LOCUS       sequence-334662-        6973 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Encodes LacI-controlled expression of surface-displayed H6L9 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: pKB254
FEATURES             Location/Qualifiers
     source          1..6973
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     970..989
                     /label=LacI-R
                     /note="LacI, reverse primer"
     promoter        complement(1009..1086)
                     /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(1176..1193)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     CDS             complement(1455..2405)
                     /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      2453..2675
                     /label=pSC101 ori
                     /note="low-copy replication origin that requires the Rep101
                     protein"
     terminator      complement(3167..3261)
                     /label=lambda t0 terminator
                     /note="transcription terminator from phage lambda"
     CDS             complement(join(3291..4082,4083..4151))
                     /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     3914..3933
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(4152..4256)
                     /gene="bla"
                     /label=AmpR promoter
     primer_bind     4321..4339
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     RBS             4537..4548
                     /note="strong bacterial ribosome binding site (Elowitz and 
                     Leibler, 2000)"
     terminator      5274..5345
                     /label=rrnB T1 terminator
                     /note="transcription terminator T1 from the E. coli rrnB 
                     gene"
     terminator      5361..5388
                     /label=T7Te terminator
                     /note="phage T7 early transcription terminator"
     primer_bind     complement(6571..6593)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    6582..6598
                     /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(6606..6612,6613..6628,6629..6634))
                     /label=tac promoter
                     /note="strong E. coli promoter; hybrid between the trp and 
                     lac UV5 promoters"
     terminator      complement(6807..6893)
                     /gene="Escherichia coli rrnB"
                     /label=rrnB T1 terminator
                     /note="transcription terminator T1 from the E. coli rrnB 
                     gene"
     CDS             complement(join(6899..6973,1..1008))
                     /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
                     YIPPLTTIKQDFRLLGQTSVDRLLQLSQDQAVKGNQLLPVSLVKRKTTLAPNTQTASPR
                     ALADSLMQLARQVSRLESGQ"
ORIGIN
        1 ggtttgcgta ttgggcgcca gggtggtttt tcttttcacc agtgagacgg gcaacagctg
       61 attgcccttc accgcctggt cctgagagag ttgcagcaag cggtccacgc tggtttgccc
      121 cagcaggcga aaatcctgtt tgatggtggt taacggcggg atataacatg agctgtcttc
      181 ggtatcgtcg tatcccacta ccgagatatc cgcaccaacg cgcagcccgg actcggtaat
      241 ggcgcgcatt gcgcccagcg ccatctgatc gttggcaacc agcatcgcag tgggaacgat
      301 gccctcattc agcatttgca tggtttgttg aaaaccggac atggcactcc agtcgccttc
      361 ccgttccgct atcggctgaa tttgattgcg agtgagatat ttatgccagc cagccagacg
      421 cagacgcgcc gagacagaac ttaatgggcc cgctaacagc gcgatttgct ggtgacccaa
      481 tgcgaccaga tgctccacgc ccagtcgcgt accgtcttca tgggagaaaa taatactgtt
      541 gatgggtgtc tggtcagaga catcaagaaa taacgccgga acattagtgc aggcagcttc
      601 cacagcaatg gcatcctggt catccagcgg atagttaatg atcagcccac tgacgcgttg
      661 cgcgagaaga ttgtgcaccg ccgctttaca ggcttcgacg ccgcttcgtt ctaccatcga
      721 caccaccacg ctggcaccca gttgatcggc gcgagattta atcgccgcga caatttgcga
      781 cggcgcgtgc agggccagac tggaggtggc aacgccaatc agcaacgact gtttgcccgc
      841 cagttgttgt gccacgcggt tgggaatgta attcagctcc gccatcgccg cttccacttt
      901 ttcccgcgtt ttcgcagaaa cgtggctggc ctggttcacc acgcgggaaa cggtctgata
      961 agagacaccg gcatactctg cgacatcgta taacgttact ggtttcacat tcaccaccct
     1021 gaattgactc tcttccgggc gctatcatgc cataccgcga aaggttttgc accattcgat
     1081 ggtgtcaacg taaatgcatg ccgcttcggc aaggtgtcac caccctgccc tttttcttta
     1141 aaaccgaaaa gattacttcg cgttatgcag gcttcctcgc tcactgactc gctgcgctcg
     1201 gtcgttcggc tgcggcgagc ggtatcagct cactcaaagg cggtaatctc gaggttacat
     1261 tgtcgatctg ttcatggtga acagctttga atgcaccaaa aactcgtaaa agctctgatg
     1321 tatctatctt ttttacaccg ttttcatctg tgcatatgga cagttttccc tttgatatgt
     1381 aacggtgaac agttgttcta cttttgtttg ttagtcttga tgcttcactg atagatacaa
     1441 gagccataag aacctcagat ccttccgtat ttagccagta tgttctctag tgtggttcgt
     1501 tgtttttgcg tgagccatga gaacgaacca ttgagatcat acttactttg catgtcactc
     1561 aaaaattttg cctcaaaact ggtgagctga atttttgcag ttaaagcatc gtgtagtgtt
     1621 tttcttagtc cgttatgtag gtaggaatct gatgtaatgg ttgttggtat tttgtcacca
     1681 ttcattttta tctggttgtt ctcaagttcg gttacgagat ccatttgtct atctagttca
     1741 acttggaaaa tcaacgtatc agtcgggcgg cctcgcttat caaccaccaa tttcatattg
     1801 ctgtaagtgt ttaaatcttt acttattggt ttcaaaaccc attggttaag ccttttaaac
     1861 tcatggtagt tattttcaag cattaacatg aacttaaatt catcaaggct aatctctata
     1921 tttgccttgt gagttttctt ttgtgttagt tcttttaata accactcata aatcctcata
     1981 gagtatttgt tttcaaaaga cttaacatgt tccagattat attttatgaa tttttttaac
     2041 tggaaaagat aaggcaatat ctcttcacta aaaactaatt ctaatttttc gcttgagaac
     2101 ttggcatagt ttgtccactg gaaaatctca aagcctttaa ccaaaggatt catgatttcc
     2161 acagttctcg tcatcagctc tctggttgct ttagctaata caccataagc attttcccta
     2221 ctgatgttca tcatctgagc gtattggtta taagtgaacg ataccgtccg ttctttcctt
     2281 gtagggtttt caatagtggg gttgagtagt gccacacagc ataaaattag cttggtttca
     2341 tgctccgtta agtcatagcg actaatcgct agttcatttg ctttgaaaac aactaattca
     2401 gacatacatc tcaattggtc taggtgattt taatcactat accaattgag atgggctagt
     2461 caatgataat tacatgtcct tttcctttga gttgtgggta tctgtaaatt ctgctagacc
     2521 tttgctggaa aacttgtaaa ttctgctaga ccctctgtaa attccgctag acctttgtgt
     2581 gttttttttg tttatattca agtggttata atttatagaa taaagaaaga ataaaaaaag
     2641 ataaaaagaa tagatcccag ccctgtgtat aactcactac tttagtcagt tccgcagtat
     2701 tacaaaagga tgtcacaaac gctgtttgct cctctacaaa acagacctta aaaccctaaa
     2761 ggcttaagta gcaccctcgc aagctcgtgc aaatcgctta atattccttt tgtctccgac
     2821 catcaggcac ctgagtcgct gcctttttcg tgacattcag ttcgctgcgc tcacggctct
     2881 ggcagtgaat gtgggtaaat ggcactacag gcgcctttta tggattcatg caaggaaact
     2941 acccataata caagaaaagc ccgtcacggg cttctcaggg cgttttatgg ctggtctgct
     3001 atgtggtgct atctgacttt ttgctgttca gcagttcctg ccctctgatt ttccagtctg
     3061 accacttcgg attatcccgt gacaggtcat tcagactggc taatgcaccc agtaaggcag
     3121 cggtatcatc aacaggctta cccgtcttac tgtccctagt gcttggattc tcaccaataa
     3181 aaaacgcccg gcggcaaccg agcgttctga acaaatccag atggagttct gatgtcatta
     3241 ctggatctat caacaggagt ccaagcgagc tcgtaaactt ggtctgacag ttaccaatgc
     3301 ttaatcagtg aggcacctat ctcagcgatc tgtctatttc gttcatccat agttgcctga
     3361 ctccccgtcg tgtagataac tacgatacgg gagggcttac catctggccc cagtgctgca
     3421 atgataccgc gtgatccacg ctcaccggct ccagatttat cagcaataaa ccagccagcc
     3481 ggaagggccg agcgcagaag tggtcctgca actttatccg cctccatcca gtctattaat
     3541 tgttgccggg aagctagagt aagtagttcg ccagttaata gtttgcgcaa cgttgttgcc
     3601 attgctacag gcatcgtggt gtcacgctcg tcgtttggta tggcttcatt cagctccggt
     3661 tcccaacgat caaggcgagt tacatgatcc cccatgttgt gcaaaaaagc ggttagctcc
     3721 ttcggtcctc cgatcgttgt cagaagtaag ttggccgcag tgttatcact catggttatg
     3781 gcagcactgc ataattctct tactgtcatg ccatccgtaa gatgcttttc tgttactggt
     3841 gagtactcaa ccaagtcatt ctgagaatag tgtatgcggc gaccgagttg ctcttgcccg
     3901 gcgtcaatac ggtataatac cgcgccacat agcagaactt taaaagtgct catcattgga
     3961 aaacgttctt cggggcgaaa actctcaagg atcttaccgc tgttgagatc cagttcgatg
     4021 taacccactc gtgcacccaa ctgatcttca gcatctttta ctttcaccag cgtttctggg
     4081 tgagcaaaaa cgggaaggca aaatgccgca aaaaagtgaa taagggcgac actgaaatgt
     4141 tgaatactca tactcttcct ttttcaatat tattgaagca tttatcaggg ttattgtctc
     4201 atgagcggat acatatttta atgtatttag aaaaataaac aaataggggt tccgcgcaca
     4261 tttcccatgg tgccacctga cgtctaataa accattatta tcatgacatt aacctataaa
     4321 aataggcgta tcacgaggca gaatttttat cgcttccgcg ttccgtctac tccctattac
     4381 gctccggaat ctccgacgcc tacggccgac gtcggatacc gccattgata aactgtttaa
     4441 caacatcgtc tgtacagacc ttcttcgttg cctttacgtt taactcaatc aggctaccgt
     4501 ctcggttata agtctatcga gtagtagagc cgtagtaaag aggagaaata ctagatggtt
     4561 tcgaaaggtg aggaggataa catggcgtcg ctgcctgcta cacatgaact gcatattttc
     4621 ggcagcatca acggagttga tttcgacatg gtgggccaag gcaccggcaa cccgaacgac
     4681 ggctatgaag aattgaattt gaaatctacc aagggcgact tgcaatttag tccgtggatt
     4741 ctggtaccgc atattggtta tggttttcat cagtatctgc cctacccaga cggaatgagc
     4801 ccatttcagg cggctatggt ggatggcagt ggctatcagg tccatcgcac catgcaattc
     4861 gaggatgggg cgagcctgac cgtgaattat cggtacactt acgaaggttc acatattaaa
     4921 ggtgaagcac aagttaaagg caccggcttc ccggcggatg gcccagtgat gaccaacagc
     4981 ctgacggcgg cggactggtg tcgtagcaaa aagacgtatc ccaatgataa aacaattatt
     5041 tctaccttta aatggagtta caccacgggt aacggtaaac gctaccgtag cacggctcgc
     5101 accacataca cctttgcgaa acccatggcc gcgaactatc tgaaaaatca gccgatgtat
     5161 gtgtttcgca aaacggaact gaaacattcg aaaactgaac tcaatttcaa agagtggcaa
     5221 aaagccttca ccgacgtaat gggcatggat gaactgtaca aatgaccagg catcaaataa
     5281 aacgaaaggc tcagtcgaaa gactgggcct ttcgttttat ctgttgtttg tcggtgaacg
     5341 ctctctacta gagtcacact ggctcacctt cgggtgggcc tttctgcgtt tatagtttca
     5401 cttctgagtt cggcatgggg tcaggtggga ccaccgcgct actgccgcca ggcagctgta
     5461 tcatctgcgt actccattag tgtgacgacc tatgaggatg gcggggtatt aactgtaacg
     5521 caagatactt ctctgcaaga tggttgttta atttataatg tgaaactgcg cggcgttaat
     5581 tttccctcaa atggtcccgt aatgcaaaag aagaccttag ggtgggaagc ttctacggaa
     5641 acgttgtatc ctgcagatgg cggtcttgag ggtcgttgtg atatggcgtt gaaactggtc
     5701 ggcggtggac atttgcattg taatctgaaa acgacttatc gtattacaat aagtgcagta
     5761 agtgatgaag taaatgatgt aaaaggtgaa gggttcctcc gatacccgca gctggagcct
     5821 cagcggcatc tgcaggagcg gcttccgcag ccggtgcttc actcgctggt gcctccgctg
     5881 ctggtgtagc ttccgcagct ggtgcagcag gttcctgact gatgccagcg gctggggctt
     5941 cggcagcatc cgctggggca gcttctgctg caggcgcttc gctagctgga gcttcagcag
     6001 caggagtcgc ctctgcggcg ggcgctgcag gctcttgact ggtaccaata ccgttgtccg
     6061 gacgagtgcc gatggtgtgt gcgtcaccga tgttgttggt ccactggtat tccagacggg
     6121 tagcgatttc aggagtgatc gcgtactcaa caccgccagc gaagaccgga gaaacgccgg
     6181 tgtcgtggtt tttaccataa acgttggatt tagtgtctgc acgccatacc atgccaccca
     6241 gacgagtgta gatgtccagg tcgtcagtga ttgggtaacc cagtttagcg gtcagttgaa
     6301 cgccctgagc tttgtatgca ccgttttcaa cgctgccttt gtacggcata cgacctaacc
     6361 agtcgtaacc catttcaaag ccaacatacg ggttaattcc ctgatcgatt ttagcgttgc
     6421 tggagcaacc tgccagcaga gtagaaccca ggattaccgc gcccagtacc agtttagtag
     6481 ctttcatcag aaatactcca tagcatgatt aagatgtttc agtacgaaaa ttgctttcat
     6541 tgttgatctc ctttttaagt gaacttgggc cctgtgtgaa attgttatcc gctcacaatt
     6601 ccacacatta tacgagccga tgattaattg tcaacagctc atttcagaat attagccaga
     6661 accgttatga tgtcggcgca aaaaacatta tccagaacgg gagtgcgcct tgagcgacac
     6721 gaattatgca gtgatttacg tattaccgcc tttgagtgag ctgataccgc tcgccgcagc
     6781 cgaacgccct aggtctaggg cggcggattt gtcctactca ggagagcgtt caccgacaaa
     6841 caacagataa aacgaaaggc ccagtctttc gactgagcct ttcgttttat ttgatgcctc
     6901 actgcccgct ttccagtcgg gaaacctgtc gtgccagctg cattaatgaa tcggccaacg
     6961 cgcggggaga ggc
//
