LOCUS       sequence-335939-        6154 bp    DNA     circular SYN 14-APR-2025
DEFINITION  Encodes PpmrD-sfgfp reporter and TetR-controlled expression of S. 
            Typhimurium PhoP.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 6154)
  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 6154)
  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: pAM002
FEATURES             Location/Qualifiers
     source          1..6154
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     RBS             463..474
                     /note="strong bacterial ribosome binding site (Elowitz and 
                     Leibler, 2000)"
     primer_bind     complement(658..676)
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     promoter        741..845
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(846..914,915..1706)
                     /codon_start=1
                     /gene="bla"
                     /product="beta-lactamase"
                     /label=AmpR
                     /note="confers resistance to ampicillin, carbenicillin, and
                     related antibiotics"
                     /translation="MSIQHFSVALIPFFAAFCLPVFAHPETLVKVKDAEDQLGARVGYI
                     ELDLNSGKILESFRPEERFPMMSTFKVLLCGAVLYRIDAGQEQLGRRIHYSQNDLVEYS
                     PVTEKHLTDGMTVRELCSAAITMSDNTAANLLLTTIGGPKELTAFLHNMGDHVTRLDRW
                     EPELNEAIPNDERDTTMPVAMATTLRKLLTGELLTLASRQQLIDWMEADKVAGPLLRSA
                     LPAGWFIADKSGAGERGSRGIIAALGPDGKPSRIVVIYTTGSQATMDERNRQIAEIGAS
                     LIKHW"
     primer_bind     complement(1064..1083)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     terminator      1736..1830
                     /label=lambda t0 terminator
                     /note="transcription terminator from phage lambda"
     rep_origin      2322..2544
                     /label=pSC101 ori
                     /note="low-copy replication origin that requires the Rep101
                     protein"
     CDS             2592..3542
                     /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     3804..3821
                     /label=L4440
                     /note="L4440 vector, forward primer"
     terminator      complement(3905..3991)
                     /gene="Escherichia coli rrnB"
                     /label=rrnB T1 terminator
                     /note="transcription terminator T1 from the E. coli rrnB 
                     gene"
     CDS             complement(3997..4623)
                     /codon_start=1
                     /gene="tetR"
                     /product="tetracycline repressor TetR"
                     /label=TetR
                     /note="TetR binds to the tetracycline operator tetO to 
                     inhibit transcription. This inhibition can be relieved by 
                     adding tetracycline or doxycycline."
                     /translation="MMSRLDKSKVINSALELLNEVGIEGLTTRKLAQKLGVEQPTLYWH
                     VKNKRALLDALAIEMLDRHHTHFCPLEGESWQDFLRNNAKSFRCALLSHRDGAKVHLGT
                     RPTEKQYETLENQLAFLCQQGFSLENALYALSAVGHFTLGCVLEDQEHQVAKEERETPT
                     TDSMPPLLRQAIELFDHQGAEPAFLFGLELIICGLEKQLKCESGS"
     primer_bind     4528..4547
                     /label=Tet-R
                     /note="Tetracycline resistance gene, reverse primer"
     promoter        complement(4658..4692)
                     /label=BBa_J23104
                     /note="Anderson constitutive promoter 
                     (https://parts.igem.org/Promoters/Catalog/Anderson). 
                     Measured strength relative to BBa_J223100: 0.72"
     promoter        complement(join(5554..5579,5580..5585,5586..5602,5603..5608,
                     5609..5627))
                     /label=PLtetO-1 promoter
                     /note="modified phage lambda PL promoter with tet operator 
                     sites (Lutz and Bujard, 1997)"
     protein_bind    5584..5602
                     /gene="tetO"
                     /label=tet operator
                     /bound_moiety="tetracycline repressor TetR"
                     /note="bacterial operator O2 for the tetR and tetA genes"
     protein_bind    5609..5627
                     /gene="tetO"
                     /label=tet operator
                     /bound_moiety="tetracycline repressor TetR"
                     /note="bacterial operator O2 for the tetR and tetA genes"
     terminator      complement(5704..5731)
                     /label=T7Te terminator
                     /note="phage T7 early transcription terminator"
     terminator      complement(5747..5818)
                     /label=rrnB T1 terminator
                     /note="transcription terminator T1 from the E. coli rrnB 
                     gene"
     CDS             complement(join(5897..6154,1..456))
                     /codon_start=1
                     /product="GFP variant that folds robustly even when fused 
                     to poorly folded proteins (Pedelacq et al., 2006)"
                     /label=superfolder GFP
                     /translation="MRKGEELFTGVVPILVELDGDVNGHKFSVRGEGEGDATNGKLTLK
                     FICTTGKLPVPWPTLVTTLTYGVQCFARYPDHMKQHDFFKSAMPEGYVQERTISFKDDG
                     TYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNFNSHNVYITADKQKNGIKA
                     NFKIRHNVEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSVLSKDPNEKRDHMVLLE
                     FVTAAGITHGMDELYK"
ORIGIN
        1 gatgtaaaca ttgtggctgt taaaattgta ttccagctta tggcccagga tattgccgtc
       61 ttctttaaag tcaatgcctt tcagctcaat gcggtttacc agggtatcgc cttcaaattt
      121 cacttccgca cgcgttttgt acgtgccgtc atccttaaag gaaatcgtgc gttcctgcac
      181 atagccttcc ggcatggcgg acttgaagaa gtcatgctgc ttcatatggt ccggataacg
      241 agcaaagcac tgaacaccat aagtcagcgt cgttaccaga gtcggccaag gtaccggcag
      301 tttaccagta gtacagatga acttcagcgt cagtttacca ttagttgcgt caccttcacc
      361 ctcgccacgc acggaaaact tatgaccgtt gacatcacca tccagttcca ccagaatagg
      421 gacgacacca gtgaacagct cttcgccttt acgcatctag tatttctcct ctttagcgcc
      481 cccgtttcac gctgattgtt cataacacgt tacacatcaa cggataaaca aaacggcaat
      541 agcgatggaa tggttgtata ttaacctaac attaattttt tccgacgtcg gccgtaggcg
      601 tcggagattc cggagcgtaa tagggagtag acggaacgcg gaagcgataa aaattctgcc
      661 tcgtgatacg cctattttta taggttaatg tcatgataat aatggtttat tagacgtcag
      721 gtggcaccat gggaaatgtg cgcggaaccc ctatttgttt atttttctaa atacattaaa
      781 atatgtatcc gctcatgaga caataaccct gataaatgct tcaataatat tgaaaaagga
      841 agagtatgag tattcaacat ttcagtgtcg cccttattcc cttttttgcg gcattttgcc
      901 ttcctgtttt tgctcaccca gaaacgctgg tgaaagtaaa agatgctgaa gatcagttgg
      961 gtgcacgagt gggttacatc gaactggatc tcaacagcgg taagatcctt gagagttttc
     1021 gccccgaaga acgttttcca atgatgagca cttttaaagt tctgctatgt ggcgcggtat
     1081 tataccgtat tgacgccggg caagagcaac tcggtcgccg catacactat tctcagaatg
     1141 acttggttga gtactcacca gtaacagaaa agcatcttac ggatggcatg acagtaagag
     1201 aattatgcag tgctgccata accatgagtg ataacactgc ggccaactta cttctgacaa
     1261 cgatcggagg accgaaggag ctaaccgctt ttttgcacaa catgggggat catgtaactc
     1321 gccttgatcg ttgggaaccg gagctgaatg aagccatacc aaacgacgag cgtgacacca
     1381 cgatgcctgt agcaatggca acaacgttgc gcaaactatt aactggcgaa ctacttactc
     1441 tagcttcccg gcaacaatta atagactgga tggaggcgga taaagttgca ggaccacttc
     1501 tgcgctcggc ccttccggct ggctggttta ttgctgataa atctggagcc ggtgagcgtg
     1561 gatcacgcgg tatcattgca gcactggggc cagatggtaa gccctcccgt atcgtagtta
     1621 tctacacgac ggggagtcag gcaactatgg atgaacgaaa tagacagatc gctgagatag
     1681 gtgcctcact gattaagcat tggtaactgt cagaccaagt ttacgagctc gcttggactc
     1741 ctgttgatag atccagtaat gacatcagaa ctccatctgg atttgttcag aacgctcggt
     1801 tgccgccggg cgttttttat tggtgagaat ccaagcacta gggacagtaa gacgggtaag
     1861 cctgttgatg ataccgctgc cttactgggt gcattagcca gtctgaatga cctgtcacgg
     1921 gataatccga agtggtcaga ctggaaaatc agagggcagg aactgctgaa cagcaaaaag
     1981 tcagatagca ccacatagca gaccagccat aaaacgccct gagaagcccg tgacgggctt
     2041 ttcttgtatt atgggtagtt tccttgcatg aatccataaa aggcgcctgt agtgccattt
     2101 acccacattc actgccagag ccgtgagcgc agcgaactga atgtcacgaa aaagacagcg
     2161 actcaggtgc ctgatggtcg gagacaaaag gaatattaag cgatttgcac gagcttgcga
     2221 gggtgctact taagccttta gggttttaag gtctgttttg tagaggagca aacagcgttt
     2281 gcgacatcct tttgtaatac tgcggaactg actaaagtag tgagttatac acagggctgg
     2341 gatctattct ttttatcttt ttttattctt tctttattct ataaattata accacttgaa
     2401 tataaacaaa aaaaacacac aaaggtctag cggaatttac agagggtcta gcagaattta
     2461 caagttttcc agcaaaggtc tagcagaatt tacagatacc cacaactcaa aggaaaagga
     2521 catgtaatta tcattgacta gcccatctca attggtatag tgattaaaat cacctagacc
     2581 aattgagatg tatgtctgaa ttagttgttt tcaaagcaaa tgaactagcg attagtcgct
     2641 atgacttaac ggagcatgaa accaagctaa ttttatgctg tgtggcacta ctcaacccca
     2701 ctattgaaaa ccctacaagg aaagaacgga cggtatcgtt cacttataac caatacgctc
     2761 agatgatgaa catcagtagg gaaaatgctt atggtgtatt agctaaagca accagagagc
     2821 tgatgacgag aactgtggaa atcatgaatc ctttggttaa aggctttgag attttccagt
     2881 ggacaaacta tgccaagttc tcaagcgaaa aattagaatt agtttttagt gaagagatat
     2941 tgccttatct tttccagtta aaaaaattca taaaatataa tctggaacat gttaagtctt
     3001 ttgaaaacaa atactctatg aggatttatg agtggttatt aaaagaacta acacaaaaga
     3061 aaactcacaa ggcaaatata gagattagcc ttgatgaatt taagttcatg ttaatgcttg
     3121 aaaataacta ccatgagttt aaaaggctta accaatgggt tttgaaacca ataagtaaag
     3181 atttaaacac ttacagcaat atgaaattgg tggttgataa gcgaggccgc ccgactgata
     3241 cgttgatttt ccaagttgaa ctagatagac aaatggatct cgtaaccgaa cttgagaaca
     3301 accagataaa aatgaatggt gacaaaatac caacaaccat tacatcagat tcctacctac
     3361 ataacggact aagaaaaaca ctacacgatg ctttaactgc aaaaattcag ctcaccagtt
     3421 ttgaggcaaa atttttgagt gacatgcaaa gtaagtatga tctcaatggt tcgttctcat
     3481 ggctcacgca aaaacaacga accacactag agaacatact ggctaaatac ggaaggatct
     3541 gaggttctta tggctcttgt atctatcagt gaagcatcaa gactaacaaa caaaagtaga
     3601 acaactgttc accgttacat atcaaaggga aaactgtcca tatgcacaga tgaaaacggt
     3661 gtaaaaaaga tagatacatc agagctttta cgagtttttg gtgcattcaa agctgttcac
     3721 catgaacaga tcgacaatgt aacctcgaga ttaccgcctt tgagtgagct gataccgctc
     3781 gccgcagccg aacgaccgag cgcagcgagt cagtgagcga ggaagcctgc ataacgcgaa
     3841 gtaatctttt cggttttaaa gaaaaagggc agggtggtga caccttgccc ttctagggcg
     3901 gcggatttgt cctactcagg agagcgttca ccgacaaaca acagataaaa cgaaaggccc
     3961 agtctttcga ctgagccttt cgttttattt gatgccttaa gacccacttt cacatttaag
     4021 ttgtttttct aatccgcata tgatcaattc aaggccgaat aagaaggctg gctctgcacc
     4081 ttggtgatca aataattcga tagcttgtcg taataatggc ggcatactat cagtagtagg
     4141 tgtttccctt tcttctttag cgacttgatg ctcttgatct tccaatacgc aacctaaagt
     4201 aaaatgcccc actgcgctga gtgcatataa tgcattctct agtgaaaaac cttgttggca
     4261 taaaaaggct aattgatttt cgagagtttc atactgtttt tctgtaggcc gtgtacctaa
     4321 atgtactttt gctccatcgc gatgacttag taaagcacat ctaaaacttt tagcgttatt
     4381 acgtaaaaaa tcttgccagc tttccccttc taaagggcaa aagtgagtat ggtgcctatc
     4441 taacatctca atggctaagg cgtcgagcaa agcccgctta ttttttacat gccaatacaa
     4501 tgtaggctgc tctacaccta gcttctgggc gagtttacgg gttgttaaac cttcgattcc
     4561 gacctcatta agcagctcta atgcgctgtt aatcacttta cttttatcta aacgagacat
     4621 catactaccc cctgttaaag agtgtttctc tggctgggct agcacaatac ctaggactga
     4681 gctagctgtc aaaagcttgc atgcctgcag gtcgactcta gaggatccac gggtaccagc
     4741 gttttgttat caataaaaaa ggccccccga tttgggaggc ctttttagtt agattagcgc
     4801 aattcaaaaa gatatccttg tccgcgtacg gtggtaatga catcgtgcgg atactgggcc
     4861 tgtatttttt tccgcagacg ccccatgaga acatcaatgg tatgactttc ccgcagttcc
     4921 gcatccggat acagctgaag catcagcgaa tctttgctga ccactttacc gttgttacgg
     4981 ataagcgttt ccataatggt gtattcgaac gccgtgagtt tgatgacctc ttcattgacg
     5041 gataattccc ggcgtgagag atccacctgg aacggcggga tgttgatcac ctgggaggcc
     5101 agaccgctat tacggcgcat taacgcctgc atacgcgcca ttacctcttc gatgtggaat
     5161 ggcttcgtca cgtagtcatc ggccccggag ctgagaacct cgactttatc ctgccagcct
     5221 tcgcgcgcgg ttaacaccag aaccggcagt gaaacatcac tgctgcgcca gcggcgtatt
     5281 aaggaaaggc cgtcttcatc cggcagacct aaatcgacaa tagcgatatc cggaaggtgt
     5341 tcattaaggt agtaatcagc ttccctggca tcttctgcgg catcgacctg gtgacctgaa
     5401 tcctggagct gaaccttcag gtggtggcgt aataatgcat tatcctctac aaccagtacg
     5461 cgcatcatta gaaaacttcc taggcatgat taagatgttt cagtacgaaa attgctttca
     5521 ttgttgatct cctttttaag tgaacttggg cccggtcagt gcgtcctgct gatgtgctca
     5581 gtatctctat cactgatagg gatgtcaatc tctatcactg atagggagtc ttcgcgtaac
     5641 ggtcgccgtt attaaacggt tacgcgggag gaaatcctta tccgtcccga aaacggatat
     5701 aaacgcagaa aggcccaccc gaaggtgagc cagtgtgact ctagtagaga gcgttcaccg
     5761 acaaacaaca gataaaacga aaggcccagt ctttcgactg agcctttcgt tttatttgat
     5821 gcctggtcta gattattagc ggccgcaacg gctgtcgtca gtacaattat gagtgcctgg
     5881 gtttatatgc gcctcatttg tacagttcat ccataccatg cgtgatgccc gctgcggtta
     5941 cgaattccag cagaaccata tgatcgcgtt tctcgttcgg atctttagac agaacgcttt
     6001 gcgtgctcag atagtgattg tctggcagca gaacaggacc atcaccgatt ggagtgtttt
     6061 gctggtagtg atcagccagc tgcacgctgc catcctccac gttgtggcga attttaaaat
     6121 tcgctttaat gccatttttt tgtttatcgg cggt
//
