LOCUS       sequence-14407-e        4308 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  synthetic circular DNA
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 4308)
  AUTHORS   Mukherji S, Ebert MS, Zheng GX, Tsang JS, Sharp PA, van Oudenaarden 
            A
  TITLE     MicroRNAs can generate thresholds in target gene expression.
  JOURNAL   Nat Genet. 2011 Aug 21;43(9):854-9. doi: 10.1038/ng.905.
  PUBMED    21857679
REFERENCE   2  (bases 1 to 4308)
  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"; doi: "10.1038/ng"; 
            journalName: "Nat Genet"; date: "2011-08-21- 21"; volume: "43"; 
            issue: "9"; pages: "854-9"
COMMENT     Sequence Label: pTRETightBI-RY-0
FEATURES             Location/Qualifiers
     source          1..4308
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     protein_bind    12..30
                     /gene="tetO"
                     /label=tet operator
                     /bound_moiety="tetracycline repressor TetR"
                     /note="bacterial operator O2 for the tetR and tetA genes"
     protein_bind    48..66
                     /gene="tetO"
                     /label=tet operator
                     /bound_moiety="tetracycline repressor TetR"
                     /note="bacterial operator O2 for the tetR and tetA genes"
     protein_bind    83..101
                     /gene="tetO"
                     /label=tet operator
                     /bound_moiety="tetracycline repressor TetR"
                     /note="bacterial operator O2 for the tetR and tetA genes"
     protein_bind    119..137
                     /gene="tetO"
                     /label=tet operator
                     /bound_moiety="tetracycline repressor TetR"
                     /note="bacterial operator O2 for the tetR and tetA genes"
     protein_bind    155..173
                     /gene="tetO"
                     /label=tet operator
                     /bound_moiety="tetracycline repressor TetR"
                     /note="bacterial operator O2 for the tetR and tetA genes"
     protein_bind    190..208
                     /gene="tetO"
                     /label=tet operator
                     /bound_moiety="tetracycline repressor TetR"
                     /note="bacterial operator O2 for the tetR and tetA genes"
     protein_bind    226..244
                     /gene="tetO"
                     /label=tet operator
                     /bound_moiety="tetracycline repressor TetR"
                     /note="bacterial operator O2 for the tetR and tetA genes"
     CDS             359..379
                     /codon_start=1
                     /product="nuclear localization signal of SV40 (simian virus
                     40) large T antigen"
                     /label=SV40 NLS
                     /translation="PKKKRKV"
     CDS             380..1090
                     /codon_start=1
                     /product="monomeric derivative of DsRed fluorescent protein
                     (Shaner et al., 2004)"
                     /label=mCherry
                     /note="mammalian codon-optimized"
                     /translation="MVSKGEEDNMAIIKEFMRFKVHMEGSVNGHEFEIEGEGEGRPYEG
                     TQTAKLKVTKGGPLPFAWDILSPQFMYGSKAYVKHPADIPDYLKLSFPEGFKWERVMNF
                     EDGGVVTVTQDSSLQDGEFIYKVKLRGTNFPSDGPVMQKKTMGWEASSERMYPEDGALK
                     GEIKQRLKLKDGGHYDAEVKTTYKAKKPVQLPGAYNVNIKLDITSHNEDYTIVEQYERA
                     EGRHSTGGMDELYK"
     primer_bind     complement(525..543)
                     /label=mCherry-R
                     /note="mCherry, reverse primer"
     primer_bind     793..812
                     /label=mCherry-F
                     /note="mCherry, forward primer"
     polyA_signal    1228..1309
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     promoter        1342..1446
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(1447..1515,1516..2307)
                     /codon_start=1
                     /gene="bla"
                     /product="beta-lactamase"
                     /label=AmpR
                     /note="confers resistance to ampicillin, carbenicillin, and
                     related antibiotics"
                     /translation="MSIQHFRVALIPFFAAFCLPVFAHPETLVKVKDAEDQLGARVGYI
                     ELDLNSGKILESFRPEERFPMMSTFKVLLCGAVLSRVDAGQEQLGRRIHYSQNDLVEYS
                     PVTEKHLTDGMTVRELCSAAITMSDNTAANLLLTTIGGPKELTAFLHNMGDHVTRLDRW
                     EPELNEAIPNDERDTTMPVAMATTLRKLLTGELLTLASRQQLIDWMEADKVAGPLLRSA
                     LPAGWFIADKSGAGERGSRGIIAALGPDGKPSRIVVIYTTGSQATMDERNRQIAEIGAS
                     LIKHW"
     primer_bind     complement(1665..1684)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      2478..3066
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     2967..2986
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     3220..3237
                     /label=L4440
                     /note="L4440 vector, forward primer"
     polyA_signal    3247..3328
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     CDS             complement(join(3479..4192,4193..4195,4196..4198))
                     /codon_start=1
                     /product="enhanced YFP"
                     /label=EYFP
                     /note="mammalian codon-optimized"
                     /translation="MVSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTL
                     KFICTTGKLPVPWPTLVTTFGYGLQCFARYPDHMKQHDFFKSAMPEGYVQERTIFFKDD
                     GNYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIK
                     VNFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSYQSALSKDPNEKRDHMVLL
                     EFVTAAGITLGMDELYK"
     primer_bind     complement(3524..3545)
                     /label=EGFP-C
                     /note="EGFP, forward primer"
     primer_bind     3873..3892
                     /label=EXFP-R
                     /note="For distinguishing EGFP variants, reverse primer"
     primer_bind     4132..4153
                     /label=EGFP-N
                     /note="EGFP, reverse primer"
     CDS             complement(4199..4219)
                     /codon_start=1
                     /product="nuclear localization signal of SV40 (simian virus
                     40) large T antigen"
                     /label=SV40 NLS
                     /translation="PKKKRKV"
     promoter        join(4239..4308,1..318)
                     /label=bidirectional TRE promoter
                     /note="Tet-responsive bidirectional promoter PTight-BI, 
                     consisting of seven tet operator sequences flanked on each 
                     side by the minimal CMV promoter"
ORIGIN
        1 ctcgagttta ctccctatca gtgatagaga acgtatgtcg agtttactcc ctatcagtga
       61 tagagaacga tgtcgagttt actccctatc agtgatagag aacgtatgtc gagtttactc
      121 cctatcagtg atagagaacg tatgtcgagt ttactcccta tcagtgatag agaacgtatg
      181 tcgagtttat ccctatcagt gatagagaac gtatgtcgag tttactccct atcagtgata
      241 gagaacgtat gtcgaggtag gcgtgtacgg tgggaggcct atataagcag agctcgttta
      301 gtgaaccgtc agatcgcctg gagatttcga gctcggtacc cggggatcca ccatgggccc
      361 taaaaagaag cgtaaagtca tggtgagcaa gggcgaggag gataacatgg ccatcatcaa
      421 ggagttcatg cgcttcaagg tgcacatgga gggctccgtg aacggccacg agttcgagat
      481 cgagggcgag ggcgagggcc gcccctacga gggcacccag accgccaagc tgaaggtgac
      541 caagggtggc cccctgccct tcgcctggga catcctgtcc cctcagttca tgtacggctc
      601 caaggcctac gtgaagcacc ccgccgacat ccccgactac ttgaagctgt ccttccccga
      661 gggcttcaag tgggagcgcg tgatgaactt cgaggacggc ggcgtggtga ccgtgaccca
      721 ggactcctcc ctgcaggacg gcgagttcat ctacaaggtg aagctgcgcg gcaccaactt
      781 cccctccgac ggccccgtaa tgcagaagaa gaccatgggc tgggaggcct cctccgagcg
      841 gatgtacccc gaggacggcg ccctgaaggg cgagatcaag cagaggctga agctgaagga
      901 cggcggccac tacgacgctg aggtcaagac cacctacaag gccaagaagc ccgtgcagct
      961 gcccggcgcc tacaacgtca acatcaagtt ggacatcacc tcccacaacg aggactacac
     1021 catcgtggaa cagtacgaac gcgccgaggg ccgccactcc accggcggca tggacgagct
     1081 gtacaagtaa atcgataagc ttgtcgacga tatctccaga ggatcataat cagccatacc
     1141 acatttgtag aggttttact tgctttaaaa aacctcccac acctccccct gaacctgaaa
     1201 cataaaatga atgcaattgt tgttgttaac ttgtttattg cagcttataa tggttacaaa
     1261 taaagcaata gcatcacaaa tttcacaaat aaagcatttt tttcactgcc gacgtcaggt
     1321 ggcacttttc ggggaaatgt gcgcggaacc cctatttgtt tatttttcta aatacattca
     1381 aatatgtatc cgctcatgag acaataaccc tgataaatgc ttcaataata ttgaaaaagg
     1441 aagagtatga gtattcaaca tttccgtgtc gcccttattc ccttttttgc ggcattttgc
     1501 cttcctgttt ttgctcaccc agaaacgctg gtgaaagtaa aagatgctga agatcagttg
     1561 ggtgcacgag tgggttacat cgaactggat ctcaacagcg gtaagatcct tgagagtttt
     1621 cgccccgaag aacgttttcc aatgatgagc acttttaaag ttctgctatg tggcgcggta
     1681 ttatcccgtg ttgacgccgg gcaagagcaa ctcggtcgcc gcatacacta ttctcagaat
     1741 gacttggttg agtactcacc agtcacagaa aagcatctta cggatggcat gacagtaaga
     1801 gaattatgca gtgctgccat aaccatgagt gataacactg cggccaactt acttctgaca
     1861 acgatcggag gaccgaagga gctaaccgct tttttgcaca acatggggga tcatgtaact
     1921 cgccttgatc gttgggaacc ggagctgaat gaagccatac caaacgacga gcgtgacacc
     1981 acgatgcctg tagcaatggc aacaacgttg cgcaaactat taactggcga actacttact
     2041 ctagcttccc ggcaacaatt aatagactgg atggaggcgg ataaagttgc aggaccactt
     2101 ctgcgctcgg cccttccggc tggctggttt attgctgata aatctggagc cggtgagcgt
     2161 gggtctcgcg gtatcattgc agcactgggg ccagatggta agccctcccg tatcgtagtt
     2221 atctacacga cggggagtca ggcaactatg gatgaacgaa atagacagat cgctgagata
     2281 ggtgcctcac tgattaagca ttggtaactg tcagaccaag tttactcata tatactttag
     2341 attgatttaa aacttcattt ttaatttaaa aggatctagg tgaagatcct ttttgataat
     2401 ctcatgacca aaatccctta acgtgagttt tcgttccact gagcgtcaga ccccgtagaa
     2461 aagatcaaag gatcttcttg agatcctttt tttctgcgcg taatctgctg cttgcaaaca
     2521 aaaaaaccac cgctaccagc ggtggtttgt ttgccggatc aagagctacc aactcttttt
     2581 ccgaaggtaa ctggcttcag cagagcgcag ataccaaata ctgtccttct agtgtagccg
     2641 tagttaggcc accacttcaa gaactctgta gcaccgccta catacctcgc tctgctaatc
     2701 ctgttaccag tggctgctgc cagtggcgat aagtcgtgtc ttaccgggtt ggactcaaga
     2761 cgatagttac cggataaggc gcagcggtcg ggctgaacgg ggggttcgtg cacacagccc
     2821 agcttggagc gaacgaccta caccgaactg agatacctac agcgtgagca ttgagaaagc
     2881 gccacgcttc ccgaagggag aaaggcggac aggtatccgg taagcggcag ggtcggaaca
     2941 ggagagcgca cgagggagct tccaggggga aacgcctggt atctttatag tcctgtcggg
     3001 tttcgccacc tctgacttga gcgtcgattt ttgtgatgct cgtcaggggg gcggagccta
     3061 tggaaaaacg ccagcaacgc ggccttttta cggttcctgg ccttttgctg gccttttgct
     3121 cacatgttct ttcctgcgtt atcccctgat tctgtggata accgtattac cgcctttgag
     3181 tgagctgata ccgctcgccg cagccgaacg accgagcgca gcgagtcagt gagcgaggaa
     3241 gctcgggcag tgaaaaaaat gctttatttg tgaaatttgt gatgctattg ctttatttgt
     3301 aaccattata agctgcaata aacaagttaa caacaacaat tgcattcatt ttatgtttca
     3361 ggttcagggg gaggtgtggg aggtttttta aagcaagtaa aacctctaca aatgtggtat
     3421 ggctgattat gatcctctag actgcagcct caggagatct gggcccccgc ggcatatgtt
     3481 acttgtacag ctcgtccatg ccgagagtga tcccggcggc ggtcacgaac tccagcagga
     3541 ccatgtgatc gcgcttctcg ttggggtctt tgctcagggc ggactggtag ctcaggtagt
     3601 ggttgtcggg cagcagcacg gggccgtcgc cgatgggggt gttctgctgg tagtggtcgg
     3661 cgagctgcac gctgccgtcc tcgatgttgt ggcggatctt gaagttcacc ttgatgccgt
     3721 tcttctgctt gtcggccatg atatagacgt tgtggctgtt gtagttgtac tccagcttgt
     3781 gccccaggat gttgccgtcc tccttgaagt cgatgccctt cagctcgatg cggttcacca
     3841 gggtgtcgcc ctcgaacttc acctcggcgc gggtcttgta gttgccgtcg tccttgaaga
     3901 agatggtgcg ctcctggacg tagccttcgg gcatggcgga cttgaagaag tcgtgctgct
     3961 tcatgtggtc ggggtagcgg gcgaagcact gcaggccgta gccgaaggtg gtcacgaggg
     4021 tgggccaggg cacgggcagc ttgccggtgg tgcagatgaa cttcagggtc agcttgccgt
     4081 aggtggcatc gccctcgccc tcgccggaca cgctgaactt gtggccgttt acgtcgccgt
     4141 ccagctcgac caggatgggc accaccccgg tgaacagctc ctcgcccttg ctcaccatga
     4201 ctttacgctt ctttttaggg cccatggtga attctccagg cgatctgacg gttcactaaa
     4261 cgagctctgc ttatataggc ctcccaccgt acacgccacc tcgacata
//
