LOCUS       sequence-336783-        6973 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Encodes LacI-controlled expression of surface-displayed cecropin P1 
            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: pKB231
FEATURES             Location/Qualifiers
     source          1..6973
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     terminator      complement(21..107)
                     /gene="Escherichia coli rrnB"
                     /label=rrnB T1 terminator
                     /note="transcription terminator T1 from the E. coli rrnB 
                     gene"
     CDS             complement(113..1195)
                     /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     1157..1176
                     /label=LacI-R
                     /note="LacI, reverse primer"
     promoter        complement(1196..1273)
                     /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(1363..1380)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     CDS             complement(1642..2592)
                     /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      2640..2862
                     /label=pSC101 ori
                     /note="low-copy replication origin that requires the Rep101
                     protein"
     terminator      complement(3354..3448)
                     /label=lambda t0 terminator
                     /note="transcription terminator from phage lambda"
     CDS             complement(join(3478..4269,4270..4338))
                     /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     4101..4120
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(4339..4443)
                     /gene="bla"
                     /label=AmpR promoter
     primer_bind     4508..4526
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     RBS             4724..4735
                     /note="strong bacterial ribosome binding site (Elowitz and 
                     Leibler, 2000)"
     terminator      5461..5532
                     /label=rrnB T1 terminator
                     /note="transcription terminator T1 from the E. coli rrnB 
                     gene"
     terminator      5548..5575
                     /label=T7Te terminator
                     /note="phage T7 early transcription terminator"
     primer_bind     complement(6758..6780)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    6769..6785
                     /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(6793..6799,6800..6815,6816..6821))
                     /label=tac promoter
                     /note="strong E. coli promoter; hybrid between the trp and 
                     lac UV5 promoters"
ORIGIN
        1 ccctaggtct agggcggcgg atttgtccta ctcaggagag cgttcaccga caaacaacag
       61 ataaaacgaa aggcccagtc tttcgactga gcctttcgtt ttatttgatg cctcactgcc
      121 cgctttccag tcgggaaacc tgtcgtgcca gctgcattaa tgaatcggcc aacgcgcggg
      181 gagaggcggt ttgcgtattg ggcgccaggg tggtttttct tttcaccagt gagacgggca
      241 acagctgatt gcccttcacc gcctggccct gagagagttg cagcaagcgg tccacgctgg
      301 tttgccccag caggcgaaaa tcctgtttga tggtggttaa cggcgggata taacatgagc
      361 tgtcttcggt atcgtcgtat cccactaccg agatatccgc accaacgcgc agcccggact
      421 cggtaatggc gcgcattgcg cccagcgcca tctgatcgtt ggcaaccagc atcgcagtgg
      481 gaacgatgcc ctcattcagc atttgcatgg tttgttgaaa accggacatg gcactccagt
      541 cgccttcccg ttccgctatc ggctgaattt gattgcgagt gagatattta tgccagccag
      601 ccagacgcag acgcgccgag acagaactta atgggcccgc taacagcgcg atttgctggt
      661 gacccaatgc gaccagatgc tccacgccca gtcgcgtacc gtcttcatgg gagaaaataa
      721 tactgttgat gggtgtctgg tcagagacat caagaaataa cgccggaaca ttagtgcagg
      781 cagcttccac agcaatggca tcctggtcat ccagcggata gttaatgatc agcccactga
      841 cgcgttgcgc gagaagattg tgcaccgccg ctttacaggc ttcgacgccg cttcgttcta
      901 ccatcgacac caccacgctg gcacccagtt gatcggcgcg agatttaatc gccgcgacaa
      961 tttgcgacgg cgcgtgcagg gccagactgg aggtggcaac gccaatcagc aacgactgtt
     1021 tgcccgccag ttgttgtgcc acgcggttgg gaatgtaatt cagctccgcc atcgccgctt
     1081 ccactttttc ccgcgttttc gcagaaacgt ggctggcctg gttcaccacg cgggaaacgg
     1141 tctgataaga gacaccggca tactctgcga catcgtataa cgttactggt ttcacattca
     1201 ccaccctgaa ttgactctct tccgggcgct atcatgccat accgcgaaag gttttgcacc
     1261 attcgatggt gtcaacgtaa atgcatgccg cttcggcaag gtgtcaccac cctgcccttt
     1321 ttctttaaaa ccgaaaagat tacttcgcgt tatgcaggct tcctcgctca ctgactcgct
     1381 gcgctcggtc gttcggctgc ggcgagcggt atcagctcac tcaaaggcgg taatctcgag
     1441 gttacattgt cgatctgttc atggtgaaca gctttgaatg caccaaaaac tcgtaaaagc
     1501 tctgatgtat ctatcttttt tacaccgttt tcatctgtgc atatggacag ttttcccttt
     1561 gatatgtaac ggtgaacagt tgttctactt ttgtttgtta gtcttgatgc ttcactgata
     1621 gatacaagag ccataagaac ctcagatcct tccgtattta gccagtatgt tctctagtgt
     1681 ggttcgttgt ttttgcgtga gccatgagaa cgaaccattg agatcatact tactttgcat
     1741 gtcactcaaa aattttgcct caaaactggt gagctgaatt tttgcagtta aagcatcgtg
     1801 tagtgttttt cttagtccgt tatgtaggta ggaatctgat gtaatggttg ttggtatttt
     1861 gtcaccattc atttttatct ggttgttctc aagttcggtt acgagatcca tttgtctatc
     1921 tagttcaact tggaaaatca acgtatcagt cgggcggcct cgcttatcaa ccaccaattt
     1981 catattgctg taagtgttta aatctttact tattggtttc aaaacccatt ggttaagcct
     2041 tttaaactca tggtagttat tttcaagcat taacatgaac ttaaattcat caaggctaat
     2101 ctctatattt gccttgtgag ttttcttttg tgttagttct tttaataacc actcataaat
     2161 cctcatagag tatttgtttt caaaagactt aacatgttcc agattatatt ttatgaattt
     2221 ttttaactgg aaaagataag gcaatatctc ttcactaaaa actaattcta atttttcgct
     2281 tgagaacttg gcatagtttg tccactggaa aatctcaaag cctttaacca aaggattcat
     2341 gatttccaca gttctcgtca tcagctctct ggttgcttta gctaatacac cataagcatt
     2401 ttccctactg atgttcatca tctgagcgta ttggttataa gtgaacgata ccgtccgttc
     2461 tttccttgta gggttttcaa tagtggggtt gagtagtgcc acacagcata aaattagctt
     2521 ggtttcatgc tccgttaagt catagcgact aatcgctagt tcatttgctt tgaaaacaac
     2581 taattcagac atacatctca attggtctag gtgattttaa tcactatacc aattgagatg
     2641 ggctagtcaa tgataattac atgtcctttt cctttgagtt gtgggtatct gtaaattctg
     2701 ctagaccttt gctggaaaac ttgtaaattc tgctagaccc tctgtaaatt ccgctagacc
     2761 tttgtgtgtt ttttttgttt atattcaagt ggttataatt tatagaataa agaaagaata
     2821 aaaaaagata aaaagaatag atcccagccc tgtgtataac tcactacttt agtcagttcc
     2881 gcagtattac aaaaggatgt cgcaaacgct gtttgctcct ctacaaaaca gaccttaaaa
     2941 ccctaaaggc ttaagtagca ccctcgcaag ctcgtgcaaa tcgcttaata ttccttttgt
     3001 ctccgaccat caggcacctg agtcgctgcc tttttcgtga cattcagttc gctgcgctca
     3061 cggctctggc agtgaatgtg ggtaaatggc actacaggcg ccttttatgg attcatgcaa
     3121 ggaaactacc cataatacaa gaaaagcccg tcacgggctt ctcagggcgt tttatggctg
     3181 gtctgctatg tggtgctatc tgactttttg ctgttcagca gttcctgccc tctgattttc
     3241 cagtctgacc acttcggatt atcccgtgac aggtcattca gactggctaa tgcacccagt
     3301 aaggcagcgg tatcatcaac aggcttaccc gtcttactgt ccctagtgct tggattctca
     3361 ccaataaaaa acgcccggcg gcaaccgagc gttctgaaca aatccagatg gagttctgat
     3421 gtcattactg gatctatcaa caggagtcca agcgagctcg taaacttggt ctgacagtta
     3481 ccaatgctta atcagtgagg cacctatctc agcgatctgt ctatttcgtt catccatagt
     3541 tgcctgactc cccgtcgtgt agataactac gatacgggag ggcttaccat ctggccccag
     3601 tgctgcaatg ataccgcgtg atccacgctc accggctcca gatttatcag caataaacca
     3661 gccagccgga agggccgagc gcagaagtgg tcctgcaact ttatccgcct ccatccagtc
     3721 tattaattgt tgccgggaag ctagagtaag tagttcgcca gttaatagtt tgcgcaacgt
     3781 tgttgccatt gctacaggca tcgtggtgtc acgctcgtcg tttggtatgg cttcattcag
     3841 ctccggttcc caacgatcaa ggcgagttac atgatccccc atgttgtgca aaaaagcggt
     3901 tagctccttc ggtcctccga tcgttgtcag aagtaagttg gccgcagtgt tatcactcat
     3961 ggttatggca gcactgcata attctcttac tgtcatgcca tccgtaagat gcttttctgt
     4021 tactggtgag tactcaacca agtcattctg agaatagtgt atgcggcgac cgagttgctc
     4081 ttgcccggcg tcaatacggt ataataccgc gccacatagc agaactttaa aagtgctcat
     4141 cattggaaaa cgttcttcgg ggcgaaaact ctcaaggatc ttaccgctgt tgagatccag
     4201 ttcgatgtaa cccactcgtg cacccaactg atcttcagca tcttttactt tcaccagcgt
     4261 ttctgggtga gcaaaaacgg gaaggcaaaa tgccgcaaaa aagtgaataa gggcgacact
     4321 gaaatgttga atactcatac tcttcctttt tcaatattat tgaagcattt atcagggtta
     4381 ttgtctcatg agcggataca tattttaatg tatttagaaa aataaacaaa taggggttcc
     4441 gcgcacattt cccatggtgc cacctgacgt ctaataaacc attattatca tgacattaac
     4501 ctataaaaat aggcgtatca cgaggcagaa tttttatcgc ttccgcgttc cgtctactcc
     4561 ctattacgct ccggaatctc cgacgcctac ggccgacgtc ggataccgcc attgataaac
     4621 tgtttaacaa catcgtctgt acagaccttc ttcgttgcct ttacgtttaa ctcaatcagg
     4681 ctaccgtctc ggttataagt ctatcgagta gtagagccgt agtaaagagg agaaatacta
     4741 gatggtttcg aaaggtgagg aggataacat ggcgtcgctg cctgctacac atgaactgca
     4801 tattttcggc agcatcaacg gagttgattt cgacatggtg ggccaaggca ccggcaaccc
     4861 gaacgacggc tatgaagaat tgaatttgaa atctaccaag ggcgacttgc aatttagtcc
     4921 gtggattctg gtaccgcata ttggttatgg ttttcatcag tatctgccct acccagacgg
     4981 aatgagccca tttcaggcgg ctatggtgga tggcagtggc tatcaggtcc atcgcaccat
     5041 gcaattcgag gatggggcga gcctgaccgt gaattatcgg tacacttacg aaggttcaca
     5101 tattaaaggt gaagcacaag ttaaaggcac cggcttcccg gcggatggcc cagtgatgac
     5161 caacagcctg acggcggcgg actggtgtcg tagcaaaaag acgtatccca atgataaaac
     5221 aattatttct acctttaaat ggagttacac cacgggtaac ggtaaacgct accgtagcac
     5281 ggctcgcacc acatacacct ttgcgaaacc catggccgcg aactatctga aaaatcagcc
     5341 gatgtatgtg tttcgcaaaa cggaactgaa acattcgaaa actgaactca atttcaaaga
     5401 gtggcaaaaa gccttcaccg acgtaatggg catggatgaa ctgtacaaat gaccaggcat
     5461 caaataaaac gaaaggctca gtcgaaagac tgggcctttc gttttatctg ttgtttgtcg
     5521 gtgaacgctc tctactagag tcacactggc tcaccttcgg gtgggccttt ctgcgtttat
     5581 agtttcactt ctgagttcgg catggggtca ggtgggacca ccgcgctact gccgccaggc
     5641 agctgtatca tctgcgtact ccattagtgt gacgacctat gaggatggcg gggtattaac
     5701 tgtaacgcaa gatacttctc tgcaagatgg ttgtttaatt tataatgtga aactgcgcgg
     5761 cgttaatttt ccctcaaatg gtcccgtaat gcaaaagaag accttagggt gggaagcttc
     5821 tacggaaacg ttgtatcctg cagatggcgg tcttgagggt cgttgtgata tggcgttgaa
     5881 acttaacgag gaccaccttg aatagcaata gcaatacctt cagaaatacg ttttttagca
     5941 gaattttcaa gttttttagc agttttagaa agccaagagg ttcctccgat acccgcagct
     6001 ggagcctcag cggcatctgc aggagcggct tccgcagccg gtgcttcact cgctggtgcc
     6061 tccgctgctg gtgtagcttc cgcagctggt gcagcaggtt cctgactgat gccagcggct
     6121 ggggcttcgg cagcatccgc tggggcagct tctgctgcag gcgcttcgct agctggagct
     6181 tcagcagcag gagtcgcctc tgcggcgggc gctgcaggct cttgactggt accaataccg
     6241 ttgtccggac gagtgccgat ggtgtgtgcg tcaccgatgt tgttggtcca ctggtattcc
     6301 agacgggtag cgatttcagg agtgatcgcg tactcaacac cgccagcgaa gaccggagaa
     6361 acgccggtgt cgtggttttt accataaacg ttggatttag tgtctgcacg ccataccatg
     6421 ccacccagac gagtgtagat gtccaggtcg tcagtgattg ggtaacccag tttagcggtc
     6481 agttgaacgc cctgagcttt gtatgcaccg ttttcaacgc tgcctttgta cggcatacga
     6541 cctaaccagt cgtaacccat ttcaaagcca acatacgggt taattccctg atcgatttta
     6601 gcgttgctgg agcaacctgc cagcagagta gaacccagga ttaccgcgcc cagtaccagt
     6661 ttagtagctt tcatcagaaa tactccatag catgattaag atgtttcagt acgaaaattg
     6721 ctttcattgt tgatctcctt tttaagtgaa cttgggccct gtgtgaaatt gttatccgct
     6781 cacaattcca cacattatac gagccgatga ttaattgtca acagctcatt tcagaatatt
     6841 agccagaacc gttatgatgt cggcgcaaaa aacattatcc agaacgggag tgcgccttga
     6901 gcgacacgaa ttatgcagtg atttacgtat taccgccttt gagtgagctg ataccgctcg
     6961 ccgcagccga acg
//
