LOCUS       sequence-336759-        6973 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Encodes LacI-controlled expression of surface-displayed Bac2a 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: pKB256
FEATURES             Location/Qualifiers
     source          1..6973
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     937..956
                     /label=LacI-R
                     /note="LacI, reverse primer"
     promoter        complement(976..1053)
                     /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(1143..1160)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     CDS             complement(1422..2372)
                     /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      2420..2642
                     /label=pSC101 ori
                     /note="low-copy replication origin that requires the Rep101
                     protein"
     terminator      complement(3134..3228)
                     /label=lambda t0 terminator
                     /note="transcription terminator from phage lambda"
     CDS             complement(join(3258..4049,4050..4118))
                     /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     3881..3900
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(4119..4223)
                     /gene="bla"
                     /label=AmpR promoter
     primer_bind     4288..4306
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     RBS             4504..4515
                     /note="strong bacterial ribosome binding site (Elowitz and 
                     Leibler, 2000)"
     terminator      5241..5312
                     /label=rrnB T1 terminator
                     /note="transcription terminator T1 from the E. coli rrnB 
                     gene"
     terminator      5328..5355
                     /label=T7Te terminator
                     /note="phage T7 early transcription terminator"
     primer_bind     complement(6538..6560)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    6549..6565
                     /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(6573..6579,6580..6595,6596..6601))
                     /label=tac promoter
                     /note="strong E. coli promoter; hybrid between the trp and 
                     lac UV5 promoters"
     terminator      complement(6774..6860)
                     /gene="Escherichia coli rrnB"
                     /label=rrnB T1 terminator
                     /note="transcription terminator T1 from the E. coli rrnB 
                     gene"
     CDS             complement(join(6866..6973,1..975))
                     /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 tttcaccagt gagacgggca acagctgatt gcccttcacc gcctggccct gagagagttg
       61 cagcaagcgg tccacgctgg tttgccccag caggcgaaaa tcctgtttga tggtggttaa
      121 cggcgggata taacatgagc tgtcttcggt atcgtcgtat cccactaccg agatatccgc
      181 accaacgcgc agcccggact cggtaatggc gcgcattgcg cccagcgcca tctgatcgtt
      241 ggcaaccagc atcgcagtgg gaacgatgcc ctcattcagc atttgcatgg tttgttgaaa
      301 accggacatg gcactccagt cgccttcccg ttccgctatc ggctgaattt gattgcgagt
      361 gagatattta tgccagccag ccagacgcag acgcgccgag acagaactta atgggcccgc
      421 taacagcgcg atttgctggt gacccaatgc gaccagatgc tccacgccca gtcgcgtacc
      481 gtcttcatgg gagaaaataa tactgttgat gggtgtctgg tcagagacat caagaaataa
      541 cgccggaaca ttagtgcagg cagcttccac agcaatggca tcctggtcat ccagcggata
      601 gttaatgatc agcccactga cgcgttgcgc gagaagattg tgcaccgccg ctttacaggc
      661 ttcgacgccg cttcgttcta ccatcgacac caccacgctg gcacccagtt gatcggcgcg
      721 agatttaatc gccgcgacaa tttgcgacgg cgcgtgcagg gccagactgg aggtggcaac
      781 gccaatcagc aacgactgtt tgcccgccag ttgttgtgcc acgcggttgg gaatgtaatt
      841 cagctccgcc atcgccgctt ccactttttc ccgcgttttc gcagaaacgt ggctggcctg
      901 gttcaccacg cgggaaacgg tctgataaga gacaccggca tactctgcga catcgtataa
      961 cgttactggt ttcacattca ccaccctgaa ttgactctct tccgggcgct atcatgccat
     1021 accgcgaaag gttttgcacc attcgatggt gtcaacgtaa atgcatgccg cttcggcaag
     1081 gtgtcaccac cctgcccttt ttctttaaaa ccgaaaagat tacttcgcgt tatgcaggct
     1141 tcctcgctca ctgactcgct gcgctcggtc gttcggctgc ggcgagcggt atcagctcac
     1201 tcaaaggcgg taatctcgag gttacattgt cgatctgttc atggtgaaca gctttgaatg
     1261 caccaaaaac tcgtaaaagc tctgatgtat ctatcttttt tacaccgttt tcatctgtgc
     1321 atatggacag ttttcccttt gatatgtaac ggtgaacagt tgttctactt ttgtttgtta
     1381 gtcttgatgc ttcactgata gatacaagag ccataagaac ctcagatcct tccgtattta
     1441 gccagtatgt tctctagtgt ggttcgttgt ttttgcgtga gccatgagaa cgaaccattg
     1501 agatcatact tactttgcat gtcactcaaa aattttgcct caaaactggt gagctgaatt
     1561 tttgcagtta aagcatcgtg tagtgttttt cttagtccgt tatgtaggta ggaatctgat
     1621 gtaatggttg ttggtatttt gtcaccattc atttttatct ggttgttctc aagttcggtt
     1681 acgagatcca tttgtctatc tagttcaact tggaaaatca acgtatcagt cgggcggcct
     1741 cgcttatcaa ccaccaattt catattgctg taagtgttta aatctttact tattggtttc
     1801 aaaacccatt ggttaagcct tttaaactca tggtagttat tttcaagcat taacatgaac
     1861 ttaaattcat caaggctaat ctctatattt gccttgtgag ttttcttttg tgttagttct
     1921 tttaataacc actcataaat cctcatagag tatttgtttt caaaagactt aacatgttcc
     1981 agattatatt ttatgaattt ttttaactgg aaaagataag gcaatatctc ttcactaaaa
     2041 actaattcta atttttcgct tgagaacttg gcatagtttg tccactggaa aatctcaaag
     2101 cctttaacca aaggattcat gatttccaca gttctcgtca tcagctctct ggttgcttta
     2161 gctaatacac cataagcatt ttccctactg atgttcatca tctgagcgta ttggttataa
     2221 gtgaacgata ccgtccgttc tttccttgta gggttttcaa tagtggggtt gagtagtgcc
     2281 acacagcata aaattagctt ggtttcatgc tccgttaagt catagcgact aatcgctagt
     2341 tcatttgctt tgaaaacaac taattcagac atacatctca attggtctag gtgattttaa
     2401 tcactatacc aattgagatg ggctagtcaa tgataattac atgtcctttt cctttgagtt
     2461 gtgggtatct gtaaattctg ctagaccttt gctggaaaac ttgtaaattc tgctagaccc
     2521 tctgtaaatt ccgctagacc tttgtgtgtt ttttttgttt atattcaagt ggttataatt
     2581 tatagaataa agaaagaata aaaaaagata aaaagaatag atcccagccc tgtgtataac
     2641 tcactacttt agtcagttcc gcagtattac aaaaggatgt cgcaaacgct gtttgctcct
     2701 ctacaaaaca gaccttaaaa ccctaaaggc ttaagtagca ccctcgcaag ctcgtgcaaa
     2761 tcgcttaata ttccttttgt ctccgaccat caggcacctg agtcgctgcc tttttcgtga
     2821 cattcagttc gctgcgctca cggctctggc agtgaatgtg ggtaaatggc actacaggcg
     2881 ccttttatgg attcatgcaa ggaaactacc cataatacaa gaaaagcccg tcacgggctt
     2941 ctcagggcgt tttatggctg gtctgctatg tggtgctatc tgactttttg ctgttcagca
     3001 gttcctgccc tctgattttc cagtctgacc acttcggatt atcccgtgac aggtcattca
     3061 gactggctaa tgcacccagt aaggcagcgg tatcatcaac aggcttaccc gtcttactgt
     3121 ccctagtgct tggattctca ccaataaaaa acgcccggcg gcaaccgagc gttctgaaca
     3181 aatccagatg gagttctgat gtcattactg gatctatcaa caggagtcca agcgagctcg
     3241 taaacttggt ctgacagtta ccaatgctta atcagtgagg cacctatctc agcgatctgt
     3301 ctatttcgtt catccatagt tgcctgactc cccgtcgtgt agataactac gatacgggag
     3361 ggcttaccat ctggccccag tgctgcaatg ataccgcgtg atccacgctc accggctcca
     3421 gatttatcag caataaacca gccagccgga agggccgagc gcagaagtgg tcctgcaact
     3481 ttatccgcct ccatccagtc tattaattgt tgccgggaag ctagagtaag tagttcgcca
     3541 gttaatagtt tgcgcaacgt tgttgccatt gctacaggca tcgtggtgtc acgctcgtcg
     3601 tttggtatgg cttcattcag ctccggttcc caacgatcaa ggcgagttac atgatccccc
     3661 atgttgtgca aaaaagcggt tagctccttc ggtcctccga tcgttgtcag aagtaagttg
     3721 gccgcagtgt tatcactcat ggttatggca gcactgcata attctcttac tgtcatgcca
     3781 tccgtaagat gcttttctgt tactggtgag tactcaacca agtcattctg agaatagtgt
     3841 atgcggcgac cgagttgctc ttgcccggcg tcaatacggt ataataccgc gccacatagc
     3901 agaactttaa aagtgctcat cattggaaaa cgttcttcgg ggcgaaaact ctcaaggatc
     3961 ttaccgctgt tgagatccag ttcgatgtaa cccactcgtg cacccaactg atcttcagca
     4021 tcttttactt tcaccagcgt ttctgggtga gcaaaaacgg gaaggcaaaa tgccgcaaaa
     4081 aagtgaataa gggcgacact gaaatgttga atactcatac tcttcctttt tcaatattat
     4141 tgaagcattt atcagggtta ttgtctcatg agcggataca tattttaatg tatttagaaa
     4201 aataaacaaa taggggttcc gcgcacattt cccatggtgc cacctgacgt ctaataaacc
     4261 attattatca tgacattaac ctataaaaat aggcgtatca cgaggcagaa tttttatcgc
     4321 ttccgcgttc cgtctactcc ctattacgct ccggaatctc cgacgcctac ggccgacgtc
     4381 ggataccgcc attgataaac tgtttaacaa catcgtctgt acagaccttc ttcgttgcct
     4441 ttacgtttaa ctcaatcagg ctaccgtctc ggttataagt ctatcgagta gtagagccgt
     4501 agtaaagagg agaaatacta gatggtttcg aaaggtgagg aggataacat ggcgtcgctg
     4561 cctgctacac atgaactgca tattttcggc agcatcaacg gagttgattt cgacatggtg
     4621 ggccaaggca ccggcaaccc gaacgacggc tatgaagaat tgaatttgaa atctaccaag
     4681 ggcgacttgc aatttagtcc gtggattctg gtaccgcata ttggttatgg ttttcatcag
     4741 tatctgccct acccagacgg aatgagccca tttcaggcgg ctatggtgga tggcagtggc
     4801 tatcaggtcc atcgcaccat gcaattcgag gatggggcga gcctgaccgt gaattatcgg
     4861 tacacttacg aaggttcaca tattaaaggt gaagcacaag ttaaaggcac cggcttcccg
     4921 gcggatggcc cagtgatgac caacagcctg acggcggcgg actggtgtcg tagcaaaaag
     4981 acgtatccca atgataaaac aattatttct acctttaaat ggagttacac cacgggtaac
     5041 ggtaaacgct accgtagcac ggctcgcacc acatacacct ttgcgaaacc catggccgcg
     5101 aactatctga aaaatcagcc gatgtatgtg tttcgcaaaa cggaactgaa acattcgaaa
     5161 actgaactca atttcaaaga gtggcaaaaa gccttcaccg acgtaatggg catggatgaa
     5221 ctgtacaaat gaccaggcat caaataaaac gaaaggctca gtcgaaagac tgggcctttc
     5281 gttttatctg ttgtttgtcg gtgaacgctc tctactagag tcacactggc tcaccttcgg
     5341 gtgggccttt ctgcgtttat agtttcactt ctgagttcgg catggggtca ggtgggacca
     5401 ccgcgctact gccgccaggc agctgtatca tctgcgtact ccattagtgt gacgacctat
     5461 gaggatggcg gggtattaac tgtaacgcaa gatacttctc tgcaagatgg ttgtttaatt
     5521 tataatgtga aactgcgcgg cgttaatttt ccctcaaatg gtcccgtaat gcaaaagaag
     5581 accttagggt gggaagcttc tacggaaacg ttgtatcctg cagatggcgg tcttgagggt
     5641 cgttgtgata tggcgttgaa actggtcggc ggtggacatt tgcattgtaa tctgaaaacg
     5701 acttatcgta gcaagaagct taacgggcta cgcgaataac aacaatacgg gcaagacgtg
     5761 ttcctccgat acccgcagct ggagcctcag cggcatctgc aggagcggct tccgcagccg
     5821 gtgcttcact cgctggtgcc tccgctgctg gtgtagcttc cgcagctggt gcagcaggtt
     5881 cctgactgat gccagcggct ggggcttcgg cagcatcagc tggggcagct tctgctgcag
     5941 gcgcttcgct agctggagct tcagcagcag gagtcgcctc tgcggcgggc gctgcaggct
     6001 cttgactggt accaataccg ttgtccggac gagtgccgat ggtgtgtgcg tcaccgatgt
     6061 tgttggtcca ctggtattcc agacgggtag cgatttcagg agtgatcgcg tactcaacac
     6121 cgccagcgaa gaccggagaa acgccggtgt cgtggttttt accataaacg ttggatttag
     6181 tgtctgcacg ccataccatg ccacccagac gagtgtagat gtccaggtcg tcagtgattg
     6241 ggtaacccag tttagcggtc agttgaacgc cctgagcttt gtatgcaccg ttttcaacgc
     6301 tgcctttgta cggcatacga cctaaccagt cgtaacccat ttcaaagcca acatacgggt
     6361 taattccctg atcgatttta gcgttgctgg agcaacctgc cagcagagta gaacccagga
     6421 ttaccgcgcc cagtaccagt ttagtagctt tcatcagaaa tactccatag catgattaag
     6481 atgtttcagt acgaaaattg ctttcattgt tgatctcctt tttaagtgaa cttgggccct
     6541 gtgtgaaatt gttatccgct cacaattcca cacattatac gagccgatga ttaattgtca
     6601 acagctcatt tcagaatatt agccagaacc gttatgatgt cggcgcaaaa aacattatcc
     6661 agaacgggag tgcgccttga gcgacacgaa ttatgcagtg atttacgtat taccgccttt
     6721 gagtgagctg ataccgctcg ccgcagccga acgccctagg tctagggcgg cggatttgtc
     6781 ctactcagga gagcgttcac cgacaaacaa cagataaaac gaaaggccca gtctttcgac
     6841 tgagcctttc gttttatttg atgcctcact gcccgctttc cagtcgggaa acctgtcgtg
     6901 ccagctgcat taatgaatcg gccaacgcgc ggggagaggc ggtttgcgta ttgggcgcca
     6961 gggtggtttt tct
//
