LOCUS       sequence-335965-        6973 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Encodes LacI-controlled expression of surface-displayed K5L7 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: pKB253
FEATURES             Location/Qualifiers
     source          1..6973
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     973..992
                     /label=LacI-R
                     /note="LacI, reverse primer"
     promoter        complement(1012..1089)
                     /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(1179..1196)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     CDS             complement(1458..2408)
                     /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      2456..2678
                     /label=pSC101 ori
                     /note="low-copy replication origin that requires the Rep101
                     protein"
     terminator      complement(3170..3264)
                     /label=lambda t0 terminator
                     /note="transcription terminator from phage lambda"
     CDS             complement(join(3294..4085,4086..4154))
                     /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     3917..3936
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(4155..4259)
                     /gene="bla"
                     /label=AmpR promoter
     primer_bind     4324..4342
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     RBS             4540..4551
                     /note="strong bacterial ribosome binding site (Elowitz and 
                     Leibler, 2000)"
     terminator      5277..5348
                     /label=rrnB T1 terminator
                     /note="transcription terminator T1 from the E. coli rrnB 
                     gene"
     terminator      5364..5391
                     /label=T7Te terminator
                     /note="phage T7 early transcription terminator"
     primer_bind     complement(6574..6596)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    6585..6601
                     /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(6609..6615,6616..6631,6632..6637))
                     /label=tac promoter
                     /note="strong E. coli promoter; hybrid between the trp and 
                     lac UV5 promoters"
     terminator      complement(6810..6896)
                     /gene="Escherichia coli rrnB"
                     /label=rrnB T1 terminator
                     /note="transcription terminator T1 from the E. coli rrnB 
                     gene"
     CDS             complement(join(6902..6973,1..1011))
                     /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 ggcggtttgc gtattgggcg ccagggtggt ttttcttttc accagtgaga cgggcaacag
       61 ctgattgccc ttcaccgcct ggccctgaga gagttgcagc aagcggtcca cgctggtttg
      121 ccccagcagg cgaaaatcct gtttgatggt ggttaacggc gggatataac atgagctgtc
      181 ttcggtatcg tcgtatccca ctaccgagat atccgcacca acgcgcagcc cggactcggt
      241 aatggcgcgc attgcgccca gcgccatctg atcgttggca accagcatcg cagtgggaac
      301 gatgccctca ttcagcattt gcatggtttg ttgaaaaccg gacatggcac tccagtcgcc
      361 ttcccgttcc gctatcggct gaatttgatt gcgagtgaga tatttatgcc agccagccag
      421 acgcagacgc gccgagacag aacttaatgg gcccgctaac agcgcgattt gctggtgacc
      481 caatgcgacc agatgctcca cgcccagtcg cgtaccgtct tcatgggaga aaataatact
      541 gttgatgggt gtctggtcag agacatcaag aaataacgcc ggaacattag tgcaggcagc
      601 ttccacagca atggcatcct ggtcatccag cggatagtta atgatcagcc cactgacgcg
      661 ttgcgcgaga agattgtgca ccgccgcttt acaggcttcg acgccgcttc gttctaccat
      721 cgacaccacc acgctggcac ccagttgatc ggcgcgagat ttaatcgccg cgacaatttg
      781 cgacggcgcg tgcagggcca gactggaggt ggcaacgcca atcagcaacg actgtttgcc
      841 cgccagttgt tgtgccacgc ggttgggaat gtaattcagc tccgccatcg ccgcttccac
      901 tttttcccgc gttttcgcag aaacgtggct ggcctggttc accacgcggg aaacggtctg
      961 ataagagaca ccggcatact ctgcgacatc gtataacgtt actggtttca cattcaccac
     1021 cctgaattga ctctcttccg ggcgctatca tgccataccg cgaaaggttt tgcaccattc
     1081 gatggtgtca acgtaaatgc atgccgcttc ggcaaggtgt caccaccctg ccctttttct
     1141 ttaaaaccga aaagattact tcgcgttatg caggcttcct cgctcactga ctcgctgcgc
     1201 tcggtcgttc ggctgcggcg agcggtatca gctcactcaa aggcggtaat ctcgaggtta
     1261 cattgtcgat ctgttcatgg tgaacagctt tgaatgcacc aaaaactcgt aaaagctctg
     1321 atgtatctat cttttttaca ccgttttcat ctgtgcatat ggacagtttt ccctttgata
     1381 tgtaacggtg aacagttgtt ctacttttgt ttgttagtct tgatgcttca ctgatagata
     1441 caagagccat aagaacctca gatccttccg tatttagcca gtatgttctc tagtgtggtt
     1501 cgttgttttt gcgtgagcca tgagaacgaa ccattgagat catacttact ttgcatgtca
     1561 ctcaaaaatt ttgcctcaaa actggtgagc tgaatttttg cagttaaagc atcgtgtagt
     1621 gtttttctta gtccgttatg taggtaggaa tctgatgtaa tggttgttgg tattttgtca
     1681 ccattcattt ttatctggtt gttctcaagt tcggttacga gatccatttg tctatctagt
     1741 tcaacttgga aaatcaacgt atcagtcggg cggcctcgct tatcaaccac caatttcata
     1801 ttgctgtaag tgtttaaatc tttacttatt ggtttcaaaa cccattggtt aagcctttta
     1861 aactcatggt agttattttc aagcattaac atgaacttaa attcatcaag gctaatctct
     1921 atatttgcct tgtgagtttt cttttgtgtt agttctttta ataaccactc ataaatcctc
     1981 atagagtatt tgttttcaaa agacttaaca tgttccagat tatattttat gaattttttt
     2041 aactggaaaa gataaggcaa tatctcttca ctaaaaacta attctaattt ttcgcttgag
     2101 aacttggcat agtttgtcca ctggaaaatc tcaaagcctt taaccaaagg attcatgatt
     2161 tccacagttc tcgtcatcag ctctctggtt gctttagcta atacaccata agcattttcc
     2221 ctactgatgt tcatcatctg agcgtattgg ttataagtga acgataccgt ccgttctttc
     2281 cttgtagggt tttcaatagt ggggttgagt agtgccacac agcataaaat tagcttggtt
     2341 tcatgctccg ttaagtcata gcgactaatc gctagttcat ttgctttgaa aacaactaat
     2401 tcagacatac atctcaattg gtctaggtga ttttaatcac tataccaatt gagatgggct
     2461 agtcaatgat aattacatgt ccttttcctt tgagttgtgg gtatctgtaa attctgctag
     2521 acctttgctg gaaaacttgt aaattctgct agaccctctg taaattccgc tagacctttg
     2581 tgtgtttttt ttgtttatat tcaagtggtt ataatttata gaataaagaa agaataaaaa
     2641 aagataaaaa gaatagatcc cagccctgtg tataactcac tactttagtc agttccgcag
     2701 tattacaaaa ggatgtcgca aacgctgttt gctcctctac aaaacagacc ttaaaaccct
     2761 aaaggcttaa gtagcaccct cgcaagctcg tgcaaatcgc ttaatattcc ttttgtctcc
     2821 gaccatcagg cacctgagtc gctgcctttt tcgtgacatt cagttcgctg cgctcacggc
     2881 tctggcagtg aatgtgggta aatggcacta caggcgcctt ttatggattc atgcaaggaa
     2941 actacccata atacaagaaa agcccgtcac gggcttctca gggcgtttta tggctggtct
     3001 gctatgtggt gctatctgac tttttgctgt tcagcagttc ctgccctctg attttccagt
     3061 ctgaccactt cggattatcc cgtgacaggt cattcagact ggctaatgca cccagtaagg
     3121 cagcggtatc atcaacaggc ttacccgtct tactgtccct agtgcttgga ttctcaccaa
     3181 taaaaaacgc ccggcggcaa ccgagcgttc tgaacaaatc cagatggagt tctgatgtca
     3241 ttactggatc tatcaacagg agtccaagcg agctcgtaaa cttggtctga cagttaccaa
     3301 tgcttaatca gtgaggcacc tatctcagcg atctgtctat ttcgttcatc catagttgcc
     3361 tgactccccg tcgtgtagat aactacgata cgggagggct taccatctgg ccccagtgct
     3421 gcaatgatac cgcgtgatcc acgctcaccg gctccagatt tatcagcaat aaaccagcca
     3481 gccggaaggg ccgagcgcag aagtggtcct gcaactttat ccgcctccat ccagtctatt
     3541 aattgttgcc gggaagctag agtaagtagt tcgccagtta atagtttgcg caacgttgtt
     3601 gccattgcta caggcatcgt ggtgtcacgc tcgtcgtttg gtatggcttc attcagctcc
     3661 ggttcccaac gatcaaggcg agttacatga tcccccatgt tgtgcaaaaa agcggttagc
     3721 tccttcggtc ctccgatcgt tgtcagaagt aagttggccg cagtgttatc actcatggtt
     3781 atggcagcac tgcataattc tcttactgtc atgccatccg taagatgctt ttctgttact
     3841 ggtgagtact caaccaagtc attctgagaa tagtgtatgc ggcgaccgag ttgctcttgc
     3901 ccggcgtcaa tacggtataa taccgcgcca catagcagaa ctttaaaagt gctcatcatt
     3961 ggaaaacgtt cttcggggcg aaaactctca aggatcttac cgctgttgag atccagttcg
     4021 atgtaaccca ctcgtgcacc caactgatct tcagcatctt ttactttcac cagcgtttct
     4081 gggtgagcaa aaacgggaag gcaaaatgcc gcaaaaaagt gaataagggc gacactgaaa
     4141 tgttgaatac tcatactctt cctttttcaa tattattgaa gcatttatca gggttattgt
     4201 ctcatgagcg gatacatatt ttaatgtatt tagaaaaata aacaaatagg ggttccgcgc
     4261 acatttccca tggtgccacc tgacgtctaa taaaccatta ttatcatgac attaacctat
     4321 aaaaataggc gtatcacgag gcagaatttt tatcgcttcc gcgttccgtc tactccctat
     4381 tacgctccgg aatctccgac gcctacggcc gacgtcggat accgccattg ataaactgtt
     4441 taacaacatc gtctgtacag accttcttcg ttgcctttac gtttaactca atcaggctac
     4501 cgtctcggtt ataagtctat cgagtagtag agccgtagta aagaggagaa atactagatg
     4561 gtttcgaaag gtgaggagga taacatggcg tcgctgcctg ctacacatga actgcatatt
     4621 ttcggcagca tcaacggagt tgatttcgac atggtgggcc aaggcaccgg caacccgaac
     4681 gacggctatg aagaattgaa tttgaaatct accaagggcg acttgcaatt tagtccgtgg
     4741 attctggtac cgcatattgg ttatggtttt catcagtatc tgccctaccc agacggaatg
     4801 agcccatttc aggcggctat ggtggatggc agtggctatc aggtccatcg caccatgcaa
     4861 ttcgaggatg gggcgagcct gaccgtgaat tatcggtaca cttacgaagg ttcacatatt
     4921 aaaggtgaag cacaagttaa aggcaccggc ttcccggcgg atggcccagt gatgaccaac
     4981 agcctgacgg cggcggactg gtgtcgtagc aaaaagacgt atcccaatga taaaacaatt
     5041 atttctacct ttaaatggag ttacaccacg ggtaacggta aacgctaccg tagcacggct
     5101 cgcaccacat acacctttgc gaaacccatg gccgcgaact atctgaaaaa tcagccgatg
     5161 tatgtgtttc gcaaaacgga actgaaacat tcgaaaactg aactcaattt caaagagtgg
     5221 caaaaagcct tcaccgacgt aatgggcatg gatgaactgt acaaatgacc aggcatcaaa
     5281 taaaacgaaa ggctcagtcg aaagactggg cctttcgttt tatctgttgt ttgtcggtga
     5341 acgctctcta ctagagtcac actggctcac cttcgggtgg gcctttctgc gtttatagtt
     5401 tcacttctga gttcggcatg gggtcaggtg ggaccaccgc gctactgccg ccaggcagct
     5461 gtatcatctg cgtactccat tagtgtgacg acctatgagg atggcggggt attaactgta
     5521 acgcaagata cttctctgca agatggttgt ttaatttata atgtgaaact gcgcggcgtt
     5581 aattttccct caaatggtcc cgtaatgcaa aagaagacct tagggtggga agcttctacg
     5641 gaaacgttgt atcctgcaga tggcggtctt gagggtcgtt gtgatatggc gttgaaactg
     5701 gtcggcggtg gacatttgca ttgtaatctg aaaacgactt atcgtagcaa gaagcttact
     5761 ttaaaagttt caataacaac ttcaacaatt ttttggttcc tccgataccc gcagctggag
     5821 cctcagcggc atctgcagga gcggcttccg cagccggtgc ttcactcgct ggtgcctccg
     5881 ctgctggtgt agcttccgca gctggtgcag caggttcctg actgatgcca gcggctgggg
     5941 cttcggcagc atccgctggg gcagcttctg ctgcaggcgc ttcgctagct ggagcttcag
     6001 cagcaggagt cgcctctgcg gcgggcgctg caggctcttg actggtacca ataccgttgt
     6061 ccggacgagt gccgatggtg tgtgcgtcac cgatgttgtt ggtccactgg tattccagac
     6121 gggtagcgat ttcaggagtg atcgcgtact caacaccgcc agcgaagacc ggagaaacgc
     6181 cggtgtcgtg gtttttacca taaacgttgg atttagtgtc tgcacgccat accatgccac
     6241 ccagacgagt gtagatgtcc aggtcgtcag tgattgggta acccagttta gcggtcagtt
     6301 gaacgccctg agctttgtat gcaccgtttt caacgctgcc tttgtacggc atacgaccta
     6361 accagtcgta acccatttca aagccaacat acgggttaat tccctgatcg attttagcgt
     6421 tgctggagca acctgccagc agagtagaac ccaggattac cgcgcccagt accagtttag
     6481 tagctttcat cagaaatact ccatagcatg attaagatgt ttcagtacga aaattgcttt
     6541 cattgttgat ctccttttta agtgaacttg ggccctgtgt gaaattgtta tccgctcaca
     6601 attccacaca ttatacgagc cgatgattaa ttgtcaacag ctcatttcag aatattagcc
     6661 agaaccgtta tgatgtcggc gcaaaaaaca ttatccagaa cgggagtgcg ccttgagcga
     6721 cacgaattat gcagtgattt acgtattacc gcctttgagt gagctgatac cgctcgccgc
     6781 agccgaacgc cctaggtcta gggcggcgga tttgtcctac tcaggagagc gttcaccgac
     6841 aaacaacaga taaaacgaaa ggcccagtct ttcgactgag cctttcgttt tatttgatgc
     6901 ctcactgccc gctttccagt cgggaaacct gtcgtgccag ctgcattaat gaatcggcca
     6961 acgcgcgggg aga
//
