LOCUS       sequence-245078-        4591 bp    DNA     circular SYN 15-APR-2025
DEFINITION  Expression of uhpA under PLtetO-1, output Promoter PuhpT99.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 4591)
  AUTHORS   Schmidl SR, Ekness F, Sofjan K, Daeffler KN, Brink KR, Landry BP, 
            Gerhardt KP, Dyulgyarov N, Sheth RU, Tabor JJ
  TITLE     Rewiring bacterial two-component systems by modular DNA-binding 
            domain swapping.
  JOURNAL   Nat Chem Biol. 2019 Jul;15(7):690-698. doi: 
            10.1038/s41589-019-0286-6. Epub 2019 May 20.
  PUBMED    31110305
REFERENCE   2  (bases 1 to 4591)
  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"; doi: 
            "10.1038/s41589-019-0286-6"; journalName: "Nat Chem Biol"; date: 
            "2019-07"; volume: "15"; issue: "7"; pages: "690-698"
COMMENT     Sequence Label: pSR300.3
FEATURES             Location/Qualifiers
     source          1..4591
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     59..77
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     promoter        join(98..116,117..122,123..139,140..145,146..171)
                     /label=PLtetO-1 promoter
                     /note="modified phage lambda PL promoter with tet operator 
                     sites (Lutz and Bujard, 1997)"
     protein_bind    98..116
                     /gene="tetO"
                     /label=tet operator
                     /bound_moiety="tetracycline repressor TetR"
                     /note="bacterial operator O2 for the tetR and tetA genes"
     protein_bind    123..141
                     /gene="tetO"
                     /label=tet operator
                     /bound_moiety="tetracycline repressor TetR"
                     /note="bacterial operator O2 for the tetR and tetA genes"
     terminator      818..889
                     /label=rrnB T1 terminator
                     /note="transcription terminator T1 from the E. coli rrnB 
                     gene"
     terminator      905..932
                     /label=T7Te terminator
                     /note="phage T7 early transcription terminator"
     RBS             1155..1166
                     /note="strong bacterial ribosome binding site (Elowitz and 
                     Leibler, 2000)"
     CDS             1173..1886
                     /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"
     terminator      1913..1984
                     /label=rrnB T1 terminator
                     /note="transcription terminator T1 from the E. coli rrnB 
                     gene"
     terminator      2000..2027
                     /label=T7Te terminator
                     /note="phage T7 early transcription terminator"
     promoter        2079..2113
                     /label=BBa_J23106
                     /note="Anderson constitutive promoter 
                     (https://parts.igem.org/Promoters/Catalog/Anderson). 
                     Measured strength relative to BBa_J223100: 0.47"
     CDS             2148..2774
                     /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     complement(2224..2243)
                     /label=Tet-R
                     /note="Tetracycline resistance gene, reverse primer"
     terminator      2780..2866
                     /gene="Escherichia coli rrnB"
                     /label=rrnB T1 terminator
                     /note="transcription terminator T1 from the E. coli rrnB 
                     gene"
     rep_origin      complement(3031..3619)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(3111..3130)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     terminator      complement(3707..3801)
                     /label=lambda t0 terminator
                     /note="transcription terminator from phage lambda"
     CDS             complement(3822..4481)
                     /codon_start=1
                     /gene="cat"
                     /product="chloramphenicol acetyltransferase"
                     /label=CmR
                     /note="confers resistance to chloramphenicol"
                     /translation="MEKKITGYTTVDISQWHRKEHFEAFQSVAQCTYNQTVQLDITAFL
                     KTVKKNKHKFYPAFIHILARLMNAHPEFRMAMKDGELVIWDSVHPCYTVFHEQTETFSS
                     LWSEYHDDFRQFLHIYSQDVACYGENLAYFPKGFIENMFFVSANPWVSFTSFDLNVANM
                     DNFFAPVFTMGKYYTQGDKVLMPLAIQVHHAVCDGFHVGRMLNELQQYCDEWQGGA"
     primer_bind     4396..4415
                     /label=CAT-R
                     /note="Chloramphenicol resistance gene, reverse primer"
ORIGIN
        1 attttcgcca gatatcgacg tctaagaaac cattattatc atgacattaa cctataaaaa
       61 taggcgtatc acgaggccct ttcgtcttca cctcgagtcc ctatcagtga tagagattga
      121 catccctatc agtgatagag atactgagca catcagcagg acgcactgac cagttctaag
      181 acgtaaaaaa gtagatagga cgccatatga tcaccgttgc ccttatagac gatcacctca
      241 tcgtccgctc cggctttgcg cagctgctgg ggctggaacc tgatttgcag gtagttgccg
      301 agtttggttc ggggcgcgag gcgctggcgg ggctgccggg gcgcggtgtg caggtgtgta
      361 tttgcgatat ctccatgccc gatatctccg gtctggagct gctaagccag ctgccgaaag
      421 gtatggcgac gattatgctc tccgttcacg acagtcctgc gctggttgag caggcgctta
      481 acgcgggggc acgcggcttt ctttccaaac gctgtagccc ggatgaactc attgctgcgg
      541 tgcatacggt tgccacgggc ggctgttatc tgacgccgga tattgccatt aaactggcat
      601 ccggtcgtca ggacccgcta accaaacgtg aacgccaggt ggcggaaaaa ctggcgcaag
      661 gaatggcggt gaaagagatt gccgccgaac tgggcttgtc accgaaaacg gtacacgtcc
      721 atcgcgccaa tctgatggaa aaactgggcg tcagtaacga cgtagagctg gcgcgccgca
      781 tgtttgatgg ctggtgataa taatctagac caggcatcaa ataaaacgaa aggctcagtc
      841 gaaagactgg gcctttcgtt ttatctgttg tttgtcggtg aacgctctct actagagtca
      901 cactggctca ccttcgggtg ggcctttctg cgtttatagt aaatcactgc ataattcgtg
      961 tcgctcaagg cgcactcccg ttctggataa tgttttttgc gccgacatca taacggttct
     1021 ggcaaatatt ctgaaatgag ctggttagct agtcatttca cttcatatcc ggcaaaacta
     1081 agaaattttc caggttttgc ctggacgcta tctcaggcct gatttgctgc tgatttttac
     1141 aatgcatgcc tcacaaagag gagaaatact agatgcgtaa aggcgaagag ctgttcactg
     1201 gtgtcgtccc tattctggtg gaactggatg gtgatgtcaa cggtcataag ttttccgtgc
     1261 gtggcgaggg tgaaggtgac gcaactaatg gtaaactgac gctgaagttc atctgtacta
     1321 ctggtaaact gccggtacct tggccgactc tggtaacgac gctgacttat ggtgttcagt
     1381 gctttgctcg ttatccggac catatgaagc agcatgactt cttcaagtcc gccatgccgg
     1441 aaggctatgt gcaggaacgc acgatttcct ttaaggatga cggcacgtac aaaacgcgtg
     1501 cggaagtgaa atttgaaggc gataccctgg taaaccgcat tgagctgaaa ggcattgact
     1561 ttaaagaaga cggcaatatc ctgggccata agctggaata caattttaac agccacaatg
     1621 tttacatcac cgccgataaa caaaaaaatg gcattaaagc gaattttaaa attcgccaca
     1681 acgtggagga tggcagcgtg cagctggctg atcactacca gcaaaacact ccaatcggtg
     1741 atggtcctgt tctgctgcca gacaatcact atctgagcac gcaaagcgtt ctgtctaaag
     1801 atccgaacga gaaacgcgat catatggttc tgctggagtt cgtaaccgca gcgggcatca
     1861 cgcatggtat ggatgaactg tacaaatgat gataataatc tagaccaggc atcaaataaa
     1921 acgaaaggct cagtcgaaag actgggcctt tcgttttatc tgttgtttgt cggtgaacgc
     1981 tctctactag agtcacactg gctcaccttc gggtgggcct ttctgcgttt ataggtaccc
     2041 gtggatcctc tagagtcgac ctgcaggcat gcaagctttt tacggctagc tcagtcctag
     2101 gtatagtgct agcccagcca gagaaacact ctttaacagg gggtagtatg atgtctcgtt
     2161 tagataaaag taaagtgatt aacagcgcat tagagctgct taatgaggtc ggaatcgaag
     2221 gtttaacaac ccgtaaactc gcccagaagc taggtgtaga gcagcctaca ttgtattggc
     2281 atgtaaaaaa taagcgggct ttgctcgacg ccttagccat tgagatgtta gataggcacc
     2341 atactcactt ttgcccttta gaaggggaaa gctggcaaga ttttttacgt aataacgcta
     2401 aaagttttag atgtgcttta ctaagtcatc gcgatggagc aaaagtacat ttaggtacac
     2461 ggcctacaga aaaacagtat gaaactctcg aaaatcaatt agccttttta tgccaacaag
     2521 gtttttcact agagaatgca ttatatgcac tcagcgcagt ggggcatttt actttaggtt
     2581 gcgtattgga agatcaagag catcaagtcg ctaaagaaga aagggaaaca cctactactg
     2641 atagtatgcc gccattatta cgacaagcta tcgaattatt tgatcaccaa ggtgcagagc
     2701 cagccttctt attcggcctt gaattgatca tatgcggatt agaaaaacaa cttaaatgtg
     2761 aaagtgggtc ttaaggcatc aaataaaacg aaaggctcag tcgaaagact gggcctttcg
     2821 ttttatctgt tgtttgtcgg tgaacgctct cctgagtagg acaaatccgc cgccctagac
     2881 ctagggcgtt cggctgcggc gagcggtatc agctcactca aaggcggtaa tacggttatc
     2941 cacagaatca ggggataacg catgcaagaa catgtgagca aaaggccagc aaaaggccag
     3001 gaaccgtaaa aaggccgcgt tgctggcgtt tttccatagg ctccgccccc ctgacgagca
     3061 tcacaaaaat cgacgctcaa gtcagaggtg gcgaaacccg acaggactat aaagatacca
     3121 ggcgtttccc cctggaagct ccctcgtgcg ctctcctgtt ccgaccctgc cgcttaccgg
     3181 atacctgtcc gcctttctcc cttcgggaag cgtggcgctt tctcatagct cacgctgtag
     3241 gtatctcagt tcggtgtagg tcgttcgctc caagctgggc tgtgtgcacg aaccccccgt
     3301 tcagcccgac cgctgcgcct tatccggtaa ctatcgtctt gagtccaacc cggtaagaca
     3361 cgacttatcg ccactggcag cagccactgg taacaggatt agcagagcga ggtatgtagg
     3421 cggtgctaca gagttcttga agtggtggcc taactacggc tacactagaa ggacagtatt
     3481 tggtatctgc gctctgctga agccagttac cttcggaaaa agagttggta gctcttgatc
     3541 cggcaaacaa accaccgctg gtagcggtgg tttttttgtt tgcaagcagc agattacgcg
     3601 cagaaaaaaa ggatctcaag aagatccttt gatcttttct acggggtctg acgctcagtg
     3661 gaacgaaaac tcacgttaag ggattttggt catgactagt gcttggattc tcaccaataa
     3721 aaaacgcccg gcggcaaccg agcgttctga acaaatccag atggagttct gaggtcatta
     3781 ctggatctat caacaggagt ccaagcgagc tcgatatcaa attacgcccc gccctgccac
     3841 tcatcgcagt actgttgtaa ttcattaagc attctgccga catggaagcc atcacaaacg
     3901 gcatgatgaa cctgaatcgc cagcggcatc agcaccttgt cgccttgcgt ataatatttg
     3961 cccatggtga aaacgggggc gaagaagttg tccatattgg ccacgtttaa atcaaaactg
     4021 gtgaaactca cccagggatt ggctgagacg aaaaacatat tctcaataaa ccctttaggg
     4081 aaataggcca ggttttcacc ataacacgcc acatcttgcg aatatatgtg tagaaactgc
     4141 cggaaatcgt cgtggtattc actccagagc gatgaaaacg tttcagtttg ctcatggaaa
     4201 acggtgtaac aagggtgaac actatcccat atcaccagct caccgtcttt cattgccata
     4261 cgaaattccg gatgagcatt catcaggcgg gcaagaatgt gaataaaggc cggataaaac
     4321 ttgtgcttat ttttctttac ggtctttaaa aaggccgtaa tatccagctg aacggtctgg
     4381 ttataggtac attgagcaac tgactgaaat gcctcaaaat gttctttacg atgccattgg
     4441 gatatatcaa cggtggtata tccagtgatt tttttctcca ttttagcttc cttagctcct
     4501 gaaaatctcg ataactcaaa aaatacgacc ggtagtgatc ttatttcatt atggtgaaag
     4561 ttggaacctc ttacgggccg gccaacgtct c
//
