LOCUS       sequence-229900-        5607 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Expresses GFPmut2 with low cell-to-cell variation on a low-copy 
            plasmid and tetracycline induction.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 5607)
  AUTHORS   Silva JPN, Lopes SV, Grilo DJ, Hensel Z
  TITLE     Plasmids for Independently Tunable, Low-Noise Expression of Two 
            Genes.
  JOURNAL   mSphere. 2019 May 29;4(3). pii: 4/3/e00340-19. doi: 
            10.1128/mSphere.00340-19.
  PUBMED    31142623
REFERENCE   2  (bases 1 to 5607)
  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: "mSphere."; 
            date: "2019-05-29"; volume: "4(3). pii: 4/3/e00340-19. doi"; pages: 
            " 10.1128/mSphere.00340-19"
COMMENT     Sequence Label: pJS101
FEATURES             Location/Qualifiers
     source          1..5607
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     CDS             complement(202..1152)
                     /codon_start=1
                     /gene="rep101"
                     /product="RepA protein needed for replication with the 
                     pSC101 origin"
                     /label=Rep101
                     /translation="MSELVVFKANELAISRYDLTEHETKLILCCVALLNPTIENPTRKE
                     RTVSFTYNQYAQMMNISRENAYGVLAKATRELMTRTVEIRNPLVKGFEIFQWTNYAKFS
                     SEKLELVFSEEILPYLFQLKKFIKYNLEHVKSFENKYSMRIYEWLLKELTQKKTHKANI
                     EISLDEFKFMLMLENNYHEFKRLNQWVLKPISKDLNTYSNMKLVVDKRGRPTDTLIFQV
                     ELDRQMDLVTELENNQIKMNGDKIPTTITSDSYLHNGLRKTLHDALTAKIQLTSFEAKF
                     LSDMQSKYDLNGSFSWLTQKQRTTLENILAKYGRI"
     rep_origin      1200..1422
                     /label=pSC101 ori
                     /note="low-copy replication origin that requires the Rep101
                     protein"
     promoter        join(1929..1947,1948..1953,1954..1970,1971..1976,
                     1977..2002)
                     /label=PLtetO-1 promoter
                     /note="modified phage lambda PL promoter with tet operator 
                     sites (Lutz and Bujard, 1997)"
     protein_bind    1929..1947
                     /gene="tetO"
                     /label=tet operator
                     /bound_moiety="tetracycline repressor TetR"
                     /note="bacterial operator O2 for the tetR and tetA genes"
     protein_bind    1954..1972
                     /gene="tetO"
                     /label=tet operator
                     /bound_moiety="tetracycline repressor TetR"
                     /note="bacterial operator O2 for the tetR and tetA genes"
     RBS             2012..2023
                     /note="strong bacterial ribosome binding site (Elowitz and 
                     Leibler, 2000)"
     CDS             2032..2745
                     /codon_start=1
                     /product="S65T variant of Aequorea victoria green 
                     fluorescent protein (Heim et al., 1995)"
                     /label=GFP (S65T)
                     /note="excitable with blue light"
                     /translation="MSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTLK
                     FICTTGKLPVPWPTLVTTFAYGLQCFARYPDHMKQHDFFKSAMPEGYVQERTIFFKDDG
                     NYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIKV
                     NFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSALSKDPNEKRDHMVLLE
                     FVTAAGITHGMDELYK"
     primer_bind     complement(2062..2090)
                     /label=GFP-R
                     /note="GFP, reverse primer. Does NOT anneal to EGFP"
     primer_bind     2685..2705
                     /label=GFP-F
                     /note="GFP, forward primer. Does NOT anneal to EGFP"
     CDS             2772..3395
                     /codon_start=1
                     /gene="tetR from transposon Tn10"
                     /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="MSRLDKSKVINSALELLNEVGIEGLTTRKLAQKLGVEQPTLYWHV
                     KNKRALLDALAIEMLDRHHTHFCPLEGESWQDFLRNNAKSFRCALLSHRDGAKVHLGTR
                     PTEKQYETLENQLAFLCQQGFSLENALYALSAVGHFTLGCVLEDQEHQVAKEERETPTT
                     DSMPPLLRQAIELFDHQGAEPAFLFGLELIICGLEKQLKCESGS"
     primer_bind     complement(2845..2864)
                     /label=Tet-R
                     /note="Tetracycline resistance gene, reverse primer"
     terminator      3440..3486
                     /label=rrnB T1 terminator
                     /note="transcription terminator T1 from the E. coli rrnB 
                     gene"
     CDS             complement(3766..4557)
                     /codon_start=1
                     /gene="aadA"
                     /product="aminoglycoside adenylyltransferase (Murphy, 
                     1985)"
                     /label=SmR
                     /note="confers resistance to spectinomycin and 
                     streptomycin"
                     /translation="MREAVIAEVSTQLSEVVGVIERHLEPTLLAVHLYGSAVDGGLKPH
                     SDIDLLVTVTVRLDETTRRALINDLLETSASPGESEILRAVEVTIVVHDDIIPWRYPAK
                     RELQFGEWQRNDILAGIFEPATIDIDLAILLTKAREHSVALVGPAAEELFDPVPEQDLF
                     EALNETLTLWNSPPDWAGDERNVVLTLSRIWYSAVTGKIAPKDVAADWAMERLPAQYQP
                     VILEARQAYLGQEEDRLASRADQLEEFVHYVKGEITKVVGK"
ORIGIN
        1 gttacattgt cgatctgttc atggtgaaca gctttgaatg caccaaaaac tcgtaaaagc
       61 tctgatgtat ctatcttttt tacaccgttt tcatctgtgc atatggacag ttttcccttt
      121 gatatgtaac ggtgaacagt tgttctactt ttgtttgtta gtcttgatgc ttcactgata
      181 gatacaagag ccataagaac ctcagatcct tccgtattta gccagtatgt tctctagtgt
      241 ggttcgttgt ttttgcgtga gccatgagaa cgaaccattg agatcatact tactttgcat
      301 gtcactcaaa aattttgcct caaaactggt gagctgaatt tttgcagtta aagcatcgtg
      361 tagtgttttt cttagtccgt tatgtaggta ggaatctgat gtaatggttg ttggtatttt
      421 gtcaccattc atttttatct ggttgttctc aagttcggtt acgagatcca tttgtctatc
      481 tagttcaact tggaaaatca acgtatcagt cgggcggcct cgcttatcaa ccaccaattt
      541 catattgctg taagtgttta aatctttact tattggtttc aaaacccatt ggttaagcct
      601 tttaaactca tggtagttat tttcaagcat taacatgaac ttaaattcat caaggctaat
      661 ctctatattt gccttgtgag ttttcttttg tgttagttct tttaataacc actcataaat
      721 cctcatagag tatttgtttt caaaagactt aacatgttcc agattatatt ttatgaattt
      781 ttttaactgg aaaagataag gcaatatctc ttcactaaaa actaattcta atttttcgct
      841 tgagaacttg gcatagtttg tccactggaa aatctcaaag cctttaacca aaggattcct
      901 gatttccaca gttctcgtca tcagctctct ggttgcttta gctaatacac cataagcatt
      961 ttccctactg atgttcatca tctgagcgta ttggttataa gtgaacgata ccgtccgttc
     1021 tttccttgta gggttttcaa tcgtggggtt gagtagtgcc acacagcata aaattagctt
     1081 ggtttcatgc tccgttaagt catagcgact aatcgctagt tcatttgctt tgaaaacaac
     1141 taattcagac atacatctca attggtctag gtgattttaa tcactatacc aattgagatg
     1201 ggctagtcaa tgataattac tagtcctttt cctttgagtt gtgggtatct gtaaattctg
     1261 ctagaccttt gctggaaaac ttgtaaattc tgctagaccc tctgtaaatt ccgctagacc
     1321 tttgtgtgtt ttttttgttt atattcaagt ggttataatt tatagaataa agaaagaata
     1381 aaaaaagata aaaagaatag atcccagccc tgtgtataac tcactacttt agtcagttcc
     1441 gcagtattac aaaaggatgt cgcaaacgct gtttgctcct ctacaaaaca gaccttaaaa
     1501 ccctaaaggc ttaagtagca ccctcgcaag ctcgggcaaa tcgctgaata ttccttttgt
     1561 ctccgaccat caggcacctg agtcgctgtc tttttcgtga cattcagttc gctgcgctca
     1621 cggctctggc agtgaatggg ggtaaatggc actacaggcg ccttttatgg attcatgcaa
     1681 ggaaactacc cataatacaa gaaaagcccg tcacgggctt ctcagggcgt tttatggcgg
     1741 gtctgctatg tggtgctatc tgactttttg ctgttcagca gttcctgccc tctgattttc
     1801 cagtctgacc acttcggatt atcccgtgac aggtcattca gactggctaa tgcacccagt
     1861 aaggcagcgg tatcatcaac aggcttaccc gtcttactgt cgggaattcg agctcggtac
     1921 ccggggattc cctatcagtg atagagattg acatccctat cagtgataga gatactgagc
     1981 acatcagcag gacgcactga ccgaattcat taaagaggag aaaggtaccg catgagtaaa
     2041 ggagaagaac ttttcactgg agttgtccca attcttgttg aattagatgg tgatgttaat
     2101 gggcacaaat tttctgtcag tggagagggt gaaggtgatg caacatacgg aaaacttacc
     2161 cttaaattta tttgcactac tggaaaacta cctgttccat ggccaacact tgtcactact
     2221 ttcgcgtatg gtcttcaatg ctttgcgaga tacccagatc atatgaaaca gcatgacttt
     2281 ttcaagagtg ccatgcccga aggttatgta caggaaagaa ctatattttt caaagatgac
     2341 gggaactaca agacacgtgc tgaagtcaag tttgaaggtg atacccttgt taatagaatc
     2401 gagttaaaag gtattgattt taaagaagat ggaaacattc ttggacacaa attggaatac
     2461 aactataact cacacaatgt atacatcatg gcagacaaac aaaagaatgg aatcaaagtt
     2521 aacttcaaaa ttagacacaa cattgaagat ggaagcgttc aactagcaga ccattatcaa
     2581 caaaatactc caattggcga tggccctgtc cttttaccag acaaccatta cctgtccaca
     2641 caatctgccc tttcgaaaga tcccaacgaa aagagagacc acatggtcct tcttgagttt
     2701 gtaacagctg ctgggattac acatggcatg gatgaactat acaaataata atctagcagg
     2761 aggatttcac catgtctaga ttagataaaa gtaaagtgat taacagcgca ttagagctgc
     2821 ttaatgaggt cggaatcgaa ggtttaacaa cccgtaaact cgcccagaag ctaggtgtag
     2881 agcagcctac attgtattgg catgtaaaaa ataagcgggc tttgctcgac gccttagcca
     2941 ttgagatgtt agataggcac catactcact tttgcccttt agaaggggaa agctggcaag
     3001 attttttacg taataacgct aaaagtttta gatgtgcttt actaagtcat cgcgatggag
     3061 caaaagtaca tttaggtaca cggcctacag aaaaacagta tgaaactctc gaaaatcaat
     3121 tagccttttt atgccaacaa ggtttttcac tagagaatgc attatatgca ctcagcgctg
     3181 tggggcattt tactttaggt tgcgtattgg aagatcaaga gcatcaagtc gctaaagaag
     3241 aaagggaaac acctactact gatagtatgc cgccattatt acgacaagct atcgaattat
     3301 ttgatcacca aggtgcagag ccagccttct tattcggcct tgaattgatc atctgcggat
     3361 tagaaaaaca acttaaatgt gaaagtgggt cttaataact gcagcccggg ggatcccatg
     3421 gtacgcgtgc tagaggcatc aaataaaacg aaaggctcag tcgaaagact gggcctttcg
     3481 ttttatgggg atccgtcgac ctgcaggcat gcaagcttag taaagccctc gctagatttt
     3541 aatgcggatg ttgcgattac ttcgccaact attgcgataa caagaaaaag ccagcctttc
     3601 atgatatatc tcccaatttg tgtagggctt attatgcacg cttaaaaata ataaaagcag
     3661 acttgacctg atagtttggc tgtgagcaat tatgtgctta gtgcatctaa cgcttgagtt
     3721 aagccgcgcc gcgaagcggc gtcggcttga acgaattgtt agacattatt tgccgactac
     3781 cttggtgatc tcgcctttca cgtagtggac aaattcttcc aactgatctg cgcgcgaggc
     3841 caagcgatct tcttcttgtc caagataagc ctgtctagct tcaagtatga cgggctgata
     3901 ctgggccggc aggcgctcca ttgcccagtc ggcagcgaca tccttcggcg cgattttgcc
     3961 ggttactgcg ctgtaccaaa tgcgggacaa cgtaagcact acatttcgct catcgccagc
     4021 ccagtcgggc ggcgagttcc atagcgttaa ggtttcattt agcgcctcaa atagatcctg
     4081 ttcaggaacc ggatcaaaga gttcctccgc cgctggacct accaaggcaa cgctatgttc
     4141 tcttgctttt gtcagcaaga tagccagatc aatgtcgatc gtggctggct cgaagatacc
     4201 tgcaagaatg tcattgcgct gccattctcc aaattgcagt tcgcgcttag ctggataacg
     4261 ccacggaatg atgtcgtcgt gcacaacaat ggtgacttct acagcgcgga gaatctcgct
     4321 ctctccaggg gaagccgaag tttccaaaag gtcgttgatc aaagctcgcc gcgttgtttc
     4381 atcaagcctt acggtcaccg taaccagcaa atcaatatca ctgtgtggct tcaggccgcc
     4441 atccactgcg gagccgtaca aatgtacggc cagcaacgtc ggttcgagat ggcgctcgat
     4501 gacgccaact acctctgata gttgagtcga tacttcggcg atcaccgctt ccctcatgat
     4561 gtttaacttt gttttagggc gactgccctg ctgcgtaaca tcgttgctgc tccataacat
     4621 caaacatcga cccacggcgt aacgcgcttg ctgcttggat gcccgaggca tagactgtac
     4681 cccaaaaaaa cagtcataac aagccatgaa aaccgccact gcgccgttac caccgctgcg
     4741 ttcggtcaag gttctggacc agttgcgtga gcgcatacgc tacttgcatt acagcttacg
     4801 aaccgaacag gcttatgtcc actgggttcg tgccttcatc cgtttccacg gtgtgcgtca
     4861 cccggcaacc ttgggcagca gcgaagtcga ggcatttctg tcctggctgg cgaacgagcg
     4921 caaggtttcg gtctccacgc atcgtcaggc attggcggcc ttgctgttct tctacggcaa
     4981 ggtgctgtgc acggatctgc cctggcttca ggagatcgga agacctcggc cgtcgcggcg
     5041 cttgccggtg gtgctgaccc cggatgaagt ggttcgcatc ctcggttttc tggaaggcga
     5101 gcatcgtttg ttcgcccagc ttctgtatgg aacgggcatg cggatcagtg agggtttgca
     5161 actgcgggtc aaggatctgg atttcgatca cggcacgatc atcgtgcggg agggcaaggg
     5221 ctccaaggat cgggccttga tgttacccga gagcttggca cccagcctgc gcgagcaggg
     5281 gaattaattc ccacgggttt tgctgcccgc aaacgggctg ttctggtgtt gctagtttgt
     5341 tatcagaatc gcagatccgg cttcagccgg tttgccggct gaaagcgcta tttcttccag
     5401 aattgccatg attttttccc cacgggaggc gtcactggct cccgtgttgt cggcagcttt
     5461 gattcgataa gcagcatcgc ctgtttcagg ctgtctatgt gtgactgttg agctgtaaca
     5521 agttgtctca ggtgttcaat ttcatgttct agttgctttg ttttactggt ttcacctgtt
     5581 ctattaggtg ttacatgctg ttcatct
//
