LOCUS       sequence-335808-        7883 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Mammalian expression vector encoding a constitutive nucleolar 
            targeted roGFP and a doxycycline inducible nucleolar targeted 
            catalase. Used to study nucleolar oxidation.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 7883)
  AUTHORS   Sapio RT, Burns CJ, Pestov DG
  TITLE     Effects of Hydrogen Peroxide Stress on the Nucleolar Redox 
            Environment and Pre-rRNA Maturation.
  JOURNAL   Front Mol Biosci. 2021 Apr 26;8:678488. doi: 
            10.3389/fmolb.2021.678488. eCollection 2021.
  PUBMED    33981726
REFERENCE   2  (bases 1 to 7883)
  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: "Front Mol 
            Biosci."; date: "2021-04-26"; volume: "8:678488. doi"; pages: " 
            10.3389/fmolb.2021.678488. eCollection 2021"
COMMENT     Sequence Label: pNoROS-CAT
FEATURES             Location/Qualifiers
     source          1..7883
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     promoter        295..607
                     /label=tight TRE promoter
                     /note="Tet-responsive promoter PTight, consisting of seven 
                     tet operator sequences followed by the minimal CMV 
                     promoter"
     protein_bind    303..321
                     /gene="tetO"
                     /label=tet operator
                     /bound_moiety="tetracycline repressor TetR"
                     /note="bacterial operator O2 for the tetR and tetA genes"
     protein_bind    339..357
                     /gene="tetO"
                     /label=tet operator
                     /bound_moiety="tetracycline repressor TetR"
                     /note="bacterial operator O2 for the tetR and tetA genes"
     protein_bind    374..392
                     /gene="tetO"
                     /label=tet operator
                     /bound_moiety="tetracycline repressor TetR"
                     /note="bacterial operator O2 for the tetR and tetA genes"
     protein_bind    410..428
                     /gene="tetO"
                     /label=tet operator
                     /bound_moiety="tetracycline repressor TetR"
                     /note="bacterial operator O2 for the tetR and tetA genes"
     protein_bind    446..464
                     /gene="tetO"
                     /label=tet operator
                     /bound_moiety="tetracycline repressor TetR"
                     /note="bacterial operator O2 for the tetR and tetA genes"
     protein_bind    481..499
                     /gene="tetO"
                     /label=tet operator
                     /bound_moiety="tetracycline repressor TetR"
                     /note="bacterial operator O2 for the tetR and tetA genes"
     protein_bind    517..535
                     /gene="tetO"
                     /label=tet operator
                     /bound_moiety="tetracycline repressor TetR"
                     /note="bacterial operator O2 for the tetR and tetA genes"
     primer_bind     580..604
                     /label=LNCX
                     /note="Human CMV promoter, forward primer"
     CDS             689..709
                     /codon_start=1
                     /product="nuclear localization signal of SV40 (simian virus
                     40) large T antigen"
                     /label=SV40 NLS
                     /translation="PKKKRKV"
     CDS             2321..2344
                     /codon_start=1
                     /product="FLAG(R) epitope tag, followed by an enterokinase 
                     cleavage site"
                     /label=FLAG
                     /translation="DYKDDDDK"
     polyA_signal    2383..2504
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(2394..2413)
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     primer_bind     2448..2467
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     CDS             3230..3250
                     /codon_start=1
                     /product="nuclear localization signal of SV40 (simian virus
                     40) large T antigen"
                     /label=SV40 NLS
                     /translation="PKKKRKV"
     CDS             join(3281..3283,3284..3286,3287..3997)
                     /codon_start=1
                     /product="the original enhanced GFP (Yang et al., 1996)"
                     /label=EGFP
                     /note="mammalian codon-optimized"
                     /translation="MVSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTL
                     KFISTTGKLPVPWPTLVTTLTYGVQCFSRYPDHMKRHDFFKSAMPEGYVQERTIFFKDD
                     GNYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNCHNVYIMADKQKNGIK
                     VNFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTCSALSKDPNEKRDHMVLL
                     EFVTAAGITLGMDELYK"
     primer_bind     complement(3326..3347)
                     /label=EGFP-N
                     /note="EGFP, reverse primer"
     primer_bind     complement(3587..3606)
                     /label=EXFP-R
                     /note="For distinguishing EGFP variants, reverse primer"
     primer_bind     3934..3955
                     /label=EGFP-C
                     /note="EGFP, forward primer"
     CDS             4007..4063
                     /codon_start=1
                     /product="2A peptide from porcine teschovirus-1 
                     polyprotein"
                     /label=P2A
                     /note="Eukaryotic ribosomes fail to insert a peptide bond 
                     between the Gly and Pro residues, yielding separate 
                     polypeptides."
                     /translation="ATNFSLLKQAGDVEENPGP"
     CDS             join(4064..4681,4682..4687,4688..4723,4724..4726,4727..4762,
                     4763..4765,4766..4801,4802..4807)
                     /codon_start=1
                     /product="improved tetracycline-controlled transactivator"
                     /label=rtTA-Advanced
                     /note="In the Tet-On(R) system, rtTA-Advanced binds to the 
                     Tet-responsive element and stimulates transcription only in
                     the presence of tetracycline or doxycycline."
                     /translation="MSRLDKSKVINGALELLNGVGIEGLTTRKLAQKLGVEQPTLYWHV
                     KNKRALLDALPIEMLDRHHTHFCPLEGESWQDFLRNNAKSFRCALLSHRDGAKVHLGTR
                     PTEKQYETLENQLAFLCQQGFSLENALYALSAVGHFTLGCVLEEQEHQVAKEERETPTT
                     DSMPPLLRQAIELFDRQGAEPAFLFGLELIICGLEKQLKCESGGPADALDDFDLDMLPA
                     DALDDFDLDMLPADALDDFDLDMLPG"
     CDS             4814..4870
                     /codon_start=1
                     /product="2A peptide from porcine teschovirus-1 
                     polyprotein"
                     /label=P2A
                     /note="Eukaryotic ribosomes fail to insert a peptide bond 
                     between the Gly and Pro residues, yielding separate 
                     polypeptides."
                     /translation="ATNFSLLKQAGDVEENPGP"
     CDS             4871..5269
                     /codon_start=1
                     /gene="Aspergillus terreus BSD"
                     /product="blasticidin S deaminase"
                     /label=BSD
                     /note="confers resistance to blasticidin"
                     /translation="MAKPLSQEESTLIERATATINSIPISEDYSVASAALSSDGRIFTG
                     VNVYHFTGGPCAELVVLGTAAAAAAGNLTCIVAIGNENRGILSPCGRCRQVLLDLHPGI
                     KAIVKDSDGQPTAVGIRELLPSGYVWEG"
     primer_bind     complement(5324..5341)
                     /label=BGH-rev
                     /note="Bovine growth hormone terminator, reverse primer. 
                     Also called BGH reverse"
     polyA_signal    5330..5554
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     primer_bind     complement(5880..5897)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(6051..6639)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(6131..6150)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(6810..7601,7602..7670))
                     /codon_start=1
                     /gene="bla"
                     /product="beta-lactamase"
                     /label=AmpR
                     /note="confers resistance to ampicillin, carbenicillin, and
                     related antibiotics"
                     /translation="MSIQHFRVALIPFFAAFCLPVFAHPETLVKVKDAEDQLGARVGYI
                     ELDLNSGKILESFRPEERFPMMSTFKVLLCGAVLSRIDAGQEQLGRRIHYSQNDLVEYS
                     PVTEKHLTDGMTVRELCSAAITMSDNTAANLLLTTIGGPKELTAFLHNMGDHVTRLDRW
                     EPELNEAIPNDERDTTMPVAMATTLRKLLTGELLTLASRQQLIDWMEADKVAGPLLRSA
                     LPAGWFIADKSGAGERGSRGIIAALGPDGKPSRIVVIYTTGSQATMDERNRQIAEIGAS
                     LIKHW"
     primer_bind     7433..7452
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(7671..7775)
                     /gene="bla"
                     /label=AmpR promoter
ORIGIN
        1 ggatccctat acagttgaag tcggaagttt acatacactt aagttggagt cattaaaact
       61 cgtttttcaa ctactccaca aatttcttgt taacaaacaa tagttttggc aagtcagtta
      121 ggacatctac tttgtgcatg acacaagtca tttttccaac aattgtttac agacagatta
      181 tttcacttat aattcactgt atcacaattc cagtgggtca gaagtttaca tacactaagt
      241 tcgactcctc tgcagaatgc ggcgatgttt cggtaagggg tccgctatct agacgagttt
      301 actccctatc agtgatagag aacgtatgtc gagtttactc cctatcagtg atagagaacg
      361 atgtcgagtt tactccctat cagtgataga gaacgtatgt cgagtttact ccctatcagt
      421 gatagagaac gtatgtcgag tttactccct atcagtgata gagaacgtat gtcgagttta
      481 tccctatcag tgatagagaa cgtatgtcga gtttactccc tatcagtgat agagaacgta
      541 tgtcgattag gcgtgtacgg tgggcgccta taaaagcaga gctcgtttag tgaaccgtca
      601 gatcgcctgg agccaattcc aactcttttg ccttatacct actttccgta ccacttccta
      661 ccctcgaaag gcctctgagg ccaccatgcc gaagaagaag cgcaaggtgc cggtgcaccg
      721 ccgccgccgc cgccgccgca tgtcggacag tcgggaccca gccagcgacc agatgaagca
      781 gtggaaggag cagcgggcct cgcagagacc tgatgtcctg accaccggag gcgggaaccc
      841 aataggagat aaacttaata tcatgaccgc ggggtcccga gggcccctcc tcgttcagga
      901 tgtggttttc actgacgaga tggcacactt tgacagagag cggattcctg agagagtggt
      961 acacgcaaaa ggagcaggtg cttttggata ctttgaggtc acccacgata tcaccagata
     1021 ctccaaggca aaggtgtttg agcatattgg aaagaggacc cctattgccg ttcgattctc
     1081 cacagtcgct ggagagtcag gctcagctga cacagttcgt gaccctcggg ggtttgcagt
     1141 gaaattttac actgaagatg gtaactggga tcttgtggga aacaacaccc ctattttctt
     1201 catcagggat gccatattgt ttccatcctt tatccatagc cagaagagaa acccacagac
     1261 tcacctgaag gatcctgaca tggtctggga cttctggagt cttcgtcccg agtctctcca
     1321 tcaggtttct ttcttgttca gtgaccgagg gattcccgat ggtcaccggc acatgaatgg
     1381 ctatggatca cacaccttca agttggttaa tgcagatgga gaggcagtct attgcaagtt
     1441 ccattacaag accgaccagg gcatcaaaaa cttgcctgtt ggagaggcag gaaggcttgc
     1501 tcaggaagat ccggattatg gcctccgaga tcttttcaat gccatcgcca atggcaatta
     1561 cccgtcctgg acgttttaca tccaggtcat gacttttaag gaggcagaaa ctttcccatt
     1621 taatccattt gatctgacca aggtttggcc tcacaaggac taccctctta taccagttgg
     1681 caaactggtt ttaaacaaaa atccagttaa ttactttgct gaagttgaac agatggcttt
     1741 tgacccaagc aatatgcccc ctggcatcga gcccagccct gacaaaatgc ttcagggccg
     1801 cctttttgcc tacccggaca ctcaccgcca ccgcctggga cccaactatc tgcagatacc
     1861 tgtgaactgt ccctaccgcg ctcgagtggc caactaccag cgtgatggcc ccatgtgcat
     1921 gcatgacaac cagggtggtg cccccaacta ttaccccaac agcttcagcg caccagagca
     1981 gcagcgctca gccctggagc acagcgtcca gtgcgctgta gatgtgaaac gcttcaacag
     2041 tgctaatgaa gacaatgtca ctcaggtgcg gacattctac acaaaggtgt tgaacgagga
     2101 ggagaggaaa cgcctgtgtg agaacattgc cggccacctg aaggacgctc agcttttcat
     2161 tcagaagaaa gcggtcaaga atttcactga cgtccaccct gactatgggg cccgcatcca
     2221 ggctcttctg gacaagtaca acgctgagaa gcctaagaac gcaattcaca cctacacgca
     2281 ggccggctct cacatggctg cgaagggaaa agctaacctg gactacaaag acgatgacga
     2341 caagtaaggc ctgtcaggcc aagcttccat cgatagacat gataagatac attgatgagt
     2401 ttggacaaac cacaacaaga atgcagtgaa aaaaatgctt tatttgtgaa atttgtgatg
     2461 ctattgcttt atttgtaacc attataagct gcaataaaca agttaacaac aacaattgca
     2521 ttcattttat gtttcaggtt cagggggaga tgtgggaggt tttttaaagc aagtaaaacc
     2581 tctacaaatg tggtacctag gcgagaccct gtctcacaaa ataaagtaag cccggactga
     2641 gtgcggaaag gcgggcctgg cgggtctggt ctccccatgc gggccaccag aggccctgca
     2701 gccttcagtc gcttgaaggg gtaatggcgc ttccactcac aaacatggcg gacagagcgt
     2761 gtgaacgaga tgaacagccc ctcaaaaata tggccgccga ggctggacgg ccgtgcccca
     2821 gcagcaccgc ctccgcgccc cacgtgatct ctcgccgggc acagcgctga ccgcggaggt
     2881 ccaaccggaa gaatgtccgg attggacatt cggaagaggg cccgccttcc ctggggaatc
     2941 tctgcgcacg cgcagaacgc ttcgaccaat gaaaacacag gaagccgtcc gcgcaaccgc
     3001 gttgcgtcac ttctgccgcc cctgtttcaa ggtatatagc cgtagacgga acttcgcctt
     3061 tctctcggcc ttagcgccat ttttttgggt gagtgttttt tggttcctgc gttgggattc
     3121 cgtgtacaat ccatagacat ctgacctcgg cacttagcat catcacagca aactaactgt
     3181 agcctttctc tctttccctg tagaaacctc tgcacctgag gccaccatgc cgaagaagaa
     3241 gcgcaaggtg ccggtgcacc gccgccgccg ccgccgccgc atggtgagca agggcgagga
     3301 gctgttcacc ggggtggtgc ccatcctggt cgagctggac ggcgacgtaa acggccacaa
     3361 gttcagcgtg tccggcgagg gcgagggcga tgccacctac ggcaagctga ccctgaagtt
     3421 catcagcacc accggcaagc tgcccgtgcc ctggcccacc ctcgtgacca ccctgaccta
     3481 cggcgtgcag tgcttcagcc gctaccccga ccacatgaag cggcacgact tcttcaagtc
     3541 cgccatgccc gaaggctacg tccaggagcg caccatcttc ttcaaggacg acggcaacta
     3601 caagacccgc gccgaggtga agttcgaggg cgacaccctg gtgaaccgca tcgagctgaa
     3661 gggcatcgac ttcaaggagg acggcaacat cctggggcac aagctggagt acaactacaa
     3721 ctgccacaac gtctatatca tggccgacaa gcagaagaac ggcatcaagg tgaacttcaa
     3781 gatccgccac aacatcgagg acggcagcgt gcagctcgcc gaccactacc agcagaacac
     3841 ccccatcggc gacggccccg tgctgctgcc cgacaaccac tacctgagca cctgctccgc
     3901 cctgagcaaa gaccccaacg agaagcgcga tcacatggtc ctgctggagt tcgtgaccgc
     3961 cgccgggatc actctcggca tggacgagct gtacaagggc agtggagcta ctaacttcag
     4021 cctgctgaag caggctggtg acgtcgagga gaatcctggt cccatgtcta gactggacaa
     4081 gagcaaagtc ataaacggcg ctctggaatt actcaatgga gtcggtatcg aaggcctgac
     4141 gacaaggaaa ctcgctcaaa agctgggagt tgagcagcct accctgtact ggcacgtgaa
     4201 gaacaagcgg gccctgctcg atgccctgcc aatcgagatg ctggacaggc atcataccca
     4261 cttctgcccc ctggaaggcg agtcatggca agactttctg cggaacaacg ccaagtcatt
     4321 ccgctgtgct ctcctctcac atcgcgacgg ggctaaagtg catctcggca cccgcccaac
     4381 agagaaacag tacgaaaccc tggaaaatca gctcgcgttc ctgtgtcagc aaggcttctc
     4441 cctggagaac gcactgtacg ctctgtccgc cgtgggccac tttacactgg gctgcgtatt
     4501 ggaggaacag gagcatcaag tagcaaaaga ggaaagagag acacctacca ccgattctat
     4561 gcccccactt ctgagacaag caattgagct gttcgaccgg cagggagccg aacctgcctt
     4621 ccttttcggc ctggaactaa tcatatgtgg cctggagaaa cagctaaagt gcgaaagcgg
     4681 cgggccggcc gacgcccttg acgattttga cttagacatg ctcccagccg atgcccttga
     4741 cgactttgac cttgatatgc tgcctgctga cgctcttgac gattttgacc ttgacatgct
     4801 ccccgggtcc ggcgctacta acttcagcct gctgaagcag gctggtgacg tcgaggagaa
     4861 tcctggtccc atggccaagc ctttgtctca agaagaatcc accctcattg aaagagcaac
     4921 ggctacaatc aacagcatcc ccatctctga agactacagc gtcgccagcg cagctctctc
     4981 tagcgacggc cgcatcttca ctggtgtcaa tgtatatcat tttactgggg gaccttgtgc
     5041 agaactcgtg gtgctgggca ctgctgctgc tgcggcagct ggcaacctga cttgtatcgt
     5101 cgcgatcgga aatgagaaca ggggcatctt gagcccctgc ggacggtgcc gacaggtgct
     5161 tctcgatctg catcctggga tcaaagccat agtgaaggac agtgatggac agccgacggc
     5221 agttgggatt cgtgaattgc tgccctctgg ttatgtgtgg gagggctaat tcgaaggcct
     5281 gtcgtgaagc ttggggatca attctctaga gctcgctgat cagcctcgac tgtgccttct
     5341 agttgccagc catctgttgt ttgcccctcc cccgtgcctt ccttgaccct ggaaggtgcc
     5401 actcccactg tcctttccta ataaaatgag gaaattgcat cgcattgtct gagtaggtgt
     5461 cattctattc tggggggtgg ggtggggcag gacagcaagg gggaggattg ggaagacaat
     5521 agcaggcatg ctggggatgc ggtgggctct atggcttctg aggcggaaag aaccagctgg
     5581 gcctactagc tactcgggac cccttaccga aacatcgccg cattctgcag aggagtcgag
     5641 tgtatgtaaa cttctgaccc actgggaatg tgatgaaaga aataaaagct gaaatgaatc
     5701 attctctcta ctattattct gatatttcac attcttaaaa taaagtggtg atcctaactg
     5761 acctaagaca gggaattttt actaggatta aatgtcagga attgtgaaaa agtgagttta
     5821 aatgtatttg gctaaggtgt atgtaaactt ccgacttcaa ctgtataggg atccgcttcc
     5881 tcgctcactg actcgctgcg ctcggtcgtt cggctgcggc gagcggtatc agctcactca
     5941 aaggcggtaa tacggttatc cacagaatca ggggataacg caggaaagaa catgtgagca
     6001 aaaggccagc aaaaggccag gaaccgtaaa aaggccgcgt tgctggcgtt tttccatagg
     6061 ctccgccccc ctgacgagca tcacaaaaat cgacgctcaa gtcagaggtg gcgaaacccg
     6121 acaggactat aaagatacca ggcgtttccc cctggaagct ccctcgtgcg ctctcctgtt
     6181 ccgaccctgc cgcttaccgg atacctgtcc gcctttctcc cttcgggaag cgtggcgctt
     6241 tctcatagct cacgctgtag gtatctcagt tcggtgtagg tcgttcgctc caagctgggc
     6301 tgtgtgcacg aaccccccgt tcagcccgac cgctgcgcct tatccggtaa ctatcgtctt
     6361 gagtccaacc cggtaagaca cgacttatcg ccactggcag cagccactgg taacaggatt
     6421 agcagagcga ggtatgtagg cggtgctaca gagttcttga agtggtggcc taactacggc
     6481 tacactagaa gaacagtatt tggtatctgc gctctgctga agccagttac cttcggaaaa
     6541 agagttggta gctcttgatc cggcaaacaa accaccgctg gtagcggtgg tttttttgtt
     6601 tgcaagcagc agattacgcg cagaaaaaaa ggatctcaag aagatccttt gatcttttct
     6661 acggggtctg acgctcagtg gaacgaaaac tcacgttaag ggattttggt catgagatta
     6721 tcaaaaagga tcttcaccta gatcctttta aattaaaaat gaagttttaa atcaatctaa
     6781 agtatatatg agtaaacttg gtctgacagt taccaatgct taatcagtga ggcacctatc
     6841 tcagcgatct gtctatttcg ttcatccata gttgcctgac tccccgtcgt gtagataact
     6901 acgatacggg agggcttacc atctggcccc agtgctgcaa tgataccgcg agacccacgc
     6961 tcaccggctc cagatttatc agcaataaac cagccagccg gaagggccga gcgcagaagt
     7021 ggtcctgcaa ctttatccgc ctccatccag tctattaatt gttgccggga agctagagta
     7081 agtagttcgc cagttaatag tttgcgcaac gttgttgcca ttgctacagg catcgtggtg
     7141 tcacgctcgt cgtttggtat ggcttcattc agctccggtt cccaacgatc aaggcgagtt
     7201 acatgatccc ccatgttgtg caaaaaagcg gttagctcct tcggtcctcc gatcgttgtc
     7261 agaagtaagt tggccgcagt gttatcactc atggttatgg cagcactgca taattctctt
     7321 actgtcatgc catccgtaag atgcttttct gtgactggtg agtactcaac caagtcattc
     7381 tgagaatagt gtatgcggcg accgagttgc tcttgcccgg cgtcaatacg ggataatacc
     7441 gcgccacata gcagaacttt aaaagtgctc atcattggaa aacgttcttc ggggcgaaaa
     7501 ctctcaagga tcttaccgct gttgagatcc agttcgatgt aacccactcg tgcacccaac
     7561 tgatcttcag catcttttac tttcaccagc gtttctgggt gagcaaaaac aggaaggcaa
     7621 aatgccgcaa aaaagggaat aagggcgaca cggaaatgtt gaatactcat actcttcctt
     7681 tttcaatatt attgaagcat ttatcagggt tattgtctca tgagcggata catatttgaa
     7741 tgtatttaga aaaataaaca aataggggtt ccgcgcacat ttccccgaaa agtgccacct
     7801 gatgcggtgt gaaataccgc acagatgcgt aaggagaaaa taccgcatca ggaaattgta
     7861 agcgttaata ttttgttaaa att
//
