1134)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     CDS             complement(1396..2346)
                     /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      2394..2616
                     /label=pSC101 ori
                     /note="low-copy replication origin that requires the Rep101
                     protein"
     terminator      complement(3108..3202)
                     /label=lambda t0 terminator
                     /note="transcription terminator from phage lambda"
     CDS             complement(join(3232..4023,4024..4092))
                     /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     3855..3874
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(4093..4197)
                     /gene="bla"
                     /label=AmpR promoter
     primer_bind     4262..4280
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     RBS             4478..4489
                     /note="strong bacterial ribosome binding site (Elowitz and 
                     Leibler, 2000)"
     terminator      5215..5286
                     /label=rrnB T1 terminator
                     /note="transcription terminator T1 from the E. coli rrnB 
                     gene"
     terminator      5302..5329
                     /label=T7Te terminator
                     /note="phage T7 early transcription terminator"
     primer_bind     complement(6512..6534)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    6523..6539
                     /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(6547..6553,6554..6569,6570..6575))
                     /label=tac promoter
                     /note="strong E. coli promoter; hybrid between the trp and 
                     lac UV5 promoters"
     terminator      complement(6748..6834)
                     /gene="Escherichia coli rrnB"
                     /label=rrnB T1 terminator
                     /note="transcription terminator T1 from the E. coli rrnB 
                     gene"
     CDS             complement(join(6840..6973,1..949))
                     /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 gattgccctt caccgcctgg ccctgagaga gttgcagcaa gcggtccacg ctggtttgcc
       61 ccagcaggcg aaaatcctgt ttgatggtgg ttaacggcgg gatataacat gagctgtctt
      121 cggtatcgtc gtatcccact accgagatat ccgcaccaac gcgcagcccg gactcggtaa
      181 tggcgcgcat tgcgcccagc gccatctgat cgttggcaac cagcatcgca gtgggaacga
      241 tgccctcatt cagcatttgc atggtttgtt gaaaaccgga catggcactc cagtcgcctt
      301 cccgttccgc tatcggctga atttgattgc gagtgagata tttatgccag ccagccagac
      361 gcagacgcgc cgagacagaa cttaatgggc ccgctaacag cgcgatttgc tggtgaccca
      421 atgcgaccag atgctccacg cccagtcgcg taccgtcttc atgggagaaa ataatactgt
      481 tgatgggtgt ctggtcagag acatcaagaa ataacgccgg aacattagtg caggcagctt
      541 ccacagcaat ggcatcctgg tcatccagcg gatagttaat gatcagccca ctgacgcgtt
      601 gcgcgagaag attgtgcacc gccgctttac aggcttcgac gccgcttcgt tctaccatcg
      661 acaccaccac gctggcaccc agttgatcgg cgcgagattt aatcgccgcg acaatttgcg
      721 acggcgcgtg cagggccaga ctggaggtgg caacgccaat cagcaacgac tgtttgcccg
      781 ccagttgttg tgccacgcgg ttgggaatgt aattcagctc cgccatcgcc gcttccactt
      841 tttcccgcgt tttcgcagaa acgtggctgg cctggttcac cacgcgggaa acggtctgat
      901 aagagacacc ggcatactct gcgacatcgt ataacgttac tggtttcaca ttcaccaccc
      961 tgaattgact ctcttccggg cgctatcatg ccataccgcg aaaggttttg caccattcga
     1021 tggtgtcaac gtaaatgcat gccgcttcgg caaggtgtca ccaccctgcc ctttttcttt
     1081 aaaaccgaaa agattacttc gcgttatgca ggcttcctcg ctcactgact cgctgcgctc
     1141 ggtcgttcgg ctgcggcgag cggtatcagc tcactcaaag gcggtaatct cgaggttaca
     1201 ttgtcgatct gttcatggtg aacagctttg aatgcaccaa aaactcgtaa aagctctgat
     1261 gtatctatct tttttacacc gttttcatct gtgcatatgg acagttttcc ctttgatatg
     1321 taacggtgaa cagttgttct acttttgttt gttagtcttg atgcttcact gatagataca
     1381 agagccataa gaacctcaga tccttccgta tttagccagt atgttctcta gtgtggttcg
     1441 ttgtttttgc gtgagccatg agaacgaacc attgagatca tacttacttt gcatgtcact
     1501 caaaaatttt gcctcaaaac tggtgagctg aatttttgca gttaaagcat cgtgtagtgt
     1561 ttttcttagt ccgttatgta ggtaggaatc tgatgtaatg gttgttggta ttttgtcacc
     1621 attcattttt atctggttgt tctcaagttc ggttacgaga tccatttgtc tatctagttc
     1681 aacttggaaa atcaacgtat cagtcgggcg gcctcgctta tcaaccacca atttcatatt
     1741 gctgtaagtg tttaaatctt tacttattgg tttcaaaacc cattggttaa gccttttaaa
     1801 ctcatggtag ttattttcaa gcattaacat gaacttaaat tcatcaaggc taatctctat
     1861 atttgccttg tgagttttct tttgtgttag ttcttttaat aaccactcat aaatcctcat
     1921 agagtatttg ttttcaaaag acttaacatg ttccagatta tattttatga atttttttaa
     1981 ctggaaaaga taaggcaata tctcttcact aaaaactaat tctaattttt cgcttgagaa
     2041 cttggcatag tttgtccact ggaaaatctc aaagccttta accaaaggat tcatgatttc
     2101 cacagttctc gtcatcagct ctctggttgc tttagctaat acaccataag cattttccct
     2161 actgatgttc atcatctgag cgtattggtt ataagtgaac gataccgtcc gttctttcct
     2221 tgtagggttt tcaatagtgg ggttgagtag tgccacacag cataaaatta gcttggtttc
     2281 atgctccgtt aagtcatagc gactaatcgc tagttcattt gctttgaaaa caactaattc
     2341 agacatacat ctcaattggt ctaggtgatt ttaatcacta taccaattga gatgggctag
     2401 tcaatgataa ttacatgtcc ttttcctttg agttgtgggt atctgtaaat tctgctagac
     2461 ctttgctgga aaacttgtaa attctgctag accctctgta aattccgcta gacctttgtg
     2521 tgtttttttt gtttatattc aagtggttat aatttataga ataaagaaag aataaaaaaa
     2581 gataaaaaga atagatccca gccctgtgta taactcacta ctttagtcag ttccgcagta
     2641 ttacaaaagg atgtcgcaaa cgctgtttgc tcctctacaa aacagacctt aaaaccctaa
     2701 aggcttaagt agcaccctcg caagctcgtg caaatcgctt aatattcctt ttgtctccga
     2761 ccatcaggca cctgagtcgc tgcctttttc gtgacattca gttcgctgcg ctcacggctc
     2821 tggcagtgaa tgtgggtaaa tggcactaca ggcgcctttt atggattcat gcaaggaaac
     2881 tacccataat acaagaaaag cccgtcacgg gcttctcagg gcgttttatg gctggtctgc
     2941 tatgtggtgc tatctgactt tttgctgttc agcagttcct gccctctgat tttccagtct
     3001 gaccacttcg gattatcccg tgacaggtca ttcagactgg ctaatgcacc cagtaaggca
     3061 gcggtatcat caacaggctt acccgtctta ctgtccctag tgcttggatt ctcaccaata
     3121 aaaaacgccc ggcggcaacc gagcgttctg aacaaatcca gatggagttc tgatgtcatt
     3181 actggatcta tcaacaggag tccaagcgag ctcgtaaact tggtctgaca gttaccaatg
     3241 cttaatcagt gaggcaccta tctcagcgat ctgtctattt cgttcatcca tagttgcctg
     3301 actccccgtc gtgtagataa ctacgatacg ggagggctta ccatctggcc ccagtgctgc
     3361 aatgataccg cgtgatccac gctcaccggc tccagattta tcagcaataa accagccagc
     3421 cggaagggcc gagcgcagaa gtggtcctgc aactttatcc gcctccatcc agtctattaa
     3481 ttgttgccgg gaagctagag taagtagttc gccagttaat agtttgcgca acgttgttgc
     3541 cattgctaca ggcatcgtgg tgtcacgctc gtcgtttggt atggcttcat tcagctccgg
     3601 ttcccaacga tcaaggcgag ttacatgatc ccccatgttg tgcaaaaaag cggttagctc
     3661 cttcggtcct ccgatcgttg tcagaagtaa gttggccgca gtgttatcac tcatggttat
     3721 ggcagcactg cataattctc ttactgtcat gccatccgta agatgctttt ctgttactgg
     3781 tgagtactca accaagtcat tctgagaata gtgtatgcgg cgaccgagtt gctcttgccc
     3841 ggcgtcaata cggtataata ccgcgccaca tagcagaact ttaaaagtgc tcatcattgg
     3901 aaaacgttct tcggggcgaa aactctcaag gatcttaccg ctgttgagat ccagttcgat
     3961 gtaacccact cgtgcaccca actgatcttc agcatctttt actttcacca gcgtttctgg
     4021 gtgagcaaaa acgggaaggc aaaatgccgc aaaaaagtga ataagggcga cactgaaatg
     4081 ttgaatactc atactcttcc tttttcaata ttattgaagc atttatcagg gttattgtct
     4141 catgagcgga tacatatttt aatgtattta gaaaaataaa caaatagggg ttccgcgcac
     4201 atttcccatg gtgccacctg acgtctaata aaccattatt atcatgacat taacctataa
     4261 aaataggcgt atcacgaggc agaattttta tcgcttccgc gttccgtcta ctccctatta
     4321 cgctccggaa tctccgacgc ctacggccga cgtcggatac cgccattgat aaactgttta
     4381 acaacatcgt ctgtacagac cttcttcgtt gcctttacgt ttaactcaat caggctaccg
     4441 tctcggttat aagtctatcg agtagtagag ccgtagtaaa gaggagaaat actagatggt
     4501 ttcgaaaggt gaggaggata acatggcgtc gctgcctgct acacatgaac tgcatatttt
     4561 cggcagcatc aacggagttg atttcgacat ggtgggccaa ggcaccggca acccgaacga
     4621 cggctatgaa gaattgaatt tgaaatctac caagggcgac ttgcaattta gtccgtggat
     4681 tctggtaccg catattggtt atggttttca tcagtatctg ccctacccag acggaatgag
     4741 cccatttcag gcggctatgg tggatggcag tggctatcag gtccatcgca ccatgcaatt
     4801 cgaggatggg gcgagcctga ccgtgaatta tcggtacact tacgaaggtt cacatattaa
     4861 aggtgaagca caagttaaag gcaccggctt cccggcggat ggcccagtga tgaccaacag
     4921 cctgacggcg gcggactggt gtcgtagcaa aaagacgtat cccaatgata aaacaattat
     4981 ttctaccttt aaatggagtt acaccacggg taacggtaaa cgctaccgta gcacggctcg
     5041 caccacatac acctttgcga aacccatggc cgcgaactat ctgaaaaatc agccgatgta
     5101 tgtgtttcgc aaaacggaac tgaaacattc gaaaactgaa ctcaatttca aagagtggca
     5161 aaaagccttc accgacgtaa tgggcatgga tgaactgtac aaatgaccag gcatcaaata
     5221 aaacgaaagg ctcagtcgaa agactgggcc tttcgtttta tctgttgttt gtcggtgaac
     5281 gctctctact agagtcacac tggctcacct tcgggtgggc ctttctgcgt ttatagtttc
     5341 acttctgagt tcggcatggg gtcaggtggg accaccgcgc tactgccgcc aggcagctgt
     5401 atcatctgcg tactccatta gtgtgacgac ctatgaggat ggcggggtat taactgtaac
     5461 gcaagatact tctctgcaag atggttgttt aatttataat gtgaaactgc gcggcgttaa
     5521 ttttccctca aatggtcccg taatgcaaaa gaagacctta gggtgggaag cttctacgga
     5581 aacgttgtat cctgcagatg gcggtcttga gggtcgttgt gatatggcgt tgaaactggt
     5641 cggcggtgga catttgcatt gtaatctgaa aacgacttat cttaaccacc tctaacgcga
     5701 cagtagtact tctggagggt gtttatgatt cctgttcctc cgatacccgc agctggagcc
     5761 tcagcggcat ctgcaggagc ggcttccgca gccggtgctt cactcgctgg tgcctccgct
     5821 gctggtgtag cttccgcagc tggtgcagca ggttcctgac tgatgccagc ggctggggct
     5881 tcggcagcat ccgctggggc agcttctgct gcaggcgctt cgctagctgg agcttcagca
     5941 gcaggagtcg cctctgcggc gggcgctgca ggctcttgac tggtaccaat accgttgtcc
     6001 ggacgagtgc cgatggtgtg tgcgtcaccg atgttgttgg tccactggta ttccagacgg
     6061 gtagcgattt caggagtgat cgcgtactca acaccgccag cgaagaccgg agaaacgccg
     6121 gtgtcgtggt ttttaccata aacgttggat ttagtgtctg cacgccatac catgccaccc
     6181 agacgagtgt agatgtccag gtcgtcagtg attgggtaac ccagtttagc ggtcagttga
     6241 acgccctgag ctttgtatgc accgttttca acgctgcctt tgtacggcat acgacctaac
     6301 cagtcgtaac ccatttcaaa gccaacatac gggttaattc cctgatcgat tttagcgttg
     6361 ctggagcaac ctgccagcag agtagaaccc aggattaccg cgcccagtac cagtttagta
     6421 gctttcatca gaaatactcc atagcatgat taagatgttt cagtacgaaa attgctttca
     6481 ttgttgatct cctttttaag tgaacttggg ccctgtgtga aattgttatc cgctcacaat
     6541 tccacacatt atacgagccg atgattaatt gtcaacagct catttcagaa tattagccag
     6601 aaccgttatg atgtcggcgc aaaaaacatt atccagaacg ggagtgcgcc ttgagcgaca
     6661 cgaattatgc agtgatttac gtattaccgc ctttgagtga gctgataccg ctcgccgcag
     6721 ccgaacgccc taggtctagg gcggcggatt tgtcctactc aggagagcgt tcaccgacaa
     6781 acaacagata aaacgaaagg cccagtcttt cgactgagcc tttcgtttta tttgatgcct
     6841 cactgcccgc tttccagtcg ggaaacctgt cgtgccagct gcattaatga atcggccaac
     6901 gcgcggggag aggcggtttg cgtattgggc gccagggtgg tttttctttt caccagtgag
     6961 acgggcaaca gct
//
