LOCUS       sequence-335956-        6168 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Encodes PvirK-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 6168)
  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 6168)
  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: pAM005
FEATURES             Location/Qualifiers
     source          1..6168
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     RBS             463..474
                     /note="strong bacterial ribosome binding site (Elowitz and 
                     Leibler, 2000)"
     primer_bind     complement(672..690)
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     promoter        755..859
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(860..928,929..1720)
                     /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(1078..1097)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     terminator      1750..1844
                     /label=lambda t0 terminator
                     /note="transcription terminator from phage lambda"
     rep_origin      2336..2558
                     /label=pSC101 ori
                     /note="low-copy replication origin that requires the Rep101
                     protein"
     CDS             2606..3556
                     /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     3818..3835
                     /label=L4440
                     /note="L4440 vector, forward primer"
     terminator      complement(3919..4005)
                     /gene="Escherichia coli rrnB"
                     /label=rrnB T1 terminator
                     /note="transcription terminator T1 from the E. coli rrnB 
                     gene"
     CDS             complement(4011..4637)
                     /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     4542..4561
                     /label=Tet-R
                     /note="Tetracycline resistance gene, reverse primer"
     promoter        complement(4672..4706)
                     /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(5568..5593,5594..5599,5600..5616,5617..5622,
                     5623..5641))
                     /label=PLtetO-1 promoter
                     /note="modified phage lambda PL promoter with tet operator 
                     sites (Lutz and Bujard, 1997)"
     protein_bind    5598..5616
                     /gene="tetO"
                     /label=tet operator
                     /bound_moiety="tetracycline repressor TetR"
                     /note="bacterial operator O2 for the tetR and tetA genes"
     protein_bind    5623..5641
                     /gene="tetO"
                     /label=tet operator
                     /bound_moiety="tetracycline repressor TetR"
                     /note="bacterial operator O2 for the tetR and tetA genes"
     terminator      complement(5718..5745)
                     /label=T7Te terminator
                     /note="phage T7 early transcription terminator"
     terminator      complement(5761..5832)
                     /label=rrnB T1 terminator
                     /note="transcription terminator T1 from the E. coli rrnB 
                     gene"
     CDS             complement(join(5911..6168,1..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 ctttactacg
      481 gctctactac tcgatagact tataaccgag acggtagcct gattgagtta aacgtaaagg
      541 caacgaagaa ggtctgtaca gacgatgttg ttaaacagtt tatcaatggc ggtatccgac
      601 gtcggccgta ggcgtcggag attccggagc gtaataggga gtagacggaa cgcggaagcg
      661 ataaaaattc tgcctcgtga tacgcctatt tttataggtt aatgtcatga taataatggt
      721 ttattagacg tcaggtggca ccatgggaaa tgtgcgcgga acccctattt gtttattttt
      781 ctaaatacat taaaatatgt atccgctcat gagacaataa ccctgataaa tgcttcaata
      841 atattgaaaa aggaagagta tgagtattca acatttcagt gtcgccctta ttcccttttt
      901 tgcggcattt tgccttcctg tttttgctca cccagaaacg ctggtgaaag taaaagatgc
      961 tgaagatcag ttgggtgcac gagtgggtta catcgaactg gatctcaaca gcggtaagat
     1021 ccttgagagt tttcgccccg aagaacgttt tccaatgatg agcactttta aagttctgct
     1081 atgtggcgcg gtattatacc gtattgacgc cgggcaagag caactcggtc gccgcataca
     1141 ctattctcag aatgacttgg ttgagtactc accagtaaca gaaaagcatc ttacggatgg
     1201 catgacagta agagaattat gcagtgctgc cataaccatg agtgataaca ctgcggccaa
     1261 cttacttctg acaacgatcg gaggaccgaa ggagctaacc gcttttttgc acaacatggg
     1321 ggatcatgta actcgccttg atcgttggga accggagctg aatgaagcca taccaaacga
     1381 cgagcgtgac accacgatgc ctgtagcaat ggcaacaacg ttgcgcaaac tattaactgg
     1441 cgaactactt actctagctt cccggcaaca attaatagac tggatggagg cggataaagt
     1501 tgcaggacca cttctgcgct cggcccttcc ggctggctgg tttattgctg ataaatctgg
     1561 agccggtgag cgtggatcac gcggtatcat tgcagcactg gggccagatg gtaagccctc
     1621 ccgtatcgta gttatctaca cgacggggag tcaggcaact atggatgaac gaaatagaca
     1681 gatcgctgag ataggtgcct cactgattaa gcattggtaa ctgtcagacc aagtttacga
     1741 gctcgcttgg actcctgttg atagatccag taatgacatc agaactccat ctggatttgt
     1801 tcagaacgct cggttgccgc cgggcgtttt ttattggtga gaatccaagc actagggaca
     1861 gtaagacggg taagcctgtt gatgataccg ctgccttact gggtgcatta gccagtctga
     1921 atgacctgtc acgggataat ccgaagtggt cagactggaa aatcagaggg caggaactgc
     1981 tgaacagcaa aaagtcagat agcaccacat agcagaccag ccataaaacg ccctgagaag
     2041 cccgtgacgg gcttttcttg tattatgggt agtttccttg catgaatcca taaaaggcgc
     2101 ctgtagtgcc atttacccac attcactgcc agagccgtga gcgcagcgaa ctgaatgtca
     2161 cgaaaaagac agcgactcag gtgcctgatg gtcggagaca aaaggaatat taagcgattt
     2221 gcacgagctt gcgagggtgc tacttaagcc tttagggttt taaggtctgt tttgtagagg
     2281 agcaaacagc gtttgcgaca tccttttgta atactgcgga actgactaaa gtagtgagtt
     2341 atacacaggg ctgggatcta ttctttttat ctttttttat tctttcttta ttctataaat
     2401 tataaccact tgaatataaa caaaaaaaac acacaaaggt ctagcggaat ttacagaggg
     2461 tctagcagaa tttacaagtt ttccagcaaa ggtctagcag aatttacaga tacccacaac
     2521 tcaaaggaaa aggacatgta attatcattg actagcccat ctcaattggt atagtgatta
     2581 aaatcaccta gaccaattga gatgtatgtc tgaattagtt gttttcaaag caaatgaact
     2641 agcgattagt cgctatgact taacggagca tgaaaccaag ctaattttat gctgtgtggc
     2701 actactcaac cccactattg aaaaccctac aaggaaagaa cggacggtat cgttcactta
     2761 taaccaatac gctcagatga tgaacatcag tagggaaaat gcttatggtg tattagctaa
     2821 agcaaccaga gagctgatga cgagaactgt ggaaatcatg aatcctttgg ttaaaggctt
     2881 tgagattttc cagtggacaa actatgccaa gttctcaagc gaaaaattag aattagtttt
     2941 tagtgaagag atattgcctt atcttttcca gttaaaaaaa ttcataaaat ataatctgga
     3001 acatgttaag tcttttgaaa acaaatactc tatgaggatt tatgagtggt tattaaaaga
     3061 actaacacaa aagaaaactc acaaggcaaa tatagagatt agccttgatg aatttaagtt
     3121 catgttaatg cttgaaaata actaccatga gtttaaaagg cttaaccaat gggttttgaa
     3181 accaataagt aaagatttaa acacttacag caatatgaaa ttggtggttg ataagcgagg
     3241 ccgcccgact gatacgttga ttttccaagt tgaactagat agacaaatgg atctcgtaac
     3301 cgaacttgag aacaaccaga taaaaatgaa tggtgacaaa ataccaacaa ccattacatc
     3361 agattcctac ctacataacg gactaagaaa aacactacac gatgctttaa ctgcaaaaat
     3421 tcagctcacc agttttgagg caaaattttt gagtgacatg caaagtaagt atgatctcaa
     3481 tggttcgttc tcatggctca cgcaaaaaca acgaaccaca ctagagaaca tactggctaa
     3541 atacggaagg atctgaggtt cttatggctc ttgtatctat cagtgaagca tcaagactaa
     3601 caaacaaaag tagaacaact gttcaccgtt acatatcaaa gggaaaactg tccatatgca
     3661 cagatgaaaa cggtgtaaaa aagatagata catcagagct tttacgagtt tttggtgcat
     3721 tcaaagctgt tcaccatgaa cagatcgaca atgtaacctc gagattaccg cctttgagtg
     3781 agctgatacc gctcgccgca gccgaacgac cgagcgcagc gagtcagtga gcgaggaagc
     3841 ctgcataacg cgaagtaatc ttttcggttt taaagaaaaa gggcagggtg gtgacacctt
     3901 gcccttctag ggcggcggat ttgtcctact caggagagcg ttcaccgaca aacaacagat
     3961 aaaacgaaag gcccagtctt tcgactgagc ctttcgtttt atttgatgcc ttaagaccca
     4021 ctttcacatt taagttgttt ttctaatccg catatgatca attcaaggcc gaataagaag
     4081 gctggctctg caccttggtg atcaaataat tcgatagctt gtcgtaataa tggcggcata
     4141 ctatcagtag taggtgtttc cctttcttct ttagcgactt gatgctcttg atcttccaat
     4201 acgcaaccta aagtaaaatg ccccactgcg ctgagtgcat ataatgcatt ctctagtgaa
     4261 aaaccttgtt ggcataaaaa ggctaattga ttttcgagag tttcatactg tttttctgta
     4321 ggccgtgtac ctaaatgtac ttttgctcca tcgcgatgac ttagtaaagc acatctaaaa
     4381 cttttagcgt tattacgtaa aaaatcttgc cagctttccc cttctaaagg gcaaaagtga
     4441 gtatggtgcc tatctaacat ctcaatggct aaggcgtcga gcaaagcccg cttatttttt
     4501 acatgccaat acaatgtagg ctgctctaca cctagcttct gggcgagttt acgggttgtt
     4561 aaaccttcga ttccgacctc attaagcagc tctaatgcgc tgttaatcac tttactttta
     4621 tctaaacgag acatcatact accccctgtt aaagagtgtt tctctggctg ggctagcaca
     4681 atacctagga ctgagctagc tgtcaaaagc ttgcatgcct gcaggtcgac tctagaggat
     4741 ccacgggtac cagcgttttg ttatcaataa aaaaggcccc ccgatttggg aggccttttt
     4801 agttagatta gcgcaattca aaaagatatc cttgtccgcg tacggtggta atgacatcgt
     4861 gcggatactg ggcctgtatt tttttccgca gacgccccat gagaacatca atggtatgac
     4921 tttcccgcag ttccgcatcc ggatacagct gaagcatcag cgaatctttg ctgaccactt
     4981 taccgttgtt acggataagc gtttccataa tggtgtattc gaacgccgtg agtttgatga
     5041 cctcttcatt gacggataat tcccggcgtg agagatccac ctggaacggc gggatgttga
     5101 tcacctggga ggccagaccg ctattacggc gcattaacgc ctgcatacgc gccattacct
     5161 cttcgatgtg gaatggcttc gtcacgtagt catcggcccc ggagctgaga acctcgactt
     5221 tatcctgcca gccttcgcgc gcggttaaca ccagaaccgg cagtgaaaca tcactgctgc
     5281 gccagcggcg tattaaggaa aggccgtctt catccggcag acctaaatcg acaatagcga
     5341 tatccggaag gtgttcatta aggtagtaat cagcttccct ggcatcttct gcggcatcga
     5401 cctggtgacc tgaatcctgg agctgaacct tcaggtggtg gcgtaataat gcattatcct
     5461 ctacaaccag tacgcgcatc attagaaaac ttcctaggca tgattaagat gtttcagtac
     5521 gaaaattgct ttcattgttg atctcctttt taagtgaact tgggcccggt cagtgcgtcc
     5581 tgctgatgtg ctcagtatct ctatcactga tagggatgtc aatctctatc actgataggg
     5641 agtcttcgcg taacggtcgc cgttattaaa cggttacgcg ggaggaaatc cttatccgtc
     5701 ccgaaaacgg atataaacgc agaaaggccc acccgaaggt gagccagtgt gactctagta
     5761 gagagcgttc accgacaaac aacagataaa acgaaaggcc cagtctttcg actgagcctt
     5821 tcgttttatt tgatgcctgg tctagattat tagcggccgc aacggctgtc gtcagtacaa
     5881 ttatgagtgc ctgggtttat atgcgcctca tttgtacagt tcatccatac catgcgtgat
     5941 gcccgctgcg gttacgaatt ccagcagaac catatgatcg cgtttctcgt tcggatcttt
     6001 agacagaacg ctttgcgtgc tcagatagtg attgtctggc agcagaacag gaccatcacc
     6061 gattggagtg ttttgctggt agtgatcagc cagctgcacg ctgccatcct ccacgttgtg
     6121 gcgaatttta aaattcgctt taatgccatt tttttgttta tcggcggt
//
