LOCUS       sequence-335287-        6973 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Encodes LacI-controlled expression of surface display protein with 
            no C-terminal peptide 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: pKB225
FEATURES             Location/Qualifiers
     source          1..6973
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     terminator      82..168
                     /gene="Escherichia coli rrnB"
                     /label=rrnB T1 terminator
                     /note="transcription terminator T1 from the E. coli rrnB 
                     gene"
     promoter        join(341..346,347..362,363..369)
                     /label=tac promoter
                     /note="strong E. coli promoter; hybrid between the trp and 
                     lac UV5 promoters"
     protein_bind    377..393
                     /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)."
     primer_bind     382..404
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     terminator      complement(1587..1614)
                     /label=T7Te terminator
                     /note="phage T7 early transcription terminator"
     terminator      complement(1630..1701)
                     /label=rrnB T1 terminator
                     /note="transcription terminator T1 from the E. coli rrnB 
                     gene"
     RBS             2427..2438
                     /note="strong bacterial ribosome binding site (Elowitz and 
                     Leibler, 2000)"
     primer_bind     complement(2636..2654)
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     promoter        2719..2823
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(2824..2892,2893..3684)
                     /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     complement(3042..3061)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     terminator      3714..3808
                     /label=lambda t0 terminator
                     /note="transcription terminator from phage lambda"
     rep_origin      4300..4522
                     /label=pSC101 ori
                     /note="low-copy replication origin that requires the Rep101
                     protein"
     CDS             4570..5520
                     /codon_start=1
                     /gene="rep101"
                     /product="RepA protein needed for replication with the 
                     pSC101 origin"
                     /label=Rep101
                     /translation="MSELVVFKANELAISRYDLTEHETKLILCCVALLNPTIENPTRKE
                     RTVSFTYNQYAQMMNISRENAYGVLAKATRELMTRTVEIMNPLVKGFEIFQWTNYAKFS
                     SEKLELVFSEEILPYLFQLKKFIKYNLEHVKSFENKYSMRIYEWLLKELTQKKTHKANI
                     EISLDEFKFMLMLENNYHEFKRLNQWVLKPISKDLNTYSNMKLVVDKRGRPTDTLIFQV
                     ELDRQMDLVTELENNQIKMNGDKIPTTITSDSYLHNGLRKTLHDALTAKIQLTSFEAKF
                     LSDMQSKYDLNGSFSWLTQKQRTTLENILAKYGRI"
     primer_bind     5782..5799
                     /label=L4440
                     /note="L4440 vector, forward primer"
     promoter        5889..5966
                     /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."
     CDS             join(5967..6973,1..76)
                     /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     complement(5986..6005)
                     /label=LacI-R
                     /note="LacI, reverse primer"
ORIGIN
        1 cgcctctccc cgcgcgttgg ccgattcatt aatgcagctg gcacgacagg tttcccgact
       61 ggaaagcggg cagtgaggca tcaaataaaa cgaaaggctc agtcgaaaga ctgggccttt
      121 cgttttatct gttgtttgtc ggtgaacgct ctcctgagta ggacaaatcc gccgccctag
      181 acctagggcg ttcggctgcg gcgagcggta tcagctcact caaaggcggt aatacgtaaa
      241 tcactgcata attcgtgtcg ctcaaggcgc actcccgttc tggataatgt tttttgcgcc
      301 gacatcataa cggttctggc taatattctg aaatgagctg ttgacaatta atcatcggct
      361 cgtataatgt gtggaattgt gagcggataa caatttcaca cagggcccaa gttcacttaa
      421 aaaggagatc aacaatgaaa gcaattttcg tactgaaaca tcttaatcat gctatggagt
      481 atttctgatg aaagctacta aactggtact gggcgcggta atcctgggtt ctactctgct
      541 ggcaggttgc tccagcaacg ctaaaatcga tcagggaatt aacccgtatg ttggctttga
      601 aatgggttac gactggttag gtcgtatgcc gtacaaaggc agcgttgaaa acggtgcata
      661 caaagctcag ggcgttcaac tgaccgctaa actgggttac ccaatcactg acgacctgga
      721 catctacact cgtctgggtg gcatggtatg gcgtgcagac actaaatcca acgtttatgg
      781 taaaaaccac gacaccggcg tttctccggt cttcgctggc ggtgttgagt acgcgatcac
      841 tcctgaaatc gctacccgtc tggaatacca gtggaccaac aacatcggtg acgcacacac
      901 catcggcact cgtccggaca acggtattgg taccagtcaa gagcctgcag cgcccgccgc
      961 agaggcgact cctgctgctg aagctccagc tagcgaagcg cctgcagcag aagctgcccc
     1021 agcggatgct gccgaagccc cagccgctgg catcagtcag gaacctgctg caccagctgc
     1081 ggaagctaca ccagcagcgg aggcaccagc gagtgaagca ccggctgcgg aagccgctcc
     1141 tgcagatgcc gctgaggctc cagctgcggg tatcggagga acataacgaa atacacccca
     1201 ggcattttca gattcttggc cggcttcttg ctacgataag tcgttttcag attacaatgc
     1261 aaatgtccac cgccgaccag tttcaacgcc atatcacaac gaccctcaag accgccatct
     1321 gcaggataca acgtttccgt agaagcttcc caccctaagg tcttcttttg cattacggga
     1381 ccatttgagg gaaaattaac gccgcgcagt ttcacattat aaattaaaca accatcttgc
     1441 agagaagtat cttgcgttac agttaatacc ccgccatcct cataggtcgt cacactaatg
     1501 gagtacgcag atgatacagc tgcctggcgg cagtagcgcg gtggtcccac ctgaccccat
     1561 gccgaactca gaagtgaaac tataaacgca gaaaggccca cccgaaggtg agccagtgtg
     1621 actctagtag agagcgttca ccgacaaaca acagataaaa cgaaaggccc agtctttcga
     1681 ctgagccttt cgttttattt gatgcctggt catttgtaca gttcatccat gcccattacg
     1741 tcggtgaagg ctttttgcca ctctttgaaa ttgagttcag ttttcgaatg tttcagttcc
     1801 gttttgcgaa acacatacat cggctgattt ttcagatagt tcgcggccat gggtttcgca
     1861 aaggtgtatg tggtgcgagc cgtgctacgg tagcgtttac cgttacccgt ggtgtaactc
     1921 catttaaagg tagaaataat tgttttatca ttgggatacg tctttttgct acgacaccag
     1981 tccgccgccg tcaggctgtt ggtcatcact gggccatccg ccgggaagcc ggtgccttta
     2041 acttgtgctt cacctttaat atgtgaacct tcgtaagtgt accgataatt cacggtcagg
     2101 ctcgccccat cctcgaattg catggtgcga tggacctgat agccactgcc atccaccata
     2161 gccgcctgaa atgggctcat tccgtctggg tagggcagat actgatgaaa accataacca
     2221 atatgcggta ccagaatcca cggactaaat tgcaagtcgc ccttggtaga tttcaaattc
     2281 aattcttcat agccgtcgtt cgggttgccg gtgccttggc ccaccatgtc gaaatcaact
     2341 ccgttgatgc tgccgaaaat atgcagttca tgtgtagcag gcagcgacgc catgttatcc
     2401 tcctcacctt tcgaaaccat ctagtatttc tcctctttac tacggctcta ctactcgata
     2461 gacttataac cgagacggta gcctgattga gttaaacgta aaggcaacga agaaggtctg
     2521 tacagacgat gttgttaaac agtttatcaa tggcggtatc cgacgtcggc cgtaggcgtc
     2581 ggagattccg gagcgtaata gggagtagac ggaacgcgga agcgataaaa attctgcctc
     2641 gtgatacgcc tatttttata ggttaatgtc atgataataa tggtttatta gacgtcaggt
     2701 ggcaccatgg gaaatgtgcg cggaacccct atttgtttat ttttctaaat acattaaaat
     2761 atgtatccgc tcatgagaca ataaccctga taaatgcttc aataatattg aaaaaggaag
     2821 agtatgagta ttcaacattt cagtgtcgcc cttattcact tttttgcggc attttgcctt
     2881 cccgtttttg ctcacccaga aacgctggtg aaagtaaaag atgctgaaga tcagttgggt
     2941 gcacgagtgg gttacatcga actggatctc aacagcggta agatccttga gagttttcgc
     3001 cccgaagaac gttttccaat gatgagcact tttaaagttc tgctatgtgg cgcggtatta
     3061 taccgtattg acgccgggca agagcaactc ggtcgccgca tacactattc tcagaatgac
     3121 ttggttgagt actcaccagt aacagaaaag catcttacgg atggcatgac agtaagagaa
     3181 ttatgcagtg ctgccataac catgagtgat aacactgcgg ccaacttact tctgacaacg
     3241 atcggaggac cgaaggagct aaccgctttt ttgcacaaca tgggggatca tgtaactcgc
     3301 cttgatcgtt gggaaccgga gctgaatgaa gccataccaa acgacgagcg tgacaccacg
     3361 atgcctgtag caatggcaac aacgttgcgc aaactattaa ctggcgaact acttactcta
     3421 gcttcccggc aacaattaat agactggatg gaggcggata aagttgcagg accacttctg
     3481 cgctcggccc ttccggctgg ctggtttatt gctgataaat ctggagccgg tgagcgtgga
     3541 tcacgcggta tcattgcagc actggggcca gatggtaagc cctcccgtat cgtagttatc
     3601 tacacgacgg ggagtcaggc aactatggat gaacgaaata gacagatcgc tgagataggt
     3661 gcctcactga ttaagcattg gtaactgtca gaccaagttt acgagctcgc ttggactcct
     3721 gttgatagat ccagtaatga catcagaact ccatctggat ttgttcagaa cgctcggttg
     3781 ccgccgggcg ttttttattg gtgagaatcc aagcactagg gacagtaaga cgggtaagcc
     3841 tgttgatgat accgctgcct tactgggtgc attagccagt ctgaatgacc tgtcacggga
     3901 taatccgaag tggtcagact ggaaaatcag agggcaggaa ctgctgaaca gcaaaaagtc
     3961 agatagcacc acatagcaga ccagccataa aacgccctga gaagcccgtg acgggctttt
     4021 cttgtattat gggtagtttc cttgcatgaa tccataaaag gcgcctgtag tgccatttac
     4081 ccacattcac tgccagagcc gtgagcgcag cgaactgaat gtcacgaaaa aggcagcgac
     4141 tcaggtgcct gatggtcgga gacaaaagga atattaagcg atttgcacga gcttgcgagg
     4201 gtgctactta agcctttagg gttttaaggt ctgttttgta gaggagcaaa cagcgtttgc
     4261 gacatccttt tgtaatactg cggaactgac taaagtagtg agttatacac agggctggga
     4321 tctattcttt ttatcttttt ttattctttc tttattctat aaattataac cacttgaata
     4381 taaacaaaaa aaacacacaa aggtctagcg gaatttacag agggtctagc agaatttaca
     4441 agttttccag caaaggtcta gcagaattta cagataccca caactcaaag gaaaaggaca
     4501 tgtaattatc attgactagc ccatctcaat tggtatagtg attaaaatca cctagaccaa
     4561 ttgagatgta tgtctgaatt agttgttttc aaagcaaatg aactagcgat tagtcgctat
     4621 gacttaacgg agcatgaaac caagctaatt ttatgctgtg tggcactact caaccccact
     4681 attgaaaacc ctacaaggaa agaacggacg gtatcgttca cttataacca atacgctcag
     4741 atgatgaaca tcagtaggga aaatgcttat ggtgtattag ctaaagcaac cagagagctg
     4801 atgacgagaa ctgtggaaat catgaatcct ttggttaaag gctttgagat tttccagtgg
     4861 acaaactatg ccaagttctc aagcgaaaaa ttagaattag tttttagtga agagatattg
     4921 ccttatcttt tccagttaaa aaaattcata aaatataatc tggaacatgt taagtctttt
     4981 gaaaacaaat actctatgag gatttatgag tggttattaa aagaactaac acaaaagaaa
     5041 actcacaagg caaatataga gattagcctt gatgaattta agttcatgtt aatgcttgaa
     5101 aataactacc atgagtttaa aaggcttaac caatgggttt tgaaaccaat aagtaaagat
     5161 ttaaacactt acagcaatat gaaattggtg gttgataagc gaggccgccc gactgatacg
     5221 ttgattttcc aagttgaact agatagacaa atggatctcg taaccgaact tgagaacaac
     5281 cagataaaaa tgaatggtga caaaatacca acaaccatta catcagattc ctacctacat
     5341 aacggactaa gaaaaacact acacgatgct ttaactgcaa aaattcagct caccagtttt
     5401 gaggcaaaat ttttgagtga catgcaaagt aagtatgatc tcaatggttc gttctcatgg
     5461 ctcacgcaaa aacaacgaac cacactagag aacatactgg ctaaatacgg aaggatctga
     5521 ggttcttatg gctcttgtat ctatcagtga agcatcaaga ctaacaaaca aaagtagaac
     5581 aactgttcac cgttacatat caaagggaaa actgtccata tgcacagatg aaaacggtgt
     5641 aaaaaagata gatacatcag agcttttacg agtttttggt gcattcaaag ctgttcacca
     5701 tgaacagatc gacaatgtaa cctcgagatt accgcctttg agtgagctga taccgctcgc
     5761 cgcagccgaa cgaccgagcg cagcgagtca gtgagcgagg aagcctgcat aacgcgaagt
     5821 aatcttttcg gttttaaaga aaaagggcag ggtggtgaca ccttgccgaa gcggcatgca
     5881 tttacgttga caccatcgaa tggtgcaaaa cctttcgcgg tatggcatga tagcgcccgg
     5941 aagagagtca attcagggtg gtgaatgtga aaccagtaac gttatacgat gtcgcagagt
     6001 atgccggtgt ctcttatcag accgtttccc gcgtggtgaa ccaggccagc cacgtttctg
     6061 cgaaaacgcg ggaaaaagtg gaagcggcga tggcggagct gaattacatt cccaaccgcg
     6121 tggcacaaca actggcgggc aaacagtcgt tgctgattgg cgttgccacc tccagtctgg
     6181 ccctgcacgc gccgtcgcaa attgtcgcgg cgattaaatc tcgcgccgat caactgggtg
     6241 ccagcgtggt ggtgtcgatg gtagaacgaa gcggcgtcga agcctgtaaa gcggcggtgc
     6301 acaatcttct cgcgcaacgc gtcagtgggc tgatcattaa ctatccgctg gatgaccagg
     6361 atgccattgc tgtggaagct gcctgcacta atgttccggc gttatttctt gatgtctctg
     6421 accagacacc catcaacagt attattttct cccatgaaga cggtacgcga ctgggcgtgg
     6481 agcatctggt cgcattgggt caccagcaaa tcgcgctgtt agcgggccca ttaagttctg
     6541 tctcggcgcg tctgcgtctg gctggctggc ataaatatct cactcgcaat caaattcagc
     6601 cgatagcgga acgggaaggc gactggagtg ccatgtccgg ttttcaacaa accatgcaaa
     6661 tgctgaatga gggcatcgtt cccactgcga tgctggttgc caacgatcag atggcgctgg
     6721 gcgcaatgcg cgccattacc gagtccgggc tgcgcgttgg tgcggatatc tcggtagtgg
     6781 gatacgacga taccgaagac agctcatgtt atatcccgcc gttaaccacc atcaaacagg
     6841 attttcgcct gctggggcaa accagcgtgg accgcttgct gcaactctct cagggccagg
     6901 cggtgaaggg caatcagctg ttgcccgtct cactggtgaa aagaaaaacc accctggcgc
     6961 ccaatacgca aac
//
