LOCUS       sequence-336784-        6973 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Encodes LacI-controlled expression of surface-displayed CCL20 
            alpha-helix fragment 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: pKB235.2
FEATURES             Location/Qualifiers
     source          1..6973
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     971..990
                     /label=LacI-R
                     /note="LacI, reverse primer"
     promoter        complement(1010..1087)
                     /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(1177..1194)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     CDS             complement(1456..2406)
                     /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      2454..2676
                     /label=pSC101 ori
                     /note="low-copy replication origin that requires the Rep101
                     protein"
     terminator      complement(3168..3262)
                     /label=lambda t0 terminator
                     /note="transcription terminator from phage lambda"
     CDS             complement(join(3292..4083,4084..4152))
                     /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     3915..3934
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(4153..4257)
                     /gene="bla"
                     /label=AmpR promoter
     primer_bind     4322..4340
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     RBS             4538..4549
                     /note="strong bacterial ribosome binding site (Elowitz and 
                     Leibler, 2000)"
     terminator      5275..5346
                     /label=rrnB T1 terminator
                     /note="transcription terminator T1 from the E. coli rrnB 
                     gene"
     terminator      5362..5389
                     /label=T7Te terminator
                     /note="phage T7 early transcription terminator"
     primer_bind     complement(6572..6594)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    6583..6599
                     /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(6607..6613,6614..6629,6630..6635))
                     /label=tac promoter
                     /note="strong E. coli promoter; hybrid between the trp and 
                     lac UV5 promoters"
     terminator      complement(6808..6894)
                     /gene="Escherichia coli rrnB"
                     /label=rrnB T1 terminator
                     /note="transcription terminator T1 from the E. coli rrnB 
                     gene"
     CDS             complement(join(6900..6973,1..1009))
                     /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 cggtttgcgt attgggcgcc agggtggttt ttcttttcac cagtgagacg ggcaacagct
       61 gattgccctt caccgcctgg ccctgagaga gttgcagcaa gcggtccacg ctggtttgcc
      121 ccagcaggcg aaaatcctgt ttgatggtgg ttaacggcgg gatataacat gagctgtctt
      181 cggtatcgtc gtatcccact accgagatat ccgcaccaac gcgcagcccg gactcggtaa
      241 tggcgcgcat tgcgcccagc gccatctgat cgttggcaac cagcatcgca gtgggaacga
      301 tgccctcatt cagcatttgc atggtttgtt gaaaaccgga catggcactc cagtcgcctt
      361 cccgttccgc tatcggctga atttgattgc gagtgagata tttatgccag ccagccagac
      421 gcagacgcgc cgagacagaa cttaatgggc ccgctaacag cgcgatttgc tggtgaccca
      481 atgcgaccag atgctccacg cccagtcgcg taccgtcttc atgggagaaa ataatactgt
      541 tgatgggtgt ctggtcagag acatcaagaa ataacgccgg aacattagtg caggcagctt
      601 ccacagcaat ggcatcctgg tcatccagcg gatagttaat gatcagccca ctgacgcgtt
      661 gcgcgagaag attgtgcacc gccgctttac aggcttcgac gccgcttcgt tctaccatcg
      721 acaccaccac gctggcaccc agttgatcgg cgcgagattt aatcgccgcg acaatttgcg
      781 acggcgcgtg cagggccaga ctggaggtgg caacgccaat cagcaacgac tgtttgcccg
      841 ccagttgttg tgccacgcgg ttgggaatgt aattcagctc cgccatcgcc gcttccactt
      901 tttcccgcgt tttcgcagaa acgtggctgg cctggttcac cacgcgggaa acggtctgat
      961 aagagacacc ggcatactct gcgacatcgt ataacgttac tggtttcaca ttcaccaccc
     1021 tgaattgact ctcttccggg cgctatcatg ccataccgcg aaaggttttg caccattcga
     1081 tggtgtcaac gtaaatgcat gccgcttcgg caaggtgtca ccaccctgcc ctttttcttt
     1141 aaaaccgaaa agattacttc gcgttatgca ggcttcctcg ctcactgact cgctgcgctc
     1201 ggtcgttcgg ctgcggcgag cggtatcagc tcactcaaag gcggtaatct cgaggttaca
     1261 ttgtcgatct gttcatggtg aacagctttg aatgcaccaa aaactcgtaa aagctctgat
     1321 gtatctatct tttttacacc gttttcatct gtgcatatgg acagttttcc ctttgatatg
     1381 taacggtgaa cagttgttct acttttgttt gttagtcttg atgcttcact gatagataca
     1441 agagccataa gaacctcaga tccttccgta tttagccagt atgttctcta gtgtggttcg
     1501 ttgtttttgc gtgagccatg agaacgaacc attgagatca tacttacttt gcatgtcact
     1561 caaaaatttt gcctcaaaac tggtgagctg aatttttgca gttaaagcat cgtgtagtgt
     1621 ttttcttagt ccgttatgta ggtaggaatc tgatgtaatg gttgttggta ttttgtcacc
     1681 attcattttt atctggttgt tctcaagttc ggttacgaga tccatttgtc tatctagttc
     1741 aacttggaaa atcaacgtat cagtcgggcg gcctcgctta tcaaccacca atttcatatt
     1801 gctgtaagtg tttaaatctt tacttattgg tttcaaaacc cattggttaa gccttttaaa
     1861 ctcatggtag ttattttcaa gcattaacat gaacttaaat tcatcaaggc taatctctat
     1921 atttgccttg tgagttttct tttgtgttag ttcttttaat aaccactcat aaatcctcat
     1981 agagtatttg ttttcaaaag acttaacatg ttccagatta tattttatga atttttttaa
     2041 ctggaaaaga taaggcaata tctcttcact aaaaactaat tctaattttt cgcttgagaa
     2101 cttggcatag tttgtccact ggaaaatctc aaagccttta accaaaggat tcatgatttc
     2161 cacagttctc gtcatcagct ctctggttgc tttagctaat acaccataag cattttccct
     2221 actgatgttc atcatctgag cgtattggtt ataagtgaac gataccgtcc gttctttcct
     2281 tgtagggttt tcaatagtgg ggttgagtag tgccacacag cataaaatta gcttggtttc
     2341 atgctccgtt aagtcatagc gactaatcgc tagttcattt gctttgaaaa caactaattc
     2401 agacatacat ctcaattggt ctaggtgatt ttaatcacta taccaattga gatgggctag
     2461 tcaatgataa ttacatgtcc ttttcctttg agttgtgggt atctgtaaat tctgctagac
     2521 ctttgctgga aaacttgtaa attctgctag accctctgta aattccgcta gacctttgtg
     2581 tgtttttttt gtttatattc aagtggttat aatttataga ataaagaaag aataaaaaaa
     2641 gataaaaaga atagatccca gccctgtgta taactcacta ctttagtcag ttccgcagta
     2701 ttacaaaagg atgtcgcaaa cgctgtttgc tcctctacaa aacagacctt aaaaccctaa
     2761 aggcttaagt agcaccctcg caagctcgtg caaatcgctt aatattcctt ttgtctccga
     2821 ccatcaggca cctgagtcgc tgcctttttc gtgacattca gttcgctgcg ctcacggctc
     2881 tggcagtgaa tgtgggtaaa tggcactaca ggcgcctttt atggattcat gcaaggaaac
     2941 tacccataat acaagaaaag cccgtcacgg gcttctcagg gcgttttatg gctggtctgc
     3001 tatgtggtgc tatctgactt tttgctgttc agcagttcct gccctctgat tttccagtct
     3061 gaccacttcg gattatcccg tgacaggtca ttcagactgg ctaatgcacc cagtaaggca
     3121 gcggtatcat caacaggctt acccgtctta ctgtccctag tgcttggatt ctcaccaata
     3181 aaaaacgccc ggcggcaacc gagcgttctg aacaaatcca gatggagttc tgatgtcatt
     3241 actggatcta tcaacaggag tccaagcgag ctcgtaaact tggtctgaca gttaccaatg
     3301 cttaatcagt gaggcaccta tctcagcgat ctgtctattt cgttcatcca tagttgcctg
     3361 actccccgtc gtgtagataa ctacgatacg ggagggctta ccatctggcc ccagtgctgc
     3421 aatgataccg cgtgatccac gctcaccggc tccagattta tcagcaataa accagccagc
     3481 cggaagggcc gagcgcagaa gtggtcctgc aactttatcc gcctccatcc agtctattaa
     3541 ttgttgccgg gaagctagag taagtagttc gccagttaat agtttgcgca acgttgttgc
     3601 cattgctaca ggcatcgtgg tgtcacgctc gtcgtttggt atggcttcat tcagctccgg
     3661 ttcccaacga tcaaggcgag ttacatgatc ccccatgttg tgcaaaaaag cggttagctc
     3721 cttcggtcct ccgatcgttg tcagaagtaa gttggccgca gtgttatcac tcatggttat
     3781 ggcagcactg cataattctc ttactgtcat gccatccgta agatgctttt ctgttactgg
     3841 tgagtactca accaagtcat tctgagaata gtgtatgcgg cgaccgagtt gctcttgccc
     3901 ggcgtcaata cggtataata ccgcgccaca tagcagaact ttaaaagtgc tcatcattgg
     3961 aaaacgttct tcggggcgaa aactctcaag gatcttaccg ctgttgagat ccagttcgat
     4021 gtaacccact cgtgcaccca actgatcttc agcatctttt actttcacca gcgtttctgg
     4081 gtgagcaaaa acgggaaggc aaaatgccgc aaaaaagtga ataagggcga cactgaaatg
     4141 ttgaatactc atactcttcc tttttcaata ttattgaagc atttatcagg gttattgtct
     4201 catgagcgga tacatatttt aatgtattta gaaaaataaa caaatagggg ttccgcgcac
     4261 atttcccatg gtgccacctg acgtctaata aaccattatt atcatgacat taacctataa
     4321 aaataggcgt atcacgaggc agaattttta tcgcttccgc gttccgtcta ctccctatta
     4381 cgctccggaa tctccgacgc ctacggccga cgtcggatac cgccattgat aaactgttta
     4441 acaacatcgt ctgtacagac cttcttcgtt gcctttacgt ttaactcaat caggctaccg
     4501 tctcggttat aagtctatcg agtagtagag ccgtagtaaa gaggagaaat actagatggt
     4561 ttcgaaaggt gaggaggata acatggcgtc gctgcctgct acacatgaac tgcatatttt
     4621 cggcagcatc aacggagttg atttcgacat ggtgggccaa ggcaccggca acccgaacga
     4681 cggctatgaa gaattgaatt tgaaatctac caagggcgac ttgcaattta gtccgtggat
     4741 tctggtaccg catattggtt atggttttca tcagtatctg ccctacccag acggaatgag
     4801 cccatttcag gcggctatgg tggatggcag tggctatcag gtccatcgca ccatgcaatt
     4861 cgaggatggg gcgagcctga ccgtgaatta tcggtacact tacgaaggtt cacatattaa
     4921 aggtgaagca caagttaaag gcaccggctt cccggcggat ggcccagtga tgaccaacag
     4981 cctgacggcg gcggactggt gtcgtagcaa aaagacgtat cccaatgata aaacaattat
     5041 ttctaccttt aaatggagtt acaccacggg taacggtaaa cgctaccgta gcacggctcg
     5101 caccacatac acctttgcga aacccatggc cgcgaactat ctgaaaaatc agccgatgta
     5161 tgtgtttcgc aaaacggaac tgaaacattc gaaaactgaa ctcaatttca aagagtggca
     5221 aaaagccttc accgacgtaa tgggcatgga tgaactgtac aaatgaccag gcatcaaata
     5281 aaacgaaagg ctcagtcgaa agactgggcc tttcgtttta tctgttgttt gtcggtgaac
     5341 gctctctact agagtcacac tggctcacct tcgggtgggc ctttctgcgt ttatagtttc
     5401 acttctgagt tcggcatggg gtcaggtggg accaccgcgc tactgccgcc aggcagctgt
     5461 atcatctgcg tactccatta gtgtgacgac ctatgaggat ggcggggtat taactgtaac
     5521 gcaagatact tctctgcaag atggttgttt aatttataat gtgaaactgc gcggcgttaa
     5581 ttttccctca aatggtcccg taatgcaaaa gaagacctta gggtgggaag cttctacgga
     5641 aacgttgtat cctgcagatg gcggtcttga gggtcgttgt gatatggcgt tgaaactggt
     5701 cggcggtgga catttgcatt gtaatcttac atgttcttga ccttcttgct cagcaaccgg
     5761 acaatgtact tcacccaggt ttgcttcgga tttgttcctc cgatacccgc agctggagcc
     5821 tcagcggcat ctgcaggagc ggcttccgca gccggtgctt cactcgctgg tgcctccgct
     5881 gctggtgtag cttccgcagc tggtgcagca ggttcctgac tgatgccagc ggctggggct
     5941 tcggcagcat ccgctggggc agcttctgct gcaggcgctt cgctagctgg agcttcagca
     6001 gcaggagtcg cctctgcggc gggcgctgca ggctcttgac tggtaccaat accgttgtcc
     6061 ggacgagtgc cgatggtgtg tgcgtcaccg atgttgttgg tccactggta ttccagacgg
     6121 gtagcgattt caggagtgat cgcgtactca acaccgccag cgaagaccgg agaaacgccg
     6181 gtgtcgtggt ttttaccata aacgttggat ttagtgtctg cacgccatac catgccaccc
     6241 agacgagtgt agatgtccag gtcgtcagtg attgggtaac ccagtttagc ggtcagttga
     6301 acgccctgag ctttgtatgc accgttttca acgctgcctt tgtacggcat acgacctaac
     6361 cagtcgtaac ccatttcaaa gccaacatac gggttaattc cctgatcgat tttagcgttg
     6421 ctggagcaac ctgccagcag agtagaaccc aggattaccg cgcccagtac cagtttagta
     6481 gctttcatca gaaatactcc atagcatgat taagatgttt cagtacgaaa attgctttca
     6541 ttgttgatct cctttttaag tgaacttggg ccctgtgtga aattgttatc cgctcacaat
     6601 tccacacatt atacgagccg atgattaatt gtcaacagct catttcagaa tattagccag
     6661 aaccgttatg atgtcggcgc aaaaaacatt atccagaacg ggagtgcgcc ttgagcgaca
     6721 cgaattatgc agtgatttac gtattaccgc ctttgagtga gctgataccg ctcgccgcag
     6781 ccgaacgccc taggtctagg gcggcggatt tgtcctactc aggagagcgt tcaccgacaa
     6841 acaacagata aaacgaaagg cccagtcttt cgactgagcc tttcgtttta tttgatgcct
     6901 cactgcccgc tttccagtcg ggaaacctgt cgtgccagct gcattaatga atcggccaac
     6961 gcgcggggag agg
//
