LOCUS       sequence-335947-        6973 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Encodes LacI-controlled expression of surface-displayed NC 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: pKB224
FEATURES             Location/Qualifiers
     source          1..6973
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     terminator      complement(935..962)
                     /label=T7Te terminator
                     /note="phage T7 early transcription terminator"
     terminator      complement(978..1049)
                     /label=rrnB T1 terminator
                     /note="transcription terminator T1 from the E. coli rrnB 
                     gene"
     RBS             1775..1786
                     /note="strong bacterial ribosome binding site (Elowitz and 
                     Leibler, 2000)"
     primer_bind     complement(1984..2002)
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     promoter        2067..2171
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(2172..2240,2241..3032)
                     /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(2390..2409)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     terminator      3062..3156
                     /label=lambda t0 terminator
                     /note="transcription terminator from phage lambda"
     rep_origin      3648..3870
                     /label=pSC101 ori
                     /note="low-copy replication origin that requires the Rep101
                     protein"
     CDS             3918..4868
                     /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     5130..5147
                     /label=L4440
                     /note="L4440 vector, forward primer"
     promoter        5237..5314
                     /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             5315..6397
                     /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(5334..5353)
                     /label=LacI-R
                     /note="LacI, reverse primer"
     terminator      6403..6489
                     /gene="Escherichia coli rrnB"
                     /label=rrnB T1 terminator
                     /note="transcription terminator T1 from the E. coli rrnB 
                     gene"
     promoter        join(6662..6667,6668..6683,6684..6690)
                     /label=tac promoter
                     /note="strong E. coli promoter; hybrid between the trp and 
                     lac UV5 promoters"
     protein_bind    6698..6714
                     /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     6703..6725
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
ORIGIN
        1 ggtgcataca aagctcaggg cgttcaactg accgctaaac tgggttaccc aatcactgac
       61 gacctggaca tctacactcg tctgggtggc atggtatggc gtgcagacac taaatccaac
      121 gtttatggta aaaaccacga caccggcgtt tctccggtct tcgctggcgg tgttgagtac
      181 gcgatcactc ctgaaatcgc tacccgtctg gaataccagt ggaccaacaa catcggtgac
      241 gcacacacca tcggcactcg tccggacaac ggtattggta ccagtcaaga gcctgcagcg
      301 cccgccgcag aggcgactcc tgctgctgaa gctccagcta gcgaagcgcc tgcagcagaa
      361 gctgccccag cggatgctgc cgaagcccca gccgctggca tcagtcagga acctgctgca
      421 ccagctgcgg aagctacacc agcagcggag gcaccagcga gtgaagcacc ggctgcggaa
      481 gccgctcctg cagatgccgc tgaggctcca gctgcgggta tcggaggaac cggggctact
      541 cctgaggact taaatcagaa gctgagctaa gcttcttgct acgataagtc gttttcagat
      601 tacaatgcaa atgtccaccg ccgaccagtt tcaacgccat atcacaacga ccctcaagac
      661 cgccatctgc aggatacaac gtttccgtag aagcttccca ccctaaggtc ttcttttgca
      721 ttacgggacc atttgaggga aaattaacgc cgcgcagttt cacattataa attaaacaac
      781 catcttgcag agaagtatct tgcgttacag ttaatacccc gccatcctca taggtcgtca
      841 cactaatgga gtacgcagat gatacagctg cctggcggca gtagcgcggt ggtcccacct
      901 gaccccatgc cgaactcaga agtgaaacta taaacgcaga aaggcccacc cgaaggtgag
      961 ccagtgtgac tctagtagag agcgttcacc gacaaacaac agataaaacg aaaggcccag
     1021 tctttcgact gagcctttcg ttttatttga tgcctggtca tttgtacagt tcatccatgc
     1081 ccattacgtc ggtgaaggct ttttgccact ctttgaaatt gagttcagtt ttcgaatgtt
     1141 tcagttccgt tttgcgaaac acatacatcg gctgattttt cagatagttc gcggccatgg
     1201 gtttcgcaaa ggtgtatgtg gtgcgagccg tgctacggta gcgtttaccg ttacccgtgg
     1261 tgtaactcca tttaaaggta gaaataattg ttttatcatt gggatacgtc tttttgctac
     1321 gacaccagtc cgccgccgtc aggctgttgg tcatcactgg gccatccgcc gggaagccgg
     1381 tgcctttaac ttgtgcttca cctttaatat gtgaaccttc gtaagtgtac cgataattca
     1441 cggtcaggct cgccccatcc tcgaattgca tggtgcgatg gacctgatag ccactgccat
     1501 ccaccatagc cgcctgaaat gggctcattc cgtctgggta gggcagatac tgatgaaaac
     1561 cataaccaat atgcggtacc agaatccacg gactaaattg caagtcgccc ttggtagatt
     1621 tcaaattcaa ttcttcatag ccgtcgttcg ggttgccggt gccttggccc accatgtcga
     1681 aatcaactcc gttgatgctg ccgaaaatat gcagttcatg tgtagcaggc agcgacgcca
     1741 tgttatcctc ctcacctttc gaaaccatct agtatttctc ctctttacta cggctctact
     1801 actcgataga cttataaccg agacggtagc ctgattgagt taaacgtaaa ggcaacgaag
     1861 aaggtctgta cagacgatgt tgttaaacag tttatcaatg gcggtatccg acgtcggccg
     1921 taggcgtcgg agattccgga gcgtaatagg gagtagacgg aacgcggaag cgataaaaat
     1981 tctgcctcgt gatacgccta tttttatagg ttaatgtcat gataataatg gtttattaga
     2041 cgtcaggtgg caccatggga aatgtgcgcg gaacccctat ttgtttattt ttctaaatac
     2101 attaaaatat gtatccgctc atgagacaat aaccatgata aatgcttcaa taatattgaa
     2161 aaaggaagag tatgagtatt caacatttca gtgtcgccct tattcacttt tttgcggcat
     2221 tttgccttcc tgtttttgct cacccagaaa cgctggtgaa agtaaaagat gctgaagatc
     2281 agttgggtgc acgagtgggt tacatcgaac tggatctcaa cagcggtaag atccttgaga
     2341 gttttcgccc cgaagaacgt tttccaatga tgagcacttt taaagttctg ctatgtggcg
     2401 cggtattata ccgtattgac gccgggcaag agcaactcgg tcgccgcata cactattctc
     2461 agaatgactt ggttgagtac tcaccagtaa cagaaaagca tcttacggat ggcatgacag
     2521 taagagaatt atgcagtgct gccataacca tgagtgataa cactgcggcc aacttacttc
     2581 tgacaacgat cggaggaccg aaggagctaa ccgctttttt gcacaacatg ggggatcatg
     2641 taactcgcct tgatcgttgg gaaccggagc tgaatgaagc cataccaaac gacgagcgtg
     2701 acaccacgat gcctgtagca atggcaacaa cgttgcgcaa actattaact ggcgaactac
     2761 ttactctagc ttcccggcaa caattaatag actggatgga ggcggataaa gttgcaggac
     2821 cacttctgcg ctcggccctt ccggctggct ggtttattgc tgataaatct ggagccggtg
     2881 agcgtggatc acgcggtatc attgcagcac tggggccaga tggtaagccc tcccgtatcg
     2941 tagttatcta cacgacgggg agtcaggcaa ctatggatga acgaaataga cagatcgctg
     3001 agataggtgc ctcactgatt aagcattggt aactgtcaga ccaagtttac gagctcgctt
     3061 ggactcctgt tgatagatcc agtaatgaca tcagaactcc atctggattt gttcagaacg
     3121 ctcggttgcc gccgggcgtt ttttattggt gagaatccaa gcactaggga cagtaagacg
     3181 ggtaagcctg ttgatgatac cgctgcctta ctgggtgcat tagccagtct gaatgacctg
     3241 tcacgggata atccgaagtg gtcagactgg aaaatcagag ggcaggaact gctgaacagc
     3301 aaaaagtcag atagcaccac atagcagacc agccataaaa cgccctgaga agcccgtgac
     3361 gggcttttct tgtattatgg gtagtttcct tgcatgaatc cataaaaggc gcctgtagtg
     3421 ccatttaccc acattcactg ccagagccgt gagcgcagcg aactgaatgt cacgaaaaag
     3481 acagcgactc aggtgcctga tggtcggaga caaaaggaat attaagcgat ttgcacgagc
     3541 ttgcgagggt gctacttaag cctttagggt tttaaggtct gttttgtaga ggagcaaaca
     3601 gcgtttgcga catccttttg taatactgcg gaactgacta aagtagtgag ttatacacag
     3661 ggctgggatc tattcttttt atcttttttt attctttctt tattctataa attataacca
     3721 cttgaatata aacaaaaaaa acacacaaag gtctagcgga atttacagag ggtctagcag
     3781 aatttacaag ttttccagca aaggtctagc agaatttaca gatacccaca actcaaagga
     3841 aaaggacatg taattatcat tgactagccc atctcaattg gtatagtgat taaaatcacc
     3901 tagaccaatt gagatgtatg tctgaattag ttgttttcaa agcaaatgaa ctagcgatta
     3961 gtcgctatga cttaacggag catgaaacca agctaatttt atgctgtgtg gcactactca
     4021 accccactat tgaaaaccct acaaggaaag aacggacggt atcgttcact tataaccaat
     4081 acgctcagat gatgaacatc agtagggaaa atgcttatgg tgtattagct aaagcaacca
     4141 gagagctgat gacgagaact gtggaaatca tgaatccttt ggttaaaggc tttgagattt
     4201 tccagtggac aaactatgcc aagttctcaa gcgaaaaatt agaattagtt tttagtgaag
     4261 agatattgcc ttatcttttc cagttaaaaa aattcataaa atataatctg gaacatgtta
     4321 agtcttttga aaacaaatac tctatgagga tttatgagtg gttattaaaa gaactaacac
     4381 aaaagaaaac tcacaaggca aatatagaga ttagccttga tgaatttaag ttcatgttaa
     4441 tgcttgaaaa taactaccat gagtttaaaa ggcttaacca atgggttttg aaaccaataa
     4501 gtaaagattt aaacacttac agcaatatga aattggtggt tgataagcga ggccgcccga
     4561 ctgatacgtt gattttccaa gttgaactag atagacaaat ggatctcgta accgaacttg
     4621 agaacaacca gataaaaatg aatggtgaca aaataccaac aaccattaca tcagattcct
     4681 acctacataa cggactaaga aaaacactac acgatgcttt aactgcaaaa attcagctca
     4741 ccagttttga ggcaaaattt ttgagtgaca tgcaaagtaa gtatgatctc aatggttcgt
     4801 tctcatggct cacgcaaaaa caacgaacca cactagagaa catactggct aaatacggaa
     4861 ggatctgagg ttcttatggc tcttgtatct atcagtgaag catcaagact aacaaacaaa
     4921 agtagaacaa ctgttcaccg ttacatatca aagggaaaac tgtccatatg cacagatgaa
     4981 aacggtgtaa aaaagataga tacatcagag cttttacgag tttttggtgc attcaaagct
     5041 gttcaccatg aacagatcga caatgtaacc tcgagattac cgcctttgag tgagctgata
     5101 ccgctcgccg cagccgaacg accgagcgca gcgagtcagt gagcgaggaa gcctgcataa
     5161 cgcgaagtaa tcttttcggt tttaaagaaa aagggcaggg tggtgacacc ttgccgaagc
     5221 ggcatgcatt tacgttgaca ccatcgaatg gtgcaaaacc tttcgcggta tggcatgata
     5281 gcgcccggaa gagagtcaat tcggggtggt gaatgtgaaa ccagtaacgt tatacgatgt
     5341 cgcagagtat gccggtgtct cttatcagac cgtttcccgc gtggtgaacc aggccagcca
     5401 cgtttctgcg aaaacgcggg aaaaagtgga agcggcgatg gcggagctga attacattcc
     5461 caaccgcgtg gcacaacaac tggcgggcaa acagtcgttg ctgattggcg ttgccacctc
     5521 cagtctggcc ctgcacgcgc cgtcgcaaat tgtcgcggcg attaaatctc gcgccgatca
     5581 actgggtgcc agcgtggtgg tgtcgatggt agaacgaagc ggcgtcgaag cctgtaaagc
     5641 ggcggtgcac aatcttctcg cgcaacgcgt cagtgggctg atcattaact atccgctgga
     5701 tgaccaggat gccattgctg tggaagctgc ctgcactaat gttccggcgt tatttcttga
     5761 tgtctctgac cagacaccca tcaacagtat tattttctcc catgaagacg gtacgcgact
     5821 gggcgtggag catctggtcg cattgggtca ccagcaaatc gcgctgttag cgggcccatt
     5881 aagttctgtc tcggcgcgtc tgcgtctggc tggctggcat aaatatctca ctcgcaatca
     5941 aattcagccg atagcggaac gggaaggcga ctggagtgcc atgtccggtt ttcaacaaac
     6001 catgcaaatg ctgaatgagg gcatcgttcc cactgcgatg ctggttgcca acgatcagat
     6061 ggcgctgggc gcaatgcgcg ccattaccga gtccgggctg cgcgttggtg cggatatctc
     6121 ggtagtggga tacgacgata ccgaagacag ctcatgttat atcccgccgt taaccaccat
     6181 caaacaggat tttcgcctgc tggggcaaac cagcgtggac cgcttgctgc aactctctca
     6241 gggccaggcg gtgaagggca atcagctgtt gcccgtctca ctggtgaaaa gaaaaaccac
     6301 cctggcgccc aatacgcaaa ccgcctctcc ccgcgcgttg gccgattcat taatgcagct
     6361 ggcacgacag gtttcccgac tggaaagcgg gcagtgaggc atcaaataaa acgaaaggct
     6421 cagtcgaaag actgggcctt tcgttttatc tgttgtttgt cggtgaacgc tctcctgagt
     6481 aggacaaatc cgccgcccta gacctagggc gttcggctgc ggcgagcggt atcagctcac
     6541 tcaaaggcgg taatacgtaa atcactgcat aattcgtgtc gctcaaggcg cactcccgtt
     6601 ctggataatg ttttttgcgc cgacatcata acggttctgg ctaatattct gaaatgagct
     6661 gttgacaatt aatcatcggc tcgtataatg tgtggaattg tgagcggata acaatttcac
     6721 acagggccca agttcactta aaaaggagat caacaatgaa agcaattttc gtactgaaac
     6781 atcttaatca tgctatggag tatttctgat gaaagctact aaactggtac tgggcgcggt
     6841 aatcctgggt tctactctgc tggcaggttg ctccagcaac gctaaaatcg atcagggaat
     6901 taacccgtat gttggctttg aaatgggtta cgactggtta ggtcgtatgc cgtacaaagg
     6961 cagcgttgaa aac
//
