LOCUS       sequence-336758-        6973 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Encodes LacI-controlled expression of surface-displayed murine CRAMP
            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: pKB232
FEATURES             Location/Qualifiers
     source          1..6973
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     917..936
                     /label=LacI-R
                     /note="LacI, reverse primer"
     promoter        complement(956..1033)
                     /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(1123..1140)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     CDS             complement(1402..2352)
                     /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      2400..2622
                     /label=pSC101 ori
                     /note="low-copy replication origin that requires the Rep101
                     protein"
     terminator      complement(3114..3208)
                     /label=lambda t0 terminator
                     /note="transcription terminator from phage lambda"
     CDS             complement(join(3238..4029,4030..4098))
                     /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     3861..3880
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(4099..4203)
                     /gene="bla"
                     /label=AmpR promoter
     primer_bind     4268..4286
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     RBS             4484..4495
                     /note="strong bacterial ribosome binding site (Elowitz and 
                     Leibler, 2000)"
     terminator      5221..5292
                     /label=rrnB T1 terminator
                     /note="transcription terminator T1 from the E. coli rrnB 
                     gene"
     terminator      5308..5335
                     /label=T7Te terminator
                     /note="phage T7 early transcription terminator"
     primer_bind     complement(6518..6540)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    6529..6545
                     /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(6553..6559,6560..6575,6576..6581))
                     /label=tac promoter
                     /note="strong E. coli promoter; hybrid between the trp and 
                     lac UV5 promoters"
     terminator      complement(6754..6840)
                     /gene="Escherichia coli rrnB"
                     /label=rrnB T1 terminator
                     /note="transcription terminator T1 from the E. coli rrnB 
                     gene"
     CDS             complement(join(6846..6973,1..955))
                     /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
                     EDGTRLGVEHLVALGRQQIALLAGPLSSVSARLRLAGWHKYLTRNQIQPIAEREGDWSA
                     MSGFQQTMQMLNEGIVPTAMLVANDQMALGAMRAITESGLRVGADISVVGYDDTEDSSC
                     YIPPLTTIKQDFRLLGQTSVDRLLQLSQGQAVKGNQLLPVSLVKRKTTLAPNTQTASPR
                     ALADSLMQLARQVSRLESGQ"
ORIGIN
        1 acagctgatt gcccttcacc gcctggccct gagagagttg cagcaagcgg tccacgctgg
       61 tttgccccag caggcgaaaa tcctgtttga tggtggttaa cggcgggata taacatgagc
      121 tgtcttcggt atcgtcgtat cccactaccg agatatccgc accaacgcgc agcccggact
      181 cggtaatggc gcgcattgcg cccagcgcca tctgatcgtt ggcaaccagc atcgcagtgg
      241 gaacgatgcc ctcattcagc atttgcatgg tttgttgaaa accggacatg gcactccagt
      301 cgccttcccg ttccgctatc ggctgaattt gattgcgagt gagatattta tgccagccag
      361 ccagacgcag acgcgccgag acagaactta atgggcccgc taacagcgcg atttgctggc
      421 gacccaatgc gaccagatgc tccacgccca gtcgcgtacc gtcttcatgg gagaaaataa
      481 tactgttgat gggtgtctgg tcagagacat caagaaataa cgccggaaca ttagtgcagg
      541 cagcttccac agcaatggca tcctggtcat ccagcggata gttaatgatc agcccactga
      601 cgcgttgcgc gagaagattg tgcaccgccg ctttacaggc ttcgacgccg cttcgttcta
      661 ccatcgacac caccacgctg gcacccagtt gatcggcgcg agatttaatc gccgcgacaa
      721 tttgcgacgg cgcgtgcagg gccagactgg aggtggcaac gccaatcagc aacgactgtt
      781 tgcccgccag ttgttgtgcc acgcggttgg gaatgtaatt cagctccgcc atcgccgctt
      841 ccactttttc ccgcgttttc gcagaaacgt ggctggcctg gttcaccacg cgggaaacgg
      901 tctgataaga gacaccggca tactctgcga catcgtataa cgttactggt ttcacattca
      961 ccaccctgaa ttgactctct tccgggcgct atcatgccat accgcgaaag gttttgcacc
     1021 attcgatggt gtcaacgtaa atgcatgccg cttcggcaag gtgtcaccac cctgcccttt
     1081 ttctttaaaa ccgaaaagat tacttcgcgt tatgcaggct tcctcgctca ctgactcgct
     1141 gcgctcggtc gttcggctgc ggcgagcggt atcagctcac tcaaaggcgg taatctcgag
     1201 gttacattgt cgatctgttc atggtgaaca gctttgaatg caccaaaaac tcgtaaaagc
     1261 tctgatgtat ctatcttttt tacaccgttt tcatctgtgc atatggacag ttttcccttt
     1321 gatatgtaac ggtgaacagt tgttctactt ttgtttgtta gtcttgatgc ttcactgata
     1381 gatacaagag ccataagaac ctcagatcct tccgtattta gccagtatgt tctctagtgt
     1441 ggttcgttgt ttttgcgtga gccatgagaa cgaaccattg agatcatact tactttgcat
     1501 gtcactcaaa aattttgcct caaaactggt gagctgaatt tttgcagtta aagcatcgtg
     1561 tagtgttttt cttagtccgt tatgtaggta ggaatctgat gtaatggttg ttggtatttt
     1621 gtcaccattc atttttatct ggttgttctc aagttcggtt acgagatcca tttgtctatc
     1681 tagttcaact tggaaaatca acgtatcagt cgggcggcct cgcttatcaa ccaccaattt
     1741 catattgctg taagtgttta aatctttact tattggtttc aaaacccatt ggttaagcct
     1801 tttaaactca tggtagttat tttcaagcat taacatgaac ttaaattcat caaggctaat
     1861 ctctatattt gccttgtgag ttttcttttg tgttagttct tttaataacc actcataaat
     1921 cctcatagag tatttgtttt caaaagactt aacatgttcc agattatatt ttatgaattt
     1981 ttttaactgg aaaagataag gcaatatctc ttcactaaaa actaattcta atttttcgct
     2041 tgagaacttg gcatagtttg tccactggaa aatctcaaag cctttaacca aaggattcat
     2101 gatttccaca gttctcgtca tcagctctct ggttgcttta gctaatacac cataagcatt
     2161 ttccctactg atgttcatca tctgagcgta ttggttataa gtgaacgata ccgtccgttc
     2221 tttccttgta gggttttcaa tagtggggtt gagtagtgcc acacagcata aaattagctt
     2281 ggtttcatgc tccgttaagt catagcgact aatcgctagt tcatttgctt tgaaaacaac
     2341 taattcagac atacatctca attggtctag gtgattttaa tcactatacc aattgagatg
     2401 ggctagtcaa tgataattac atgtcctttt cctttgagtt gtgggtatct gtaaattctg
     2461 ctagaccttt gctggaaaac ttgtaaattc tgctagaccc tctgtaaatt ccgctagacc
     2521 tttgtgtgtt ttttttgttt atattcaagt ggttataatt tatagaataa agaaagaata
     2581 aaaaaagata aaaagaatag atcccagccc tgtgtataac tcactacttt agtcagttcc
     2641 gcagtattac aaaaggatgt cgcaaacgct gtttgctcct ctacaaaaca gaccttaaaa
     2701 ccctaaaggc ttaagtagca ccctcgcaag ctcgtgcaaa tcgcttaata ttccttttgt
     2761 ctccgaccat caggcacctg agtcgctgcc tttttcgtga cattcagttc gctgcgctca
     2821 cggctctggc agtgaatgtg ggtaaatggc actacaggcg ccttttatgg attcatgcaa
     2881 ggaaactacc cataatacaa gaaaagcccg tcacgggctt ctcagggcgt tttatggctg
     2941 gtctgctatg tggtgctatc tgactttttg ctgttcagca gttcctgccc tctgattttc
     3001 cagtctgacc acttcggatt atcccgtgac aggtcattca gactggctaa tgcacccagt
     3061 aaggcagcgg tatcatcaac aggcttaccc gtcttactgt ccctagtgct tggattctca
     3121 ccaataaaaa acgcccggcg gcaaccgagc gttctgaaca aatccagatg gagttctgat
     3181 gtcattactg gatctatcaa caggagtcca agcgagctcg taaacttggt ctgacagtta
     3241 ccaatgctta atcagtgagg cacctatctc agcgatctgt ctatttcgtt catccatagt
     3301 tgcctgactc cccgtcgtgt agataactac gatacgggag ggcttaccat ctggccccag
     3361 tgctgcaatg ataccgcgtg atccacgctc accggctcca gatttatcag caataaacca
     3421 gccagccgga agggccgagc gcagaagtgg tcctgcaact ttatccgcct ccatccagtc
     3481 tattaattgt tgccgggaag ctagagtaag tagttcgcca gttaatagtt tgcgcaacgt
     3541 tgttgccatt gctacaggca tcgtggtgtc acgctcgtcg tttggtatgg cttcattcag
     3601 ctccggttcc caacgatcaa ggcgagttac atgatccccc atgttgtgca aaaaagcggt
     3661 tagctccttc ggtcctccga tcgttgtcag aagtaagttg gccgcagtgt tatcactcat
     3721 ggttatggca gcactgcata attctcttac tgtcatgcca tccgtaagat gcttttctgt
     3781 tactggtgag tactcaacca agtcattctg agaatagtgt atgcggcgac cgagttgctc
     3841 ttgcccggcg tcaatacggt ataataccgc gccacatagc agaactttaa aagtgctcat
     3901 cattggaaaa cgttcttcgg ggcgaaaact ctcaaggatc ttaccgctgt tgagatccag
     3961 ttcgatgtaa cccactcgtg cacccaactg atcttcagca tcttttactt tcaccagcgt
     4021 ttctgggtga gcaaaaacgg gaaggcaaaa tgccgcaaaa aagtgaataa gggcgacact
     4081 gaaatgttga atactcatac tcttcctttt tcaatattat tgaagcattt atcagggtta
     4141 ttgtctcatg agcggataca tattttaatg tatttagaaa aataaacaaa taggggttcc
     4201 gcgcacattt cccatggtgc cacctgacgt ctaataaacc attattatca tgacattaac
     4261 ctataaaaat aggcgtatca cgaggcagaa tttttatcgc ttccgcgttc cgtctactcc
     4321 ctattacgct ccggaatctc cgacgcctac ggccgacgtc ggataccgcc attgataaac
     4381 tgtttaacaa catcgtctgt acagaccttc ttcgttgcct ttacgtttaa ctcaatcagg
     4441 ctaccgtctc ggttataagt ctatcgagta gtagagccgt agtaaagagg agaaatacta
     4501 gatggtttcg aaaggtgagg aggataacat ggcgtcgctg cctgctacac atgaactgca
     4561 tattttcggc agcatcaacg gagttgattt cgacatggtg ggccaaggca ccggcaaccc
     4621 gaacgacggc tatgaagaat tgaatttgaa atctaccaag ggcgacttgc aatttagtcc
     4681 gtggattctg gtaccgcata ttggttatgg ttttcatcag tatctgccct acccagacgg
     4741 aatgagccca tttcaggcgg ctatggtgga tggcagtggc tatcaggtcc atcgcaccat
     4801 gcaattcgag gatggggcga gcctgaccgt gaattatcgg tacacttacg aaggttcaca
     4861 tattaaaggt gaagcacaag ttaaaggcac cggcttcccg gcggatggcc cagtgatgac
     4921 caacagcctg acggcggcgg actggtgtcg tagcaaaaag acgtatccca atgataaaac
     4981 aattatttct acctttaaat ggagttacac cacgggtaac ggtaaacgct accgtagcac
     5041 ggctcgcacc acatacacct ttgcgaaacc catggccgcg aactatctga aaaatcagcc
     5101 gatgtatgtg tttcgcaaaa cggaactgaa acattcgaaa actgaactca atttcaaaga
     5161 gtggcaaaaa gccttcaccg acgtaatggg catggatgaa ctgtacaaat gaccaggcat
     5221 caaataaaac gaaaggctca gtcgaaagac tgggcctttc gttttatctg ttgtttgtcg
     5281 gtgaacgctc tctactagag tcacactggc tcaccttcgg gtgggccttt ctgcgtttat
     5341 agtttcactt ctgagttcgg catggggtca ggtgggacca ccgcgctact gccgccaggc
     5401 agctgtatca tctgcgtact ccattagtgt gacgacctat gaggatggcg gggtattaac
     5461 tgtaacgcaa gatacttctc tgcaagatgg ttgtttaatt tataatgtga aactgcgcgg
     5521 cgttaatttt ccctcaaatg gtcccgtaat gcaaaagaag accttagggt gggaagcttc
     5581 tacggaaacg ttgtatcctg cagatggcgg tcttgagggt cgttgtgata tggttattgc
     5641 tctggttgcg gaacaagttt ttgaaaaaag ttcttgattt tctgaccgat ttttttcaac
     5701 ttctctccga tcttctctcc acctttgcgt aacagcccgg ttcctccgat acccgcagct
     5761 ggagcctcag cggcatctgc aggagcggct tccgcagccg gtgcttcact cgctggtgcc
     5821 tccgctgctg gtgtagcttc cgcagctggt gcagcaggtt cctgactgat gccagcggct
     5881 ggggcttcgg cagcatccgc tggggcagct tctgctgcag gcgcttcgct agctggagct
     5941 tcagcagcag gagtcgcctc tgcggcgggc gctgcaggct cttgactggt accaataccg
     6001 ttgtccggac gagtgccgat ggtgtgtgcg tcaccgatgt tgttggtcca ctggtattcc
     6061 agacgggtag cgatttcagg agtgatcgcg tactcaacac cgccagcgaa gaccggagaa
     6121 acgccggtgt cgtggttttt accataaacg ttggatttag tgtctgcacg ccataccatg
     6181 ccacccagac gagtgtagat gtccaggtcg tcagtgattg ggtaacccag tttagcggtc
     6241 agttgaacgc cctgagcttt gtatgcaccg ttttcaacgc tgcctttgta cggcatacga
     6301 cctaaccagt cgtaacccat ttcaaagcca acatacgggt taattccctg atcgatttta
     6361 gcgttgctgg agcaacctgc cagcagagta gaacccagga ttaccgcgcc cagtaccagt
     6421 ttagtagctt tcatcagaaa tactccatag catgattaag atgtttcagt acgaaaattg
     6481 ctttcattgt tgatctcctt tttaagtgaa cttgggccct gtgtgaaatt gttatccgct
     6541 cacaattcca cacattatac gagccgatga ttaattgtca acagctcatt tcagaatatt
     6601 agccagaacc gttatgatgt cggcgcaaaa aacattatcc agaacgggag tgcgccttga
     6661 gcgacacgaa ttatgcagtg atttacgtat taccgccttt gagtgagctg ataccgctcg
     6721 ccgcagccga acgccctagg tctagggcgg cggatttgtc ctactcagga gagcgttcac
     6781 cgacaaacaa cagataaaac gaaaggccca gtctttcgac tgagcctttc gttttatttg
     6841 atgcctcact gcccgctttc cagtcgggaa acctgtcgtg ccagctgcat taatgaatcg
     6901 gccaacgcgc ggggagaggc ggtttgcgta ttgggcgcca gggtggtttt tcttttcacc
     6961 agtgagacgg gca
//
