LOCUS       sequence-335942-        6973 bp    DNA     circular SYN 14-APR-2025
DEFINITION  Encodes LacI-controlled expression of surface-displayed C18G 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 14, 2025 from SnapGene Server 8.0.1
            https://www.snapgene.com
COMMENT     SGRef: number: 1; type: "Journal Article"; journalName: "bioRxiv"
COMMENT     Sequence Label: pKB223
FEATURES             Location/Qualifiers
     source          1..6973
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     893..912
                     /label=LacI-R
                     /note="LacI, reverse primer"
     promoter        complement(932..1009)
                     /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(1099..1116)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     CDS             complement(1378..2328)
                     /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      2376..2598
                     /label=pSC101 ori
                     /note="low-copy replication origin that requires the Rep101
                     protein"
     terminator      complement(3090..3184)
                     /label=lambda t0 terminator
                     /note="transcription terminator from phage lambda"
     CDS             complement(join(3214..4005,4006..4074))
                     /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     3837..3856
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(4075..4179)
                     /gene="bla"
                     /label=AmpR promoter
     primer_bind     4244..4262
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     RBS             4460..4471
                     /note="strong bacterial ribosome binding site (Elowitz and 
                     Leibler, 2000)"
     terminator      5197..5268
                     /label=rrnB T1 terminator
                     /note="transcription terminator T1 from the E. coli rrnB 
                     gene"
     terminator      5284..5311
                     /label=T7Te terminator
                     /note="phage T7 early transcription terminator"
     primer_bind     complement(6494..6516)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    6505..6521
                     /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(6529..6535,6536..6551,6552..6557))
                     /label=tac promoter
                     /note="strong E. coli promoter; hybrid between the trp and 
                     lac UV5 promoters"
     terminator      complement(6730..6816)
                     /gene="Escherichia coli rrnB"
                     /label=rrnB T1 terminator
                     /note="transcription terminator T1 from the E. coli rrnB 
                     gene"
     CDS             complement(join(6822..6973,1..931))
                     /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 ggccctgaga gagttgcagc aagcggtcca cgctggtttg ccccagcagg cgaaaatcct
       61 gtttgatggt ggttaacggc gggatataac atgagctgtc ttcggtatcg tcgtatccca
      121 ctaccgagat atccgcacca acgcgcagcc cggactcggt aatggcgcgc attgcgccca
      181 gcgccatctg atcgttggca accagcatcg cagtgggaac gatgccctca ttcagcattt
      241 gcatggtttg ttgaaaaccg gacatggcac tccagtcgcc ttcccgttcc gctatcggct
      301 gaatttgatt gcgagtgaga tatttatgcc agccagccag acgcagacgc gccgagacag
      361 aacttaatgg gcccgctaac agcgcgattt gctggtgacc caatgcgacc agatgctcca
      421 cgcccagtcg cgtaccgtct tcatgggaga aaataatact gttgatgggt gtctggtcag
      481 agacatcaag aaataacgcc ggaacattag tgcaggcagc ttccacagca atggcatcct
      541 ggtcatccag cggatagtta atgatcagcc cactgacgcg ttgcgcgaga agattgtgca
      601 ccgccgcttt acaggcttcg acgccgcttc gttctaccat cgacaccacc acgctggcac
      661 ccagttgatc ggcgcgagat ttaatcgccg cgacaatttg cgacggcgcg tgcagggcca
      721 gactggaggt ggcaacgcca atcagcaacg actgtttgcc cgccagttgt tgtgccacgc
      781 ggttgggaat gtaattcagc tccgccatcg ccgcttccac tttttcccgc gttttcgcag
      841 aaacgtggct ggcctggttc accacgcggg aaacggtctg ataagagaca ccggcatact
      901 ctgcgacatc gtataacgtt actggtttca cattcaccac cctgaattga ctctcttccg
      961 ggcgctatca tgccataccg cgaaaggttt tgcaccattc gatggtgtca acgtaaatgc
     1021 atgccgcttc ggcaaggtgt caccaccctg ccctttttct ttaaaaccga aaagattact
     1081 tcgcgttatg caggcttcct cgctcactga ctcgctgcgc tcggtcgttc ggctgcggcg
     1141 agcggtatca gctcactcaa aggcggtaat ctcgaggtta cattgtcgat ctgttcatgg
     1201 tgaacagctt tgaatgcacc aaaaactcgt aaaagctctg atgtatctat cttttttaca
     1261 ccgttttcat ctgtgcatat ggacagtttt ccctttgata tgtaacggtg aacagttgtt
     1321 ctacttttgt ttgttagtct tgatgcttca ctgatagata caagagccat aagaacctca
     1381 gatccttccg tatttagcca gtatgttctc tagtgtggtt cgttgttttt gcgtgagcca
     1441 tgagaacgaa ccattgagat catacttact ttgcatgtca ctcaaaaatt ttgcctcaaa
     1501 actggtgagc tgaatttttg cagttaaagc atcgtgtagt gtttttctta gtccgttatg
     1561 taggtaggaa tctgatgtaa tggttgttgg tattttgtca ccattcattt ttatctggtt
     1621 gttctcaagt tcggttacga gatccatttg tctatctagt tcaacttgga aaatcaacgt
     1681 atcagtcggg cggcctcgct tatcaaccac caatttcata ttgctgtaag tgtttaaatc
     1741 tttacttatt ggtttcaaaa cccattggtt aagcctttta aactcatggt agttattttc
     1801 aagcattaac atgaacttaa attcatcaag gctaatctct atatttgcct tgtgagtttt
     1861 cttttgtgtt agttctttta ataaccactc ataaatcctc atagagtatt tgttttcaaa
     1921 agacttaaca tgttccagat tatattttat gaattttttt aactggaaaa gataaggcaa
     1981 tatctcttca ctaaaaacta attctaattt ttcgcttgag aacttggcat agtttgtcca
     2041 ctggaaaatc tcaaagcctt taaccaaagg attcatgatt tccacagttc tcgtcatcag
     2101 ctctctggtt gctttagcta atacaccata agcattttcc ctactgatgt tcatcatctg
     2161 agcgtattgg ttataagtga acgataccgt ccgttctttc cttgtagggt tttcaatagt
     2221 ggggttgagt agtgccacac agcataaaat tagcttggtt tcatgctccg ttaagtcata
     2281 gcgactaatc gctagttcat ttgctttgaa aacaactaat tcagacatac atctcaattg
     2341 gtctaggtga ttttaatcac tataccaatt gagatgggct agtcaatgat aattacatgt
     2401 ccttttcctt tgagttgtgg gtatctgtaa attctgctag acctttgctg gaaaacttgt
     2461 aaattctgct agaccctctg taaattccgc tagacctttg tgtgtttttt ttgtttatat
     2521 tcaagtggtt ataatttata gaataaagaa agaataaaaa aagataaaaa gaatagatcc
     2581 cagccctgtg tataactcac tactttagtc agttccgcag tattacaaaa ggatgtcgca
     2641 aacgctgttt gctcctctac aaaacagacc ttaaaaccct aaaggcttaa gtagcaccat
     2701 cgcaagctcg tgcaaatcgc ttaatattcc ttttgtctcc gaccatcagg cacctgagtc
     2761 gctgtctttt tcgtgacatt cagttcgctg cgctcacggc tctggcagtg aatgtgggta
     2821 aatggcacta caggcgcctt ttatggattc atgcaaggaa actacccata atacaagaaa
     2881 agcccgtcac gggcttctca gggcgtttta tggctggtct gctatgtggt gctatctgac
     2941 tttttgctgt tcagcagttc ctgccctctg attttccagt ctgaccactt cggattatcc
     3001 cgtgacaggt cattcagact ggctaatgca cccagtaagg cagcggtatc atcaacaggc
     3061 ttacccgtct tactgtccct agtgcttgga ttctcaccaa taaaaaacgc ccggcggcaa
     3121 ccgagcgttc tgaacaaatc cagatggagt tctgatgtca ttactggatc tatcaacagg
     3181 agtccaagcg agctcgtaaa cttggtctga cagttaccaa tgcttaatca gtgaggcacc
     3241 tatctcagcg atctgtctat ttcgttcatc catagttgcc tgactccccg tcgtgtagat
     3301 aactacgata cgggagggct taccatctgg ccccagtgct gcaatgatac cgcgtgatcc
     3361 acgctcaccg gctccagatt tatcagcaat aaaccagcca gccggaaggg ccgagcgcag
     3421 aagtggtcct gcaactttat ccgcctccat ccagtctatt aattgttgcc gggaagctag
     3481 agtaagtagt tcgccagtta atagtttgcg caacgttgtt gccattgcta caggcatcgt
     3541 ggtgtcacgc tcgtcgtttg gtatggcttc attcagctcc ggttcccaac gatcaaggcg
     3601 agttacatga tcccccatgt tgtgcaaaaa agcggttagc tccttcggtc ctccgatcgt
     3661 tgtcagaagt aagttggccg cagtgttatc actcatggtt atggcagcac tgcataattc
     3721 tcttactgtc atgccatccg taagatgctt ttctgttact ggtgagtact caaccaagtc
     3781 attctgagaa tagtgtatgc ggcgaccgag ttgctcttgc ccggcgtcaa tacggtataa
     3841 taccgcgcca catagcagaa ctttaaaagt gctcatcatt ggaaaacgtt cttcggggcg
     3901 aaaactctca aggatcttac cgctgttgag atccagttcg atgtaaccca ctcgtgcacc
     3961 caactgatct tcagcatctt ttactttcac cagcgtttct gggtgagcaa aaacaggaag
     4021 gcaaaatgcc gcaaaaaagt gaataagggc gacactgaaa tgttgaatac tcatactctt
     4081 cctttttcaa tattattgaa gcatttatca gggttattgt ctcatgagcg gatacatatt
     4141 ttaatgtatt tagaaaaata aacaaatagg ggttccgcgc acatttccca tggtgccacc
     4201 tgacgtctaa taaaccatta ttatcatgac attaacctat aaaaataggc gtatcacgag
     4261 gcagaatttt tatcgcttcc gcgttccgtc tactccctat tacgctccgg aatctccgac
     4321 gcctacggcc gacgtcggat accgccattg ataaactgtt taacaacatc gtctgtacag
     4381 accttcttcg ttgcctttac gtttaactca atcaggctac cgtctcggtt ataagtctat
     4441 cgagtagtag agccgtagta aagaggagaa atactagatg gtttcgaaag gtgaggagga
     4501 taacatggcg tcgctgcctg ctacacatga actgcatatt ttcggcagca tcaacggagt
     4561 tgatttcgac atggtgggcc aaggcaccgg caacccgaac gacggctatg aagaattgaa
     4621 tttgaaatct accaagggcg acttgcaatt tagtccgtgg attctggtac cgcatattgg
     4681 ttatggtttt catcagtatc tgccctaccc agacggaatg agcccatttc aggcggctat
     4741 ggtggatggc agtggctatc aggtccatcg caccatgcaa ttcgaggatg gggcgagcct
     4801 gaccgtgaat tatcggtaca cttacgaagg ttcacatatt aaaggtgaag cacaagttaa
     4861 aggcaccggc ttcccggcgg atggcccagt gatgaccaac agcctgacgg cggcggactg
     4921 gtgtcgtagc aaaaagacgt atcccaatga taaaacaatt atttctacct ttaaatggag
     4981 ttacaccacg ggtaacggta aacgctaccg tagcacggct cgcaccacat acacctttgc
     5041 gaaacccatg gccgcgaact atctgaaaaa tcagccgatg tatgtgtttc gcaaaacgga
     5101 actgaaacat tcgaaaactg aactcaattt caaagagtgg caaaaagcct tcaccgacgt
     5161 aatgggcatg gatgaactgt acaaatgacc aggcatcaaa taaaacgaaa ggctcagtcg
     5221 aaagactggg cctttcgttt tatctgttgt ttgtcggtga acgctctcta ctagagtcac
     5281 actggctcac cttcgggtgg gcctttctgc gtttatagtt tcacttctga gttcggcatg
     5341 gggtcaggtg ggaccaccgc gctactgccg ccaggcagct gtatcatctg cgtactccat
     5401 tagtgtgacg acctatgagg atggcggggt attaactgta acgcaagata cttctctgca
     5461 agatggttgt ttaatttata atgtgaaact gcgcggcgtt aattttccct caaatggtcc
     5521 cgtaatgcaa aagaagacct tagggtggga agcttctacg gaaacgttgt atcctgcaga
     5581 tggcggtctt gagggtcgtt gtgatatggc gttgaaactg gtcggcggtg gacatttgca
     5641 ttgtaatctg ctgagtctta gcccaacttt ttagcggatt tcaacaactt ctttaacaac
     5701 ttcttatata acgcggttcc tccgataccc gcagctggag cctcagcggc atctgcagga
     5761 gcggcttcag cagccggtgc ttcactcgct ggtgcctccg ctgctggtgt agcttccgca
     5821 gctggtgcag caggttcctg actgatgcca gcggctgggg cttcggcagc atccgctggg
     5881 gcagcttctg ctgcaggcgc ttcgctagct ggagcttcag cagcaggagt cgcctctgcg
     5941 gcgggcgctg caggctcttg actggtacca ataccgttgt ccggacgagt gccgatggtg
     6001 tgtgcgtcac cgatgttgtt ggtccactgg tattccagac gggtagcgat ttcaggagtg
     6061 atcgcgtact caacaccgcc agcgaagacc ggagaaacgc cggtgtcgtg gtttttacca
     6121 taaacgttgg atttagtgtc tgcacgccat accatgccac ccagacgagt gtagatgtcc
     6181 aggtcgtcag tgattgggta acccagttta gcggtcagtt gaacgccctg agctttgtat
     6241 gcaccgtttt caacgctgcc tttgtacggc atacgaccta accagtcgta acccatttca
     6301 aagccaacat acgggttaat tccctgatcg attttagcgt tgctggagca acctgccagc
     6361 agagtagaac ccaggattac cgcgcccagt accagtttag tagctttcat cagaaatact
     6421 ccatagcatg attaagatgt ttcagtacga aaattgcttt cattgttgat ctccttttta
     6481 agtgaacttg ggccctgtgt gaaattgtta tccgctcaca attccacaca ttatacgagc
     6541 cgatgattaa ttgtcaacag ctcatttcag aatattagcc agaaccgtta tgatgtcggc
     6601 gcaaaaaaca ttatccagaa cgggagtgcg ccttgagcga cacgaattat gcagtgattt
     6661 acgtattacc gcctttgagt gagctgatac cgctcgccgc agccgaacgc cctaggtcta
     6721 gggcggcgga tttgtcctac tcaggagagc gttcaccgac aaacaacaga taaaacgaaa
     6781 ggcccagtct ttcgactgag cctttcgttt tatttgatgc ctcactgccc gctttccagt
     6841 cgggaaacct gtcgtgccag ctgcattaat gaatcggcca acgcgcgggg agaggcggtt
     6901 tgcgtattgg gcgccagggt ggtttttctt ttcaccagtg agacgggcaa cagctgattg
     6961 cccttcaccg cct
//
