LOCUS       sequence-335294-        6315 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Encodes PugtL-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 6315)
  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 6315)
  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: pAM004
FEATURES             Location/Qualifiers
     source          1..6315
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     RBS             466..477
                     /note="strong bacterial ribosome binding site (Elowitz and 
                     Leibler, 2000)"
     primer_bind     complement(822..840)
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     promoter        905..1009
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(1010..1078,1079..1870)
                     /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(1228..1247)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     terminator      1900..1994
                     /label=lambda t0 terminator
                     /note="transcription terminator from phage lambda"
     rep_origin      2486..2708
                     /label=pSC101 ori
                     /note="low-copy replication origin that requires the Rep101
                     protein"
     CDS             2756..3706
                     /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     3968..3985
                     /label=L4440
                     /note="L4440 vector, forward primer"
     terminator      complement(4069..4155)
                     /gene="Escherichia coli rrnB"
                     /label=rrnB T1 terminator
                     /note="transcription terminator T1 from the E. coli rrnB 
                     gene"
     CDS             complement(4161..4787)
                     /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     4692..4711
                     /label=Tet-R
                     /note="Tetracycline resistance gene, reverse primer"
     promoter        complement(4822..4856)
                     /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(5718..5743,5744..5749,5750..5766,5767..5772,
                     5773..5791))
                     /label=PLtetO-1 promoter
                     /note="modified phage lambda PL promoter with tet operator 
                     sites (Lutz and Bujard, 1997)"
     protein_bind    5748..5766
                     /gene="tetO"
                     /label=tet operator
                     /bound_moiety="tetracycline repressor TetR"
                     /note="bacterial operator O2 for the tetR and tetA genes"
     protein_bind    5773..5791
                     /gene="tetO"
                     /label=tet operator
                     /bound_moiety="tetracycline repressor TetR"
                     /note="bacterial operator O2 for the tetR and tetA genes"
     terminator      complement(5868..5895)
                     /label=T7Te terminator
                     /note="phage T7 early transcription terminator"
     terminator      complement(5911..5982)
                     /label=rrnB T1 terminator
                     /note="transcription terminator T1 from the E. coli rrnB 
                     gene"
     CDS             complement(join(6061..6315,1..459))
                     /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 ggtgatgtaa acattgtggc tgttaaaatt gtattccagc ttatggccca ggatattgcc
       61 gtcttcttta aagtcaatgc ctttcagctc aatgcggttt accagggtat cgccttcaaa
      121 tttcacttcc gcacgcgttt tgtacgtgcc gtcatcctta aaggaaatcg tgcgttcctg
      181 cacatagcct tccggcatgg cggacttgaa gaagtcatgc tgcttcatat ggtccggata
      241 acgagcaaag cactgaacac cataagtcag cgtcgttacc agagtcggcc aaggtaccgg
      301 cagtttacca gtagtacaga tgaacttcag cgtcagttta ccattagttg cgtcaccttc
      361 accctcgcca cgcacggaaa acttatgacc gttgacatca ccatccagtt ccaccagaat
      421 agggacgaca ccagtgaaca gctcttcgcc tttacgcatc tagtatttct cctctttttt
      481 gagcctcctc gcaggttttt ataattttat cgcccaactg gaaacaaagc cgtcagctaa
      541 tcgttacaac aaatataatt aagacaaaaa ctaaagagta agatatttat atcataagca
      601 ctatcagtat tggccttctg ccctaccgct aaacatctca ttgttgttag cctaataata
      661 cttttagttt aacttcttat aagacaattt ctacacggtt gagcaactat ttactttctc
      721 taaaaataat atagtgcgta attatccgac gtcggccgta ggcgtcggag attccggagc
      781 gtaataggga gtagacggaa cgcggaagcg ataaaaattc tgcctcgtga tacgcctatt
      841 tttataggtt aatgtcatga taataatggt ttattagacg tcaggtggca ccatgggaaa
      901 tgtgcgcgga acccctattt gtttattttt ctaaatacat taaaatatgt atccgctcat
      961 gagacaataa ccctgataaa tgcttcaata atattgaaaa aggaagagta tgagtattca
     1021 acatttcagt gtcgccctta ttcccttttt tgcggcattt tgccttcctg tttttgctca
     1081 cccagaaacg ctggtgaaag taaaagatgc tgaagatcag ttgggtgcac gagtgggtta
     1141 catcgaactg gatctcaaca gcggtaagat ccttgagagt tttcgccccg aagaacgttt
     1201 tccaatgatg agcactttta aagttctgct atgtggcgcg gtattatacc gtattgacgc
     1261 cgggcaagag caactcggtc gccgcataca ctattctcag aatgacttgg ttgagtactc
     1321 accagtaaca gaaaagcatc ttacggatgg catgacagta agagaattat gcagtgctgc
     1381 cataaccatg agtgataaca ctgcggccaa cttacttctg acaacgatcg gaggaccgaa
     1441 ggagctaacc gcttttttgc acaacatggg ggatcatgta actcgccttg atcgttggga
     1501 accggagctg aatgaagcca taccaaacga cgagcgtgac accacgatgc ctgtagcaat
     1561 ggcaacaacg ttgcgcaaac tattaactgg cgaactactt actctagctt cccggcaaca
     1621 attaatagac tggatggagg cggataaagt tgcaggacca cttctgcgct cggcccttcc
     1681 ggctggctgg tttattgctg ataaatctgg agccggtgag cgtggatcac gcggtatcat
     1741 tgcagcactg gggccagatg gtaagccctc ccgtatcgta gttatctaca cgacggggag
     1801 tcaggcaact atggatgaac gaaatagaca gatcgctgag ataggtgcct cactgattaa
     1861 gcattggtaa ctgtcagacc aagtttacga gctcgcttgg actcctgttg atagatccag
     1921 taatgacatc agaactccat ctggatttgt tcagaacgct cggttgccgc cgggcgtttt
     1981 ttattggtga gaatccaagc actagggaca gtaagacggg taagcctgtt gatgataccg
     2041 ctgccttact gggtgcatta gccagtctga atgacctgtc acgggataat ccgaagtggt
     2101 cagactggaa aatcagaggg caggaactgc tgaacagcaa aaagtcagat agcaccacat
     2161 agcagaccag ccataaaacg ccctgagaag cccgtgacgg gcttttcttg tattatgggt
     2221 agtttccttg catgaatcca taaaaggcgc ctgtagtgcc atttacccac attcactgcc
     2281 agagccgtga gcgcagcgaa ctgaatgtca cgaaaaagac agcgactcag gtgcctgatg
     2341 gtcggagaca aaaggaatat taagcgattt gcacgagctt gcgagggtgc tacttaagcc
     2401 tttagggttt taaggtctgt tttgtagagg agcaaacagc gtttgcgaca tccttttgta
     2461 atactgcgga actgactaaa gtagtgagtt atacacaggg ctgggatcta ttctttttat
     2521 ctttttttat tctttcttta ttctataaat tataaccact tgaatataaa caaaaaaaac
     2581 acacaaaggt ctagcggaat ttacagaggg tctagcagaa tttacaagtt ttccagcaaa
     2641 ggtctagcag aatttacaga tacccacaac tcaaaggaaa aggacatgta attatcattg
     2701 actagcccat ctcaattggt atagtgatta aaatcaccta gaccaattga gatgtatgtc
     2761 tgaattagtt gttttcaaag caaatgaact agcgattagt cgctatgact taacggagca
     2821 tgaaaccaag ctaattttat gctgtgtggc actactcaac cccactattg aaaaccctac
     2881 aaggaaagaa cggacggtat cgttcactta taaccaatac gctcagatga tgaacatcag
     2941 tagggaaaat gcttatggtg tattagctaa agcaaccaga gagctgatga cgagaactgt
     3001 ggaaatcatg aatcctttgg ttaaaggctt tgagattttc cagtggacaa actatgccaa
     3061 gttctcaagc gaaaaattag aattagtttt tagtgaagag atattgcctt atcttttcca
     3121 gttaaaaaaa ttcataaaat ataatctgga acatgttaag tcttttgaaa acaaatactc
     3181 tatgaggatt tatgagtggt tattaaaaga actaacacaa aagaaaactc acaaggcaaa
     3241 tatagagatt agccttgatg aatttaagtt catgttaatg cttgaaaata actaccatga
     3301 gtttaaaagg cttaaccaat gggttttgaa accaataagt aaagatttaa acacttacag
     3361 caatatgaaa ttggtggttg ataagcgagg ccgcccgact gatacgttga ttttccaagt
     3421 tgaactagat agacaaatgg atctcgtaac cgaacttgag aacaaccaga taaaaatgaa
     3481 tggtgacaaa ataccaacaa ccattacatc agattcctac ctacataacg gactaagaaa
     3541 aacactacac gatgctttaa ctgcaaaaat tcagctcacc agttttgagg caaaattttt
     3601 gagtgacatg caaagtaagt atgatctcaa tggttcgttc tcatggctca cgcaaaaaca
     3661 acgaaccaca ctagagaaca tactggctaa atacggaagg atctgaggtt cttatggctc
     3721 ttgtatctat cagtgaagca tcaagactaa caaacaaaag tagaacaact gttcaccgtt
     3781 acatatcaaa gggaaaactg tccatatgca cagatgaaaa cggtgtaaaa aagatagata
     3841 catcagagct tttacgagtt tttggtgcat tcaaagctgt tcaccatgaa cagatcgaca
     3901 atgtaacctc gagattaccg cctttgagtg agctgatacc gctcgccgca gccgaacgac
     3961 cgagcgcagc gagtcagtga gcgaggaagc ctgcataacg cgaagtaatc ttttcggttt
     4021 taaagaaaaa gggcagggtg gtgacacctt gcccttctag ggcggcggat ttgtcctact
     4081 caggagagcg ttcaccgaca aacaacagat aaaacgaaag gcccagtctt tcgactgagc
     4141 ctttcgtttt atttgatgcc ttaagaccca ctttcacatt taagttgttt ttctaatccg
     4201 catatgatca attcaaggcc gaataagaag gctggctctg caccttggtg atcaaataat
     4261 tcgatagctt gtcgtaataa tggcggcata ctatcagtag taggtgtttc cctttcttct
     4321 ttagcgactt gatgctcttg atcttccaat acgcaaccta aagtaaaatg ccccactgcg
     4381 ctgagtgcat ataatgcatt ctctagtgaa aaaccttgtt ggcataaaaa ggctaattga
     4441 ttttcgagag tttcatactg tttttctgta ggccgtgtac ctaaatgtac ttttgctcca
     4501 tcgcgatgac ttagtaaagc acatctaaaa cttttagcgt tattacgtaa aaaatcttgc
     4561 cagctttccc cttctaaagg gcaaaagtga gtatggtgcc tatctaacat ctcaatggct
     4621 aaggcgtcga gcaaagcccg cttatttttt acatgccaat acaatgtagg ctgctctaca
     4681 cctagcttct gggcgagttt acgggttgtt aaaccttcga ttccgacctc attaagcagc
     4741 tctaatgcgc tgttaatcac tttactttta tctaaacgag acatcatact accccctgtt
     4801 aaagagtgtt tctctggctg ggctagcaca atacctagga ctgagctagc tgtcaaaagc
     4861 ttgcatgcct gcaggtcgac tctagaggat ccacgggtac cagcgttttg ttatcaataa
     4921 aaaaggcccc ccgatttggg aggccttttt agttagatta gcgcaattca aaaagatatc
     4981 cttgtccgcg tacggtggta atgacatcgt gcggatactg ggcctgtatt tttttccgca
     5041 gacgccccat gagaacatca atggtatgac tttcccgcag ttccgcatcc ggatacagct
     5101 gaagcatcag cgaatctttg ctgaccactt taccgttgtt acggataagc gtttccataa
     5161 tggtgtattc gaacgccgtg agtttgatga cctcttcatt gacggataat tcccggcgtg
     5221 agagatccac ctggaacggc gggatgttga tcacctggga ggccagaccg ctattacggc
     5281 gcattaacgc ctgcatacgc gccattacct cttcgatgtg gaatggcttc gtcacgtagt
     5341 catcggcccc ggagctgaga acctcgactt tatcctgcca gccttcgcgc gcggttaaca
     5401 ccagaaccgg cagtgaaaca tcactgctgc gccagcggcg tattaaggaa aggccgtctt
     5461 catccggcag acctaaatcg acaatagcga tatccggaag gtgttcatta aggtagtaat
     5521 cagcttccct ggcatcttct gcggcatcga cctggtgacc tgaatcctgg agctgaacct
     5581 tcaggtggtg gcgtaataat gcattatcct ctacaaccag tacgcgcatc attagaaaac
     5641 ttcctaggca tgattaagat gtttcagtac gaaaattgct ttcattgttg atctcctttt
     5701 taagtgaact tgggcccggt cagtgcgtcc tgctgatgtg ctcagtatct ctatcactga
     5761 tagggatgtc aatctctatc actgataggg agtcttcgcg taacggtcgc cgttattaaa
     5821 cggttacgcg ggaggaaatc cttatccgtc ccgaaaacgg atataaacgc agaaaggccc
     5881 acccgaaggt gagccagtgt gactctagta gagagcgttc accgacaaac aacagataaa
     5941 acgaaaggcc cagtctttcg actgagcctt tcgttttatt tgatgcctgg tctagattat
     6001 tagcggccgc aacggctgtc gtcagtacaa ttatgagtgc ctgggtttat atgcgcctca
     6061 tttgtacagt tcatccatac catgcgtgat gcccgctgcg gttacgaatt ccagcagaac
     6121 catatgatcg cgtttctcgt tcggatcttt agacagaacg ctttgcgtgc tcagatagtg
     6181 attgtctggc agcagaacag gaccatcacc gattggagtg ttttgctggt agtgatcagc
     6241 cagctgcacg ctgccatcct ccacgttgtg gcgaatttta aaattcgctt taatgccatt
     6301 tttttgttta tcggc
//
