LOCUS       sequence-335944-        6973 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Encodes LacI-controlled expression of surface-displayed hBD3 
            fragment lacking 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: pKB234.2
FEATURES             Location/Qualifiers
     source          1..6973
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     terminator      487..573
                     /gene="Escherichia coli rrnB"
                     /label=rrnB T1 terminator
                     /note="transcription terminator T1 from the E. coli rrnB 
                     gene"
     promoter        join(746..751,752..767,768..774)
                     /label=tac promoter
                     /note="strong E. coli promoter; hybrid between the trp and 
                     lac UV5 promoters"
     protein_bind    782..798
                     /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     787..809
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     terminator      complement(1992..2019)
                     /label=T7Te terminator
                     /note="phage T7 early transcription terminator"
     terminator      complement(2035..2106)
                     /label=rrnB T1 terminator
                     /note="transcription terminator T1 from the E. coli rrnB 
                     gene"
     RBS             2832..2843
                     /note="strong bacterial ribosome binding site (Elowitz and 
                     Leibler, 2000)"
     primer_bind     complement(3041..3059)
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     promoter        3124..3228
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(3229..3297,3298..4089)
                     /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(3447..3466)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     terminator      4119..4213
                     /label=lambda t0 terminator
                     /note="transcription terminator from phage lambda"
     rep_origin      4705..4927
                     /label=pSC101 ori
                     /note="low-copy replication origin that requires the Rep101
                     protein"
     CDS             4975..5925
                     /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     6187..6204
                     /label=L4440
                     /note="L4440 vector, forward primer"
     promoter        6294..6371
                     /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(6372..6973,1..481)
                     /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(6391..6410)
                     /label=LacI-R
                     /note="LacI, reverse primer"
ORIGIN
        1 gcataaatat ctcactcgca atcaaattca gccgatagcg gaacgggaag gcgactggag
       61 tgccatgtcc ggttttcaac aaaccatgca aatgctgaat gagggcatcg ttcccactgc
      121 gatgctggtt gccaacgatc agatggcgct gggcgcaatg cgcgccatta ccgagtccgg
      181 gctgcgcgtt ggtgcggata tctcggtagt gggatacgac gataccgaag acagctcatg
      241 ttatatcccg ccgttaacca ccatcaaaca ggattttcgc ctgctggggc aaaccagcgt
      301 ggaccgcttg ctgcaactct ctcagggcca ggcggtgaag ggcaatcagc tgttgcccgt
      361 ctcactggtg aaaagaaaaa ccaccctggc gcccaatacg caaaccgcct ctccccgcgc
      421 gttggccgat tcattaatgc agctggcacg acaggtttcc cgactggaaa gcgggcagtg
      481 aggcatcaaa taaaacgaaa ggctcagtcg aaagactggg cctttcgttt tatctgttgt
      541 ttgtcggtga acgctctcct gagtaggaca aatccgccgc cctagaccta gggcgttcgg
      601 ctgcggcgag cggtatcagc tcactcaaag gcggtaatac gtaaatcact gcataattcg
      661 tgtcgctcaa ggcgcactcc cgttctggat aatgtttttt gcgccgacat cataacggtt
      721 ctggctaata ttctgaaatg agctgttgac aattaatcat cggctcgtat aatgtgtgga
      781 attgtgagcg gataacaatt tcacacaggg cccaagttca cttaaaaagg agatcaacaa
      841 tgaaagcaat tttcgtactg aaacatctta atcatgctat ggagtatttc tgatgaaagc
      901 tactaaactg gtactgggcg cggtaatcct gggttctact ctgctggcag gttgctccag
      961 caacgctaaa atcgatcagg gaattaaccc gtatgttggc tttgaaatgg gttacgactg
     1021 gttaggtcgt atgccgtaca aaggcagcgt tgaaaacggt gcatacaaag ctcagggcgt
     1081 tcaactgacc gctaaactgg gttacccaat cactgacgac ctggacatct acactcgtct
     1141 gggtggcatg gtatggcgtg cagacactaa atccaacgtt tatggtaaaa accacgacac
     1201 cggcgtttct ccggtcttcg ctggcggtgt tgagtacgcg atcactcctg aaatcgctac
     1261 ccgtctggaa taccagtgga ccaacaacat cggtgacgca cacaccatcg gcactcgtcc
     1321 ggacaacggt attggtacca gtcaagagcc tgcagcgccc gccgcagagg cgactcctgc
     1381 tgctgaagct ccagctagcg aagcgcctgc agcagaagct gccccagcgg atgctgccga
     1441 agccccagcc gctggcatca gtcaggaacc tgctgcacca gctgcggaag ctacaccagc
     1501 agcggaggca ccagcgagtg aagcaccggc tgcggaagcc gctcctgcag atgccgctga
     1561 ggctccagct gcgggtatcg gaggaacagg tggtcgttgt gctgtcctgt cttgtttgcc
     1621 taaggaggag caaattggta agtgttctac tcgtggtaga aagtgttgcc gtcgtaagaa
     1681 gtaagtttca acgccatatc acaacgaccc tcaagaccgc catctgcagg atacaacgtt
     1741 tccgtagaag cttcccaccc taaggtcttc ttttgcatta cgggaccatt tgagggaaaa
     1801 ttaacgccgc gcagtttcac attataaatt aaacaaccat cttgcagaga agtatcttgc
     1861 gttacagtta ataccccgcc atcctcatag gtcgtcacac taatggagta cgcagatgat
     1921 acagctgcct ggcggcagta gcgcggtggt cccacctgac cccatgccga actcagaagt
     1981 gaaactataa acgcagaaag gcccacccga aggtgagcca gtgtgactct agtagagagc
     2041 gttcaccgac aaacaacaga taaaacgaaa ggcccagtct ttcgactgag cctttcgttt
     2101 tatttgatgc ctggtcattt gtacagttca tccatgccca ttacgtcggt gaaggctttt
     2161 tgccactctt tgaaattgag ttcagttttc gaatgtttca gttccgtttt gcgaaacaca
     2221 tacatcggct gatttttcag atagttcgcg gccatgggtt tcgcaaaggt gtatgtggtg
     2281 cgagccgtgc tacggtagcg tttaccgtta cccgtggtgt aactccattt aaaggtagaa
     2341 ataattgttt tatcattggg atacgtcttt ttgctacgac accagtccgc cgccgtcagg
     2401 ctgttggtca tcactgggcc atccgccggg aagccggtgc ctttaacttg tgcttcacct
     2461 ttaatatgtg aaccttcgta agtgtaccga taattcacgg tcaggctcgc cccatcctcg
     2521 aattgcatgg tgcgatggac ctgatagcca ctgccatcca ccatagccgc ctgaaatggg
     2581 ctcattccgt ctgggtaggg cagatactga tgaaaaccat aaccaatatg cggtaccaga
     2641 atccacggac taaattgcaa gtcgcccttg gtagatttca aattcaattc ttcatagccg
     2701 tcgttcgggt tgccggtgcc ttggcccacc atgtcgaaat caactccgtt gatgctgccg
     2761 aaaatatgca gttcatgtgt agcaggcagc gacgccatgt tatcctcctc acctttcgaa
     2821 accatctagt atttctcctc tttactacgg ctctactact cgatagactt ataaccgaga
     2881 cggtagcctg attgagttaa acgtaaaggc aacgaagaag gtctgtacag acgatgttgt
     2941 taaacagttt atcaatggcg gtatccgacg tcggccgtag gcgtcggaga ttccggagcg
     3001 taatagggag tagacggaac gcggaagcga taaaaattct gcctcgtgat acgcctattt
     3061 ttataggtta atgtcatgat aataatggtt tattagacgt caggtggcac catgggaaat
     3121 gtgcgcggaa cccctatttg tttatttttc taaatacatt aaaatatgta tccgctcatg
     3181 agacaataac cctgataaat gcttcaataa tattgaaaaa ggaagagtat gagtattcaa
     3241 catttcagtg tcgcccttat tcactttttt gcggcatttt gccttcccgt ttttgctcac
     3301 ccagaaacgc tggtgaaagt aaaagatgct gaagatcagt tgggtgcacg agtgggttac
     3361 atcgaactgg atctcaacag cggtaagatc cttgagagtt ttcgccccga agaacgtttt
     3421 ccaatgatga gcacttttaa agttctgcta tgtggcgcgg tattataccg tattgacgcc
     3481 gggcaagagc aactcggtcg ccgcatacac tattctcaga atgacttggt tgagtactca
     3541 ccagtaacag aaaagcatct tacggatggc atgacagtaa gagaattatg cagtgctgcc
     3601 ataaccatga gtgataacac tgcggccaac ttacttctga caacgatcgg aggaccgaag
     3661 gagctaaccg cttttttgca caacatgggg gatcatgtaa ctcgccttga tcgttgggaa
     3721 ccggagctga atgaagccat accaaacgac gagcgtgaca ccacgatgcc tgtagcaatg
     3781 gcaacaacgt tgcgcaaact attaactggc gaactactta ctctagcttc ccggcaacaa
     3841 ttaatagact ggatggaggc ggataaagtt gcaggaccac ttctgcgctc ggcccttccg
     3901 gctggctggt ttattgctga taaatctgga gccggtgagc gtggatcacg cggtatcatt
     3961 gcagcactgg ggccagatgg taagccctcc cgtatcgtag ttatctacac gacggggagt
     4021 caggcaacta tggatgaacg aaatagacag atcgctgaga taggtgcctc actgattaag
     4081 cattggtaac tgtcagacca agtttacgag ctcgcttgga ctcctgttga tagatccagt
     4141 aatgacatca gaactccatc tggatttgtt cagaacgctc ggttgccgcc gggcgttttt
     4201 tattggtgag aatccaagca ctagggacag taagacgggt aagcctgttg atgataccgc
     4261 tgccttactg ggtgcattag ccagtctgaa tgacctgtca cgggataatc cgaagtggtc
     4321 agactggaaa atcagagggc aggaactgct gaacagcaaa aagtcagata gcaccacata
     4381 gcagaccagc cataaaacgc cctgagaagc ccgtgacggg cttttcttgt attatgggta
     4441 gtttccttgc atgaatccat aaaaggcgcc tgtagtgcca tttacccaca ttcactgcca
     4501 gagccgtgag cgcagcgaac tgaatgtcac gaaaaaggca gcgactcagg tgcctgatgg
     4561 tcggagacaa aaggaatatt aagcgatttg cacgagcttg cgagggtgct acttaagcct
     4621 ttagggtttt aaggtctgtt ttgtagagga gcaaacagcg tttgcgacat ccttttgtaa
     4681 tactgcggaa ctgactaaag tagtgagtta tacacagggc tgggatctat tctttttatc
     4741 tttttttatt ctttctttat tctataaatt ataaccactt gaatataaac aaaaaaaaca
     4801 cacaaaggtc tagcggaatt tacagagggt ctagcagaat ttacaagttt tccagcaaag
     4861 gtctagcaga atttacagat acccacaact caaaggaaaa ggacatgtaa ttatcattga
     4921 ctagcccatc tcaattggta tagtgattaa aatcacctag accaattgag atgtatgtct
     4981 gaattagttg ttttcaaagc aaatgaacta gcgattagtc gctatgactt aacggagcat
     5041 gaaaccaagc taattttatg ctgtgtggca ctactcaacc ccactattga aaaccctaca
     5101 aggaaagaac ggacggtatc gttcacttat aaccaatacg ctcagatgat gaacatcagt
     5161 agggaaaatg cttatggtgt attagctaaa gcaaccagag agctgatgac gagaactgtg
     5221 gaaatcatga atcctttggt taaaggcttt gagattttcc agtggacaaa ctatgccaag
     5281 ttctcaagcg aaaaattaga attagttttt agtgaagaga tattgcctta tcttttccag
     5341 ttaaaaaaat tcataaaata taatctggaa catgttaagt cttttgaaaa caaatactct
     5401 atgaggattt atgagtggtt attaaaagaa ctaacacaaa agaaaactca caaggcaaat
     5461 atagagatta gccttgatga atttaagttc atgttaatgc ttgaaaataa ctaccatgag
     5521 tttaaaaggc ttaaccaatg ggttttgaaa ccaataagta aagatttaaa cacttacagc
     5581 aatatgaaat tggtggttga taagcgaggc cgcccgactg atacgttgat tttccaagtt
     5641 gaactagata gacaaatgga tctcgtaacc gaacttgaga acaaccagat aaaaatgaat
     5701 ggtgacaaaa taccaacaac cattacatca gattcctacc tacataacgg actaagaaaa
     5761 acactacacg atgctttaac tgcaaaaatt cagctcacca gttttgaggc aaaatttttg
     5821 agtgacatgc aaagtaagta tgatctcaat ggttcgttct catggctcac gcaaaaacaa
     5881 cgaaccacac tagagaacat actggctaaa tacggaagga tctgaggttc ttatggctct
     5941 tgtatctatc agtgaagcat caagactaac aaacaaaagt agaacaactg ttcaccgtta
     6001 catatcaaag ggaaaactgt ccatatgcac agatgaaaac ggtgtaaaaa agatagatac
     6061 atcagagctt ttacgagttt ttggtgcatt caaagctgtt caccatgaac agatcgacaa
     6121 tgtaacctcg agattaccgc ctttgagtga gctgataccg ctcgccgcag ccgaacgacc
     6181 gagcgcagcg agtcagtgag cgaggaagcc tgcataacgc gaagtaatct tttcggtttt
     6241 aaagaaaaag ggcagggtgg tgacaccttg ccgaagcggc atgcatttac gttgacacca
     6301 tcgaatggtg caaaaccttt cgcggtatgg catgatagcg cccggaagag agtcaattca
     6361 gggtggtgaa tgtgaaacca gtaacgttat acgatgtcgc agagtatgcc ggtgtctctt
     6421 atcagaccgt ttcccgcgtg gtgaaccagg ccagccacgt ttctgcgaaa acgcgggaaa
     6481 aagtggaagc ggcgatggcg gagctgaatt acattcccaa ccgcgtggca caacaactgg
     6541 cgggcaaaca gtcgttgctg attggcgttg ccacctccag tctggccctg cacgcgccgt
     6601 cgcaaattgt cgcggcgatt aaatctcgcg ccgatcaact gggtgccagc gtggtggtgt
     6661 cgatggtaga acgaagcggc gtcgaagcct gtaaagcggc ggtgcacaat cttctcgcgc
     6721 aacgcgtcag tgggctgatc attaactatc cgctggatga ccaggatgcc attgctgtgg
     6781 aagctgcctg cactaatgtt ccggcgttat ttcttgatgt ctctgaccag acacccatca
     6841 acagtattat tttctcccat gaagacggta cgcgactggg cgtggagcat ctggtcgcat
     6901 tgggtcacca gcaaatcgcg ctgttagcgg gcccattaag ttctgtctcg gcgcgtctgc
     6961 gtctggctgg ctg
//
