LOCUS       sequence-339960-        6973 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Encodes LacI-controlled expression of surface-displayed protegrin-1 
            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: pKB230
FEATURES             Location/Qualifiers
     source          1..6973
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     969..988
                     /label=LacI-R
                     /note="LacI, reverse primer"
     promoter        complement(1008..1085)
                     /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(1175..1192)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     CDS             complement(1454..2404)
                     /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      2452..2674
                     /label=pSC101 ori
                     /note="low-copy replication origin that requires the Rep101
                     protein"
     terminator      complement(3166..3260)
                     /label=lambda t0 terminator
                     /note="transcription terminator from phage lambda"
     CDS             complement(join(3290..4081,4082..4150))
                     /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     3913..3932
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(4151..4255)
                     /gene="bla"
                     /label=AmpR promoter
     primer_bind     4320..4338
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     RBS             4536..4547
                     /note="strong bacterial ribosome binding site (Elowitz and 
                     Leibler, 2000)"
     terminator      5273..5344
                     /label=rrnB T1 terminator
                     /note="transcription terminator T1 from the E. coli rrnB 
                     gene"
     terminator      5360..5387
                     /label=T7Te terminator
                     /note="phage T7 early transcription terminator"
     primer_bind     complement(6570..6592)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    6581..6597
                     /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(6605..6611,6612..6627,6628..6633))
                     /label=tac promoter
                     /note="strong E. coli promoter; hybrid between the trp and 
                     lac UV5 promoters"
     terminator      complement(6806..6892)
                     /gene="Escherichia coli rrnB"
                     /label=rrnB T1 terminator
                     /note="transcription terminator T1 from the E. coli rrnB 
                     gene"
     CDS             complement(join(6898..6973,1..1007))
                     /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 gtttgcgtat tgggcgccag ggtggttttt cttttcacca gtgagacggg caacagctga
       61 ttgcccttca ccgcctggcc ctgagagagt tgcagcaagc ggtccacgct ggtttgcccc
      121 agcaggcgaa aatcctgttt gatggtggtt aacggcggga tataacatga gctgtcttcg
      181 gtatcgtcgt atcccactac cgagatatcc gcaccaacgc gcagcccgga ctcggtaatg
      241 gcgcgcattg cgcccagcgc catctgatcg ttggcaacca gcatcgcagt gggaacgatg
      301 ccctcattca gcatttgcat ggtttgttga aaaccggaca tggcactcca gtcgccttcc
      361 cgttccgcta tcggctgaat ttgattgcga gtgagatatt tatgccagcc agccagacgc
      421 agacgcgccg agacagaact taatgggccc gctaacagcg cgatttgctg gtgacccaat
      481 gcgaccagat gctccacgcc cagtcgcgta ccgtcttcat gggagaaaat aatactgttg
      541 atgggtgtct ggtcagagac atcaagaaat aacgccggaa cattagtgca ggcagcttcc
      601 acagcaatgg catcctggtc atccagcgga tagttaatga tcagcccact gacgcgttgc
      661 gcgagaagat tgtgcaccgc cgctttacag gcttcgacgc cgcttcgttc taccatcgac
      721 accaccacgc tggcacccag ttgatcggcg cgagatttaa tcgccgcgac aatttgcgac
      781 ggcgcgtgca gggccagact ggaggtggca acgccaatca gcaacgactg tttgcccgcc
      841 agttgttgtg ccacgcggtt gggaatgtaa ttcagctccg ccatcgccgc ttccactttt
      901 tcccgcgttt tcgcagaaac gtggctggcc tggttcacca cgcgggaaac ggtctgataa
      961 gagacaccgg catactctgc gacatcgtat aacgttactg gtttcacatt caccaccctg
     1021 aattgactct cttccgggcg ctatcatgcc ataccgcgaa aggttttgca ccattcgatg
     1081 gtgtcaacgt aaatgcatgc cgcttcggca aggtgtcacc accctgccct ttttctttaa
     1141 aaccgaaaag attacttcgc gttatgcagg cttcctcgct cactgactcg ctgcgctcgg
     1201 tcgttcggct gcggcgagcg gtatcagctc actcaaaggc ggtaatctcg aggttacatt
     1261 gtcgatctgt tcatggtgaa cagctttgaa tgcaccaaaa actcgtaaaa gctctgatgt
     1321 atctatcttt tttacaccgt tttcatctgt gcatatggac agttttccct ttgatatgta
     1381 acggtgaaca gttgttctac ttttgtttgt tagtcttgat gcttcactga tagatacaag
     1441 agccataaga acctcagatc cttccgtatt tagccagtat gttctctagt gtggttcgtt
     1501 gtttttgcgt gagccatgag aacgaaccat tgagatcata cttactttgc atgtcactca
     1561 aaaattttgc ctcaaaactg gtgagctgaa tttttgcagt taaagcatcg tgtagtgttt
     1621 ttcttagtcc gttatgtagg taggaatctg atgtaatggt tgttggtatt ttgtcaccat
     1681 tcatttttat ctggttgttc tcaagttcgg ttacgagatc catttgtcta tctagttcaa
     1741 cttggaaaat caacgtatca gtcgggcggc ctcgcttatc aaccaccaat ttcatattgc
     1801 tgtaagtgtt taaatcttta cttattggtt tcaaaaccca ttggttaagc cttttaaact
     1861 catggtagtt attttcaagc attaacatga acttaaattc atcaaggcta atctctatat
     1921 ttgccttgtg agttttcttt tgtgttagtt cttttaataa ccactcataa atcctcatag
     1981 agtatttgtt ttcaaaagac ttaacatgtt ccagattata ttttatgaat ttttttaact
     2041 ggaaaagata aggcaatatc tcttcactaa aaactaattc taatttttcg cttgagaact
     2101 tggcatagtt tgtccactgg aaaatctcaa agcctttaac caaaggattc atgatttcca
     2161 cagttctcgt catcagctct ctggttgctt tagctaatac accataagca ttttccctac
     2221 tgatgttcat catctgagcg tattggttat aagtgaacga taccgtccgt tctttccttg
     2281 tagggttttc aatagtgggg ttgagtagtg ccacacagca taaaattagc ttggtttcat
     2341 gctccgttaa gtcatagcga ctaatcgcta gttcatttgc tttgaaaaca actaattcag
     2401 acatacatct caattggtct aggtgatttt aatcactata ccaattgaga tgggctagtc
     2461 aatgataatt acatgtcctt ttcctttgag ttgtgggtat ctgtaaattc tgctagacct
     2521 ttgctggaaa acttgtaaat tctgctagac cctctgtaaa ttccgctaga cctttgtgtg
     2581 ttttttttgt ttatattcaa gtggttataa tttatagaat aaagaaagaa taaaaaaaga
     2641 taaaaagaat agatcccagc cctgtgtata actcactact ttagtcagtt ccgcagtatt
     2701 acaaaaggat gtcgcaaacg ctgtttgctc ctctacaaaa cagaccttaa aaccctaaag
     2761 gcttaagtag caccctcgca agctcgtgca aatcgcttaa tattcctttt gtctccgacc
     2821 atcaggcacc tgagtcgctg cctttttcgt gacattcagt tcgctgcgct cacggctctg
     2881 gcagtgaatg tgggtaaatg gcactacagg cgccttttat ggattcatgc aaggaaacta
     2941 cccataatac aagaaaagcc cgtcacgggc ttctcagggc gttttatggc tggtctgcta
     3001 tgtggtgcta tctgactttt tgctgttcag cagttcctgc cctctgattt tccagtctga
     3061 ccacttcgga ttatcccgtg acaggtcatt cagactggct aatgcaccca gtaaggcagc
     3121 ggtatcatca acaggcttac ccgtcttact gtccctagtg cttggattct caccaataaa
     3181 aaacgcccgg cggcaaccga gcgttctgaa caaatccaga tggagttctg atgtcattac
     3241 tggatctatc aacaggagtc caagcgagct cgtaaacttg gtctgacagt taccaatgct
     3301 taatcagtga ggcacctatc tcagcgatct gtctatttcg ttcatccata gttgcctgac
     3361 tccccgtcgt gtagataact acgatacggg agggcttacc atctggcccc agtgctgcaa
     3421 tgataccgcg tgatccacgc tcaccggctc cagatttatc agcaataaac cagccagccg
     3481 gaagggccga gcgcagaagt ggtcctgcaa ctttatccgc ctccatccag tctattaatt
     3541 gttgccggga agctagagta agtagttcgc cagttaatag tttgcgcaac gttgttgcca
     3601 ttgctacagg catcgtggtg tcacgctcgt cgtttggtat ggcttcattc agctccggtt
     3661 cccaacgatc aaggcgagtt acatgatccc ccatgttgtg caaaaaagcg gttagctcct
     3721 tcggtcctcc gatcgttgtc agaagtaagt tggccgcagt gttatcactc atggttatgg
     3781 cagcactgca taattctctt actgtcatgc catccgtaag atgcttttct gttactggtg
     3841 agtactcaac caagtcattc tgagaatagt gtatgcggcg accgagttgc tcttgcccgg
     3901 cgtcaatacg gtataatacc gcgccacata gcagaacttt aaaagtgctc atcattggaa
     3961 aacgttcttc ggggcgaaaa ctctcaagga tcttaccgct gttgagatcc agttcgatgt
     4021 aacccactcg tgcacccaac tgatcttcag catcttttac tttcaccagc gtttctgggt
     4081 gagcaaaaac gggaaggcaa aatgccgcaa aaaagtgaat aagggcgaca ctgaaatgtt
     4141 gaatactcat actcttcctt tttcaatatt attgaagcat ttatcagggt tattgtctca
     4201 tgagcggata catattttaa tgtatttaga aaaataaaca aataggggtt ccgcgcacat
     4261 ttcccatggt gccacctgac gtctaataaa ccattattat catgacatta acctataaaa
     4321 ataggcgtat cacgaggcag aatttttatc gcttccgcgt tccgtctact ccctattacg
     4381 ctccggaatc tccgacgcct acggccgacg tcggataccg ccattgataa actgtttaac
     4441 aacatcgtct gtacagacct tcttcgttgc ctttacgttt aactcaatca ggctaccgtc
     4501 tcggttataa gtctatcgag tagtagagcc gtagtaaaga ggagaaatac tagatggttt
     4561 cgaaaggtga ggaggataac atggcgtcgc tgcctgctac acatgaactg catattttcg
     4621 gcagcatcaa cggagttgat ttcgacatgg tgggccaagg caccggcaac ccgaacgacg
     4681 gctatgaaga attgaatttg aaatctacca agggcgactt gcaatttagt ccgtggattc
     4741 tggtaccgca tattggttat ggttttcatc agtatctgcc ctacccagac ggaatgagcc
     4801 catttcaggc ggctatggtg gatggcagtg gctatcaggt ccatcgcacc atgcaattcg
     4861 aggatggggc gagcctgacc gtgaattatc ggtacactta cgaaggttca catattaaag
     4921 gtgaagcaca agttaaaggc accggcttcc cggcggatgg cccagtgatg accaacagcc
     4981 tgacggcggc ggactggtgt cgtagcaaaa agacgtatcc caatgataaa acaattattt
     5041 ctacctttaa atggagttac accacgggta acggtaaacg ctaccgtagc acggctcgca
     5101 ccacatacac ctttgcgaaa cccatggccg cgaactatct gaaaaatcag ccgatgtatg
     5161 tgtttcgcaa aacggaactg aaacattcga aaactgaact caatttcaaa gagtggcaaa
     5221 aagccttcac cgacgtaatg ggcatggatg aactgtacaa atgaccaggc atcaaataaa
     5281 acgaaaggct cagtcgaaag actgggcctt tcgttttatc tgttgtttgt cggtgaacgc
     5341 tctctactag agtcacactg gctcaccttc gggtgggcct ttctgcgttt atagtttcac
     5401 ttctgagttc ggcatggggt caggtgggac caccgcgcta ctgccgccag gcagctgtat
     5461 catctgcgta ctccattagt gtgacgacct atgaggatgg cggggtatta actgtaacgc
     5521 aagatacttc tctgcaagat ggttgtttaa tttataatgt gaaactgcgc ggcgttaatt
     5581 ttccctcaaa tggtcccgta atgcaaaaga agaccttagg gtgggaagct tctacggaaa
     5641 cgttgtatcc tgcagatggc ggtcttgagg gtcgttgtga tatggcgttg aaactggtcg
     5701 gcggtggaca tttgcattgt aatctgaaaa cgattaacgt cctacgcaaa cacagaaacg
     5761 gcgacggcag taacaaagac gcccaccgcg ggttcctccg atacccgcag ctggagcctc
     5821 agcggcatct gcaggagcgg cttccgcagc cggtgcttca ctcgctggtg cctccgctgc
     5881 tggtgtagct tccgcagctg gtgcagcagg ttcctgactg atgccagcgg ctggggcttc
     5941 ggcagcatcc gctggggcag cttctgctgc aggcgcttcg ctagctggag cttcagcagc
     6001 aggagtcgcc tctgcggcgg gcgctgcagg ctcttgactg gtaccaatac cgttgtccgg
     6061 acgagtgccg atggtgtgtg cgtcaccgat gttgttggtc cactggtatt ccagacgggt
     6121 agcgatttca ggagtgatcg cgtactcaac accgccagcg aagaccggag aaacgccggt
     6181 gtcgtggttt ttaccataaa cgttggattt agtgtctgca cgccatacca tgccacccag
     6241 acgagtgtag atgtccaggt cgtcagtgat tgggtaaccc agtttagcgg tcagttgaac
     6301 gccctgagct ttgtatgcac cgttttcaac gctgcctttg tacggcatac gacctaacca
     6361 gtcgtaaccc atttcaaagc caacatacgg gttaattccc tgatcgattt tagcgttgct
     6421 ggagcaacct gccagcagag tagaacccag gattaccgcg cccagtacca gtttagtagc
     6481 tttcatcaga aatactccat agcatgatta agatgtttca gtacgaaaat tgctttcatt
     6541 gttgatctcc tttttaagtg aacttgggcc ctgtgtgaaa ttgttatccg ctcacaattc
     6601 cacacattat acgagccgat gattaattgt caacagctca tttcagaata ttagccagaa
     6661 ccgttatgat gtcggcgcaa aaaacattat ccagaacggg agtgcgcctt gagcgacacg
     6721 aattatgcag tgatttacgt attaccgcct ttgagtgagc tgataccgct cgccgcagcc
     6781 gaacgcccta ggtctagggc ggcggatttg tcctactcag gagagcgttc accgacaaac
     6841 aacagataaa acgaaaggcc cagtctttcg actgagcctt tcgttttatt tgatgcctca
     6901 ctgcccgctt tccagtcggg aaacctgtcg tgccagctgc attaatgaat cggccaacgc
     6961 gcggggagag gcg
//
