LOCUS       sequence-334656-        6973 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Encodes LacI-controlled expression of surface-displayed melittin 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: pKB255
FEATURES             Location/Qualifiers
     source          1..6973
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     959..978
                     /label=LacI-R
                     /note="LacI, reverse primer"
     promoter        complement(998..1075)
                     /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(1165..1182)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     CDS             complement(1444..2394)
                     /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      2442..2664
                     /label=pSC101 ori
                     /note="low-copy replication origin that requires the Rep101
                     protein"
     terminator      complement(3156..3250)
                     /label=lambda t0 terminator
                     /note="transcription terminator from phage lambda"
     CDS             complement(join(3280..4071,4072..4140))
                     /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     3903..3922
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(4141..4245)
                     /gene="bla"
                     /label=AmpR promoter
     primer_bind     4310..4328
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     RBS             4526..4537
                     /note="strong bacterial ribosome binding site (Elowitz and 
                     Leibler, 2000)"
     terminator      5263..5334
                     /label=rrnB T1 terminator
                     /note="transcription terminator T1 from the E. coli rrnB 
                     gene"
     terminator      5350..5377
                     /label=T7Te terminator
                     /note="phage T7 early transcription terminator"
     primer_bind     complement(6560..6582)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    6571..6587
                     /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(6595..6601,6602..6617,6618..6623))
                     /label=tac promoter
                     /note="strong E. coli promoter; hybrid between the trp and 
                     lac UV5 promoters"
     terminator      complement(6796..6882)
                     /gene="Escherichia coli rrnB"
                     /label=rrnB T1 terminator
                     /note="transcription terminator T1 from the E. coli rrnB 
                     gene"
     CDS             complement(join(6888..6973,1..997))
                     /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 tgggcgccag ggtggttttt cttttcacca gtgagacggg caacagctga ttgcccttca
       61 ccgcctggcc ctgagagagt tgcagcaagc ggtccacgct ggtttgcccc agcaggcgaa
      121 aatcctgttt gatggtggtt aacggcggga tataacatga gctgtcttcg gtatcgtcgt
      181 atcccactac cgagatatcc gcaccaacgc gcagcccgga ctcggtaatg gcgcgcattg
      241 cgcccagcgc catctgatcg ttggcaacca gcatcgcagt gggaacgatg ccctcattca
      301 gcatttgcat ggtttgttga aaaccggaca tggcactcca gtcgccttcc cgttccgcta
      361 tcggctgaat ttgattgcga gtgagatatt tatgccagcc agccagacgc agacgcgccg
      421 agacagaact taatgggccc gctaacagcg cgatttgctg gtgacccaat gcgaccagat
      481 gctccacgcc cagtcgcgta ccgtcttcat gggagaaaat aatactgttg atgggtgtct
      541 ggtcagagac atcaagaaat aacgccggaa cattagtgca ggcagcttcc acagcaatgg
      601 catcctggtc atccagcgga tagttaatga tcagcccact gacgcgttgc gcgagaagat
      661 tgtgcaccgc cgctttacag gcttcgacgc cgcttcgttc taccatcgac accaccacgc
      721 tggcacccag ttgatcggcg cgagatttaa tcgccgcgac aatttgcgac ggcgcgtgca
      781 gggccagact ggaggtggca acgccaatca gcaacgactg tttgcccgcc agttgttgtg
      841 ccacgcggtt gggaatgtaa ttcagctccg ccatcgccgc ttccactttt tcccgcgttt
      901 tcgcagaaac gtggctggcc tggttcacca cgcgggaaac ggtctgataa gagacaccgg
      961 catactctgc gacatcgtat aacgttactg gtttcacatt caccaccctg aattgactct
     1021 cttccgggcg ctatcatgcc ataccgcgaa aggttttgca ccattcgatg gtgtcaacgt
     1081 aaatgcatgc cgcttcggca aggtgtcacc accctgccct ttttctttaa aaccgaaaag
     1141 attacttcgc gttatgcagg cttcctcgct cactgactcg ctgcgctcgg tcgttcggct
     1201 gcggcgagcg gtatcagctc actcaaaggc ggtaatctcg aggttacatt gtcgatctgt
     1261 tcatggtgaa cagctttgaa tgcaccaaaa actcgtaaaa gctctgatgt atctatcttt
     1321 tttacaccgt tttcatctgt gcatatggac agttttccct ttgatatgta acggtgaaca
     1381 gttgttctac ttttgtttgt tagtcttgat gcttcactga tagatacaag agccataaga
     1441 acctcagatc cttccgtatt tagccagtat gttctctagt gtggttcgtt gtttttgcgt
     1501 gagccatgag aacgaaccat tgagatcata cttactttgc atgtcactca aaaattttgc
     1561 ctcaaaactg gtgagctgaa tttttgcagt taaagcatcg tgtagtgttt ttcttagtcc
     1621 gttatgtagg taggaatctg atgtaatggt tgttggtatt ttgtcaccat tcatttttat
     1681 ctggttgttc tcaagttcgg ttacgagatc catttgtcta tctagttcaa cttggaaaat
     1741 caacgtatca gtcgggcggc ctcgcttatc aaccaccaat ttcatattgc tgtaagtgtt
     1801 taaatcttta cttattggtt tcaaaaccca ttggttaagc cttttaaact catggtagtt
     1861 attttcaagc attaacatga acttaaattc atcaaggcta atctctatat ttgccttgtg
     1921 agttttcttt tgtgttagtt cttttaataa ccactcataa atcctcatag agtatttgtt
     1981 ttcaaaagac ttaacatgtt ccagattata ttttatgaat ttttttaact ggaaaagata
     2041 aggcaatatc tcttcactaa aaactaattc taatttttcg cttgagaact tggcatagtt
     2101 tgtccactgg aaaatctcaa agcctttaac caaaggattc atgatttcca cagttctcgt
     2161 catcagctct ctggttgctt tagctaatac accataagca ttttccctac tgatgttcat
     2221 catctgagcg tattggttat aagtgaacga taccgtccgt tctttccttg tagggttttc
     2281 aatagtgggg ttgagtagtg ccacacagca taaaattagc ttggtttcat gctccgttaa
     2341 gtcatagcga ctaatcgcta gttcatttgc tttgaaaaca actaattcag acatacatct
     2401 caattggtct aggtgatttt aatcactata ccaattgaga tgggctagtc aatgataatt
     2461 acatgtcctt ttcctttgag ttgtgggtat ctgtaaattc tgctagacct ttgctggaaa
     2521 acttgtaaat tctgctagac cctctgtaaa ttccgctaga cctttgtgtg ttttttttgt
     2581 ttatattcaa gtggttataa tttatagaat aaagaaagaa taaaaaaaga taaaaagaat
     2641 agatcccagc cctgtgtata actcactact ttagtcagtt ccgcagtatt acaaaaggat
     2701 gtcgcaaacg ctgtttgctc ctctacaaaa cagaccttaa aaccctaaag gcttaagtag
     2761 caccctcgca agctcgtgca aatcgcttaa tattcctttt gtctccgacc atcaggcacc
     2821 tgagtcgctg cctttttcgt gacattcagt tcgctgcgct cacggctctg gcagtgaatg
     2881 tgggtaaatg gcactacagg cgccttttat ggattcatgc aaggaaacta cccataatac
     2941 aagaaaagcc cgtcacgggc ttctcagggc gttttatggc tggtctgcta tgtggtgcta
     3001 tctgactttt tgctgttcag cagttcctgc cctctgattt tccagtctga ccacttcgga
     3061 ttatcccgtg acaggtcatt cagactggct aatgcaccca gtaaggcagc ggtatcatca
     3121 acaggcttac ccgtcttact gtccctagtg cttggattct caccaataaa aaacgcccgg
     3181 cggcaaccga gcgttctgaa caaatccaga tggagttctg atgtcattac tggatctatc
     3241 aacaggagtc caagcgagct cgtaaacttg gtctgacagt taccaatgct taatcagtga
     3301 ggcacctatc tcagcgatct gtctatttcg ttcatccata gttgcctgac tccccgtcgt
     3361 gtagataact acgatacggg agggcttacc atctggcccc agtgctgcaa tgataccgcg
     3421 tgatccacgc tcaccggctc cagatttatc agcaataaac cagccagccg gaagggccga
     3481 gcgcagaagt ggtcctgcaa ctttatccgc ctccatccag tctattaatt gttgccggga
     3541 agctagagta agtagttcgc cagttaatag tttgcgcaac gttgttgcca ttgctacagg
     3601 catcgtggtg tcacgctcgt cgtttggtat ggcttcattc agctccggtt cccaacgatc
     3661 aaggcgagtt acatgatccc ccatgttgtg caaaaaagcg gttagctcct tcggtcctcc
     3721 gatcgttgtc agaagtaagt tggccgcagt gttatcactc atggttatgg cagcactgca
     3781 taattctctt actgtcatgc catccgtaag atgcttttct gttactggtg agtactcaac
     3841 caagtcattc tgagaatagt gtatgcggcg accgagttgc tcttgcccgg cgtcaatacg
     3901 gtataatacc gcgccacata gcagaacttt aaaagtgctc atcattggaa aacgttcttc
     3961 ggggcgaaaa ctctcaagga tcttaccgct gttgagatcc agttcgatgt aacccactcg
     4021 tgcacccaac tgatcttcag catcttttac tttcaccagc gtttctgggt gagcaaaaac
     4081 gggaaggcaa aatgccgcaa aaaagtgaat aagggcgaca ctgaaatgtt gaatactcat
     4141 actcttcctt tttcaatatt attgaagcat ttatcagggt tattgtctca tgagcggata
     4201 catattttaa tgtatttaga aaaataaaca aataggggtt ccgcgcacat ttcccatggt
     4261 gccacctgac gtctaataaa ccattattat catgacatta acctataaaa ataggcgtat
     4321 cacgaggcag aatttttatc gcttccgcgt tccgtctact ccctattacg ctccggaatc
     4381 tccgacgcct acggccgacg tcggataccg ccattgataa actgtttaac aacatcgtct
     4441 gtacagacct tcttcgttgc ctttacgttt aactcaatca ggctaccgtc tcggttataa
     4501 gtctatcgag tagtagagcc gtagtaaaga ggagaaatac tagatggttt cgaaaggtga
     4561 ggaggataac atggcgtcgc tgcctgctac acatgaactg catattttcg gcagcatcaa
     4621 cggagttgat ttcgacatgg tgggccaagg caccggcaac ccgaacgacg gctatgaaga
     4681 attgaatttg aaatctacca agggcgactt gcaatttagt ccgtggattc tggtaccgca
     4741 tattggttat ggttttcatc agtatctgcc ctacccagac ggaatgagcc catttcaggc
     4801 ggctatggtg gatggcagtg gctatcaggt ccatcgcacc atgcaattcg aggatggggc
     4861 gagcctgacc gtgaattatc ggtacactta cgaaggttca catattaaag gtgaagcaca
     4921 agttaaaggc accggcttcc cggcggatgg cccagtgatg accaacagcc tgacggcggc
     4981 ggactggtgt cgtagcaaaa agacgtatcc caatgataaa acaattattt ctacctttaa
     5041 atggagttac accacgggta acggtaaacg ctaccgtagc acggctcgca ccacatacac
     5101 ctttgcgaaa cccatggccg cgaactatct gaaaaatcag ccgatgtatg tgtttcgcaa
     5161 aacggaactg aaacattcga aaactgaact caatttcaaa gagtggcaaa aagccttcac
     5221 cgacgtaatg ggcatggatg aactgtacaa atgaccaggc atcaaataaa acgaaaggct
     5281 cagtcgaaag actgggcctt tcgttttatc tgttgtttgt cggtgaacgc tctctactag
     5341 agtcacactg gctcaccttc gggtgggcct ttctgcgttt atagtttcac ttctgagttc
     5401 ggcatggggt caggtgggac caccgcgcta ctgccgccag gcagctgtat catctgcgta
     5461 ctccattagt gtgacgacct atgaggatgg cggggtatta actgtaacgc aagatacttc
     5521 tctgcaagat ggttgtttaa tttataatgt gaaactgcgc ggcgttaatt ttccctcaaa
     5581 tggtcccgta atgcaaaaga agaccttagg gtgggaagct tctacggaaa cgttgtatcc
     5641 tgcagatggc ggtcttgagg gtcgttgtga tatggcgttg aaactggtcg gcggtgttac
     5701 ccctgttgac gtttacgttt gatccaacta attaaggctg gcaaccccgt ggtcagaact
     5761 ttcagtaccg ctccaatacc tgttcctccg atacccgcag ctggagcctc agcggcatct
     5821 gcaggagcgg cttccgcagc cggtgcttca ctcgctggtg cctccgctgc tggtgtagct
     5881 tccgcagctg gtgcagcagg ttcctgactg atgccagcgg ctggggcttc ggcagcatcc
     5941 gctggggcag cttctgctgc aggcgcttcg ctagctggag cttcagcagc aggagtcgcc
     6001 tctgcggcgg gcgctgcagg ctcttgactg gtaccaatac cgttgtccgg acgagtgccg
     6061 atggtgtgtg cgtcaccgat gttgttggtc cactggtatt ccagacgggt agcgatttca
     6121 ggagtgatcg cgtactcaac accgccagcg aagaccggag aaacgccggt gtcgtggttt
     6181 ttaccataaa cgttggattt agtgtctgca cgccatacca tgccacccag acgagtgtag
     6241 atgtccaggt cgtcagtgat tgggtaaccc agtttagcgg tcagttgaac gccctgagct
     6301 ttgtatgcac cgttttcaac gctgcctttg tacggcatac gacctaacca gtcgtaaccc
     6361 atttcaaagc caacatacgg gttaattccc tgatcgattt tagcgttgct ggagcaacct
     6421 gccagcagag tagaacccag gattaccgcg cccagtacca gtttagtagc tttcatcaga
     6481 aatactccat agcatgatta agatgtttca gtacgaaaat tgctttcatt gttgatctcc
     6541 tttttaagtg aacttgggcc ctgtgtgaaa ttgttatccg ctcacaattc cacacattat
     6601 acgagccgat gattaattgt caacagctca tttcagaata ttagccagaa ccgttatgat
     6661 gtcggcgcaa aaaacattat ccagaacggg agtgcgcctt gagcgacacg aattatgcag
     6721 tgatttacgt attaccgcct ttgagtgagc tgataccgct cgccgcagcc gaacgcccta
     6781 ggtctagggc ggcggatttg tcctactcag gagagcgttc accgacaaac aacagataaa
     6841 acgaaaggcc cagtctttcg actgagcctt tcgttttatt tgatgcctca ctgcccgctt
     6901 tccagtcggg aaacctgtcg tgccagctgc attaatgaat cggccaacgc gcggggagag
     6961 gcggtttgcg tat
//
