LOCUS       sequence-335295-        6973 bp    DNA     circular SYN 15-APR-2025
DEFINITION  Encodes LacI-controlled expression of surface-displayed CCL19 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 Apr 15, 2025 from SnapGene Server 8.0.1
            https://www.snapgene.com
COMMENT     SGRef: number: 1; type: "Journal Article"; journalName: "bioRxiv"
COMMENT     Sequence Label: pKB237
FEATURES             Location/Qualifiers
     source          1..6973
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     951..970
                     /label=LacI-R
                     /note="LacI, reverse primer"
     promoter        complement(990..1067)
                     /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(1157..1174)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     CDS             complement(1436..2386)
                     /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      2434..2656
                     /label=pSC101 ori
                     /note="low-copy replication origin that requires the Rep101
                     protein"
     terminator      complement(3148..3242)
                     /label=lambda t0 terminator
                     /note="transcription terminator from phage lambda"
     CDS             complement(join(3272..4063,4064..4132))
                     /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     3895..3914
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(4133..4237)
                     /gene="bla"
                     /label=AmpR promoter
     primer_bind     4302..4320
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     RBS             4518..4529
                     /note="strong bacterial ribosome binding site (Elowitz and 
                     Leibler, 2000)"
     terminator      5255..5326
                     /label=rrnB T1 terminator
                     /note="transcription terminator T1 from the E. coli rrnB 
                     gene"
     terminator      5342..5369
                     /label=T7Te terminator
                     /note="phage T7 early transcription terminator"
     primer_bind     complement(6552..6574)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    6563..6579
                     /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(6587..6593,6594..6609,6610..6615))
                     /label=tac promoter
                     /note="strong E. coli promoter; hybrid between the trp and 
                     lac UV5 promoters"
     terminator      complement(6788..6874)
                     /gene="Escherichia coli rrnB"
                     /label=rrnB T1 terminator
                     /note="transcription terminator T1 from the E. coli rrnB 
                     gene"
     CDS             complement(join(6880..6973,1..989))
                     /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 agggtggttt ttcttttcac cagtgagacg ggcaacagct gattgccctt caccgcctgg
       61 ccctgagaga gttgcagcaa gcggtccacg ctggtttgcc ccagcaggcg aaaatcctgt
      121 ttgatggtgg ttaacggcgg gatataacat gagctgtctt cggtatcgtc gtatcccact
      181 accgagatat ccgcaccaac gcgcagcccg gactcggtaa tggcgcgcat tgcgcccagc
      241 gccatctgat cgttggcaac cagcatcgca gtgggaacga tgccctcatt cagcatttgc
      301 atggtttgtt gaaaaccgga catggcactc cagtcgcctt cccgttccgc tatcggctga
      361 atttgattgc gagtgagata tttatgccag ccagccagac gcagacgcgc cgagacagaa
      421 cttaatgggc ccgctaacag cgcgatttgc tggtgaccca atgcgaccag atgctccacg
      481 cccagtcgcg taccgtcttc atgggagaaa ataatactgt tgatgggtgt ctggtcagag
      541 acatcaagaa ataacgccgg aacattagtg caggcagctt ccacagcaat ggcatcctgg
      601 tcatccagcg gatagttaat gatcagccca ctgacgcgtt gcgcgagaag attgtgcacc
      661 gccgctttac aggcttcgac gccgcttcgt tctaccatcg acaccaccac gctggcaccc
      721 agttgatcgg cgcgagattt aatcgccgcg acaatttgcg acggcgcgtg cagggccaga
      781 ctggaggtgg caacgccaat cagcaacgac tgtttgcccg ccagttgttg tgccacgcgg
      841 ttgggaatgt aattcagctc cgccatcgcc gcttccactt tttcccgcgt tttcgcagaa
      901 acgtggctgg cctggttcac cacgcgggaa acggtctgat aagagacacc ggcatactct
      961 gcgacatcgt ataacgttac tggtttcaca ttcaccaccc tgaattgact ctcttccggg
     1021 cgctatcatg ccataccgcg aaaggttttg caccattcga tggtgtcaac gtaaatgcat
     1081 gccgcttcgg caaggtgtca ccaccctgcc ctttttcttt aaaaccgaaa agattacttc
     1141 gcgttatgca ggcttcctcg ctcactgact cgctgcgctc ggtcgttcgg ctgcggcgag
     1201 cggtatcagc tcactcaaag gcggtaatct cgaggttaca ttgtcgatct gttcatggtg
     1261 aacagctttg aatgcaccaa aaactcgtaa aagctctgat gtatctatct tttttacacc
     1321 gttttcatct gtgcatatgg acagttttcc ctttgatatg taacggtgaa cagttgttct
     1381 acttttgttt gttagtcttg atgcttcact gatagataca agagccataa gaacctcaga
     1441 tccttccgta tttagccagt atgttctcta gtgtggttcg ttgtttttgc gtgagccatg
     1501 agaacgaacc attgagatca tacttacttt gcatgtcact caaaaatttt gcctcaaaac
     1561 tggtgagctg aatttttgca gttaaagcat cgtgtagtgt ttttcttagt ccgttatgta
     1621 ggtaggaatc tgatgtaatg gttgttggta ttttgtcacc attcattttt atctggttgt
     1681 tctcaagttc ggttacgaga tccatttgtc tatctagttc aacttggaaa atcaacgtat
     1741 cagtcgggcg gcctcgctta tcaaccacca atttcatatt gctgtaagtg tttaaatctt
     1801 tacttattgg tttcaaaacc cattggttaa gccttttaaa ctcatggtag ttattttcaa
     1861 gcattaacat gaacttaaat tcatcaaggc taatctctat atttgccttg tgagttttct
     1921 tttgtgttag ttcttttaat aaccactcat aaatcctcat agagtatttg ttttcaaaag
     1981 acttaacatg ttccagatta tattttatga atttttttaa ctggaaaaga taaggcaata
     2041 tctcttcact aaaaactaat tctaattttt cgcttgagaa cttggcatag tttgtccact
     2101 ggaaaatctc aaagccttta accaaaggat tcatgatttc cacagttctc gtcatcagct
     2161 ctctggttgc tttagctaat acaccataag cattttccct actgatgttc atcatctgag
     2221 cgtattggtt ataagtgaac gataccgtcc gttctttcct tgtagggttt tcaatagtgg
     2281 ggttgagtag tgccacacag cataaaatta gcttggtttc atgctccgtt aagtcatagc
     2341 gactaatcgc tagttcattt gctttgaaaa caactaattc agacatacat ctcaattggt
     2401 ctaggtgatt ttaatcacta taccaattga gatgggctag tcaatgataa ttacatgtcc
     2461 ttttcctttg agttgtgggt atctgtaaat tctgctagac ctttgctgga aaacttgtaa
     2521 attctgctag accctctgta aattccgcta gacctttgtg tgtttttttt gtttatattc
     2581 aagtggttat aatttataga ataaagaaag aataaaaaaa gataaaaaga atagatccca
     2641 gccctgtgta taactcacta ctttagtcag ttccgcagta ttacaaaagg atgtcgcaaa
     2701 cgctgtttgc tcctctacaa aacagacctt aaaaccctaa aggcttaagt agcaccctcg
     2761 caagctcgtg caaatcgctt aatattcctt ttgtctccga ccatcaggca cctgagtcgc
     2821 tgcctttttc gtgacattca gttcgctgcg ctcacggctc tggcagtgaa tgtgggtaaa
     2881 tggcactaca ggcgcctttt atggattcat gcaaggaaac tacccataat acaagaaaag
     2941 cccgtcacgg gcttctcagg gcgttttatg gctggtctgc tatgtggtgc tatctgactt
     3001 tttgctgttc agcagttcct gccctctgat tttccagtct gaccacttcg gattatcccg
     3061 tgacaggtca ttcagactgg ctaatgcacc cagtaaggca gcggtatcat caacaggctt
     3121 acccgtctta ctgtccctag tgcttggatt ctcaccaata aaaaacgccc ggcggcaacc
     3181 gagcgttctg aacaaatcca gatggagttc tgatgtcatt actggatcta tcaacaggag
     3241 tccaagcgag ctcgtaaact tggtctgaca gttaccaatg cttaatcagt gaggcaccta
     3301 tctcagcgat ctgtctattt cgttcatcca tagttgcctg actccccgtc gtgtagataa
     3361 ctacgatacg ggagggctta ccatctggcc ccagtgctgc aatgataccg cgtgatccac
     3421 gctcaccggc tccagattta tcagcaataa accagccagc cggaagggcc gagcgcagaa
     3481 gtggtcctgc aactttatcc gcctccatcc agtctattaa ttgttgccgg gaagctagag
     3541 taagtagttc gccagttaat agtttgcgca acgttgttgc cattgctaca ggcatcgtgg
     3601 tgtcacgctc gtcgtttggt atggcttcat tcagctccgg ttcccaacga tcaaggcgag
     3661 ttacatgatc ccccatgttg tgcaaaaaag cggttagctc cttcggtcct ccgatcgttg
     3721 tcagaagtaa gttggccgca gtgttatcac tcatggttat ggcagcactg cataattctc
     3781 ttactgtcat gccatccgta agatgctttt ctgttactgg tgagtactca accaagtcat
     3841 tctgagaata gtgtatgcgg cgaccgagtt gctcttgccc ggcgtcaata cggtataata
     3901 ccgcgccaca tagcagaact ttaaaagtgc tcatcattgg aaaacgttct tcggggcgaa
     3961 aactctcaag gatcttaccg ctgttgagat ccagttcgat gtaacccact cgtgcaccca
     4021 actgatcttc agcatctttt actttcacca gcgtttctgg gtgagcaaaa acgggaaggc
     4081 aaaatgccgc aaaaaagtga ataagggcga cactgaaatg ttgaatactc atactcttcc
     4141 tttttcaata ttattgaagc atttatcagg gttattgtct catgagcgga tacatatttt
     4201 aatgtattta gaaaaataaa caaatagggg ttccgcgcac atttcccatg gtgccacctg
     4261 acgtctaata aaccattatt atcatgacat taacctataa aaataggcgt atcacgaggc
     4321 agaattttta tcgcttccgc gttccgtcta ctccctatta cgctccggaa tctccgacgc
     4381 ctacggccga cgtcggatac cgccattgat aaactgttta acaacatcgt ctgtacagac
     4441 cttcttcgtt gcctttacgt ttaactcaat caggctaccg tctcggttat aagtctatcg
     4501 agtagtagag ccgtagtaaa gaggagaaat actagatggt ttcgaaaggt gaggaggata
     4561 acatggcgtc gctgcctgct acacatgaac tgcatatttt cggcagcatc aacggagttg
     4621 atttcgacat ggtgggccaa ggcaccggca acccgaacga cggctatgaa gaattgaatt
     4681 tgaaatctac caagggcgac ttgcaattta gtccgtggat tctggtaccg catattggtt
     4741 atggttttca tcagtatctg ccctacccag acggaatgag cccatttcag gcggctatgg
     4801 tggatggcag tggctatcag gtccatcgca ccatgcaatt cgaggatggg gcgagcctga
     4861 ccgtgaatta tcggtacact tacgaaggtt cacatattaa aggtgaagca caagttaaag
     4921 gcaccggctt cccggcggat ggcccagtga tgaccaacag cctgacggcg gcggactggt
     4981 gtcgtagcaa aaagacgtat cccaatgata aaacaattat ttctaccttt aaatggagtt
     5041 acaccacggg taacggtaaa cgctaccgta gcacggctcg caccacatac acctttgcga
     5101 aacccatggc cgcgaactat ctgaaaaatc agccgatgta tgtgtttcgc aaaacggaac
     5161 tgaaacattc gaaaactgaa ctcaatttca aagagtggca aaaagccttc accgacgtaa
     5221 tgggcatgga tgaactgtac aaatgaccag gcatcaaata aaacgaaagg ctcagtcgaa
     5281 agactgggcc tttcgtttta tctgttgttt gtcggtgaac gctctctact agagtcacac
     5341 tggctcacct tcgggtgggc ctttctgcgt ttatagtttc acttctgagt tcggcatggg
     5401 gtcaggtggg accaccgcgc tactgccgcc aggcagctgt atcatctgcg tactccatta
     5461 gtgtgacgac ctatgaggat ggcggggtat taactgtaac gcaagatact tctctgcaag
     5521 atggttgttt aatttatatt agctactacg acgcttcatc ttagcagacg tacgctgcaa
     5581 acgctgaata atacgctcaa cccaaggctg atcaggagga gcacataact gacgacctcg
     5641 caaagtagtg aagacgacgg caggaactct acacccatcc ttgatcagca agtagtggaa
     5701 gttacggacg atgtaaccag gaataggctt ctgagtcaca gaaaggcagc aatcctcagc
     5761 atcatttgtt cctgttcctc cgatacccgc agctggagcc tcagcggcat ctgcaggagc
     5821 ggcttccgca gccggtgctt cactcgctgg tgcctccgct gctggtgtag cttccgcagc
     5881 tggtgcagca ggttcctgac tgatgccagc ggctggggct tcggcagcat ccgctggggc
     5941 agcttctgct gcaggcgctt cgctagctgg agcttcagca gcaggagtcg cctctgcggc
     6001 gggcgctgca ggctcttgac tggtaccaat accgttgtcc ggacgagtgc cgatggtgtg
     6061 tgcgtcaccg atgttgttgg tccactggta ttccagacgg gtagcgattt caggagtgat
     6121 cgcgtactca acaccgccag cgaagaccgg agaaacgccg gtgtcgtggt ttttaccata
     6181 aacgttggat ttagtgtctg cacgccatac catgccaccc agacgagtgt agatgtccag
     6241 gtcgtcagtg attgggtaac ccagtttagc ggtcagttga acgccctgag ctttgtatgc
     6301 accgttttca acgctgcctt tgtacggcat acgacctaac cagtcgtaac ccatttcaaa
     6361 gccaacatac gggttaattc cctgatcgat tttagcgttg ctggagcaac ctgccagcag
     6421 agtagaaccc aggattaccg cgcccagtac cagtttagta gctttcatca gaaatactcc
     6481 atagcatgat taagatgttt cagtacgaaa attgctttca ttgttgatct cctttttaag
     6541 tgaacttggg ccctgtgtga aattgttatc cgctcacaat tccacacatt atacgagccg
     6601 atgattaatt gtcaacagct catttcagaa tattagccag aaccgttatg atgtcggcgc
     6661 aaaaaacatt atccagaacg ggagtgcgcc ttgagcgaca cgaattatgc agtgatttac
     6721 gtattaccgc ctttgagtga gctgataccg ctcgccgcag ccgaacgccc taggtctagg
     6781 gcggcggatt tgtcctactc aggagagcgt tcaccgacaa acaacagata aaacgaaagg
     6841 cccagtcttt cgactgagcc tttcgtttta tttgatgcct cactgcccgc tttccagtcg
     6901 ggaaacctgt cgtgccagct gcattaatga atcggccaac gcgcggggag aggcggtttg
     6961 cgtattgggc gcc
//
