LOCUS       sequence-308574-        6953 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Tet-On YFP-tagged Gateway destination vector, to be used together 
            with pT2-TetR-neoR transposon plasmid.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 6953)
  AUTHORS   Laidou S, Alanis-Lobato G, Pribyl J, Rasko T, Tichy B, Mikulasek K, 
            Tsagiopoulou M, Oppelt J, Kastrinaki G, Lefaki M, Singh M, Zink A, 
            Chondrogianni N, Psomopoulos F, Prigione A, Ivics Z, Pospisilova S, 
            Skladal P, Izsvak Z, Andrade-Navarro MA, Petrakis S
  TITLE     Nuclear inclusions of pathogenic ataxin-1 induce oxidative stress 
            and perturb the protein synthesis machinery.
  JOURNAL   Redox Biol. 2020 Feb 11;32:101458. doi: 10.1016/j.redox.2020.101458.
  PUBMED    32145456
REFERENCE   2  (bases 1 to 6953)
  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: "Redox 
            Biol."; date: "2020-02-11"; volume: "32:101458. doi"; pages: " 
            10.1016/j.redox.2020.101458"
COMMENT     Sequence Label: pT2-CMV/TetO2-EYFP-GW
FEATURES             Location/Qualifiers
     source          1..6953
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     34..56
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     primer_bind     48..65
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     49..65
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     75..94
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        75..93
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     enhancer        477..780
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
     promoter        781..984
                     /label=CMV promoter
                     /note="human cytomegalovirus (CMV) immediate early 
                     promoter"
     primer_bind     935..955
                     /label=CMV-F
                     /note="Human CMV immediate early promoter, forward primer"
     protein_bind    986..1004
                     /gene="tetO"
                     /label=tet operator
                     /bound_moiety="tetracycline repressor TetR"
                     /note="bacterial operator O2 for the tetR and tetA genes"
     protein_bind    1007..1025
                     /gene="tetO"
                     /label=tet operator
                     /bound_moiety="tetracycline repressor TetR"
                     /note="bacterial operator O2 for the tetR and tetA genes"
     primer_bind     1035..1059
                     /label=LNCX
                     /note="Human CMV promoter, forward primer"
     regulatory      1140..1149
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     CDS             join(1146..1148,1149..1151,1152..1862)
                     /codon_start=1
                     /product="enhanced YFP"
                     /label=EYFP
                     /note="mammalian codon-optimized"
                     /translation="MVSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTL
                     KFICTTGKLPVPWPTLVTTFGYGLQCFARYPDHMKQHDFFKSAMPEGYVQERTIFFKDD
                     GNYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIK
                     VNFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSYQSALSKDPNEKRDHMVLL
                     EFVTAAGITLGMDELYK"
     primer_bind     complement(1191..1212)
                     /label=EGFP-N
                     /note="EGFP, reverse primer"
     primer_bind     complement(1452..1471)
                     /label=EXFP-R
                     /note="For distinguishing EGFP variants, reverse primer"
     primer_bind     1799..1820
                     /label=EGFP-C
                     /note="EGFP, forward primer"
     protein_bind    1881..2005
                     /gene="mutant version of attR"
                     /label=attR1
                     /bound_moiety="LR Clonase(TM)"
                     /note="recombination site for the Gateway(R) LR reaction"
     promoter        join(2030..2035,2036..2053,2054..2060)
                     /label=lac UV5 promoter
                     /note="E. coli lac promoter with an ""up"" mutation"
     CDS             2114..2773
                     /codon_start=1
                     /gene="cat"
                     /product="chloramphenicol acetyltransferase"
                     /label=CmR
                     /note="confers resistance to chloramphenicol"
                     /translation="MEKKITGYTTVDISQWHRKEHFEAFQSVAQCTYNQTVQLDITAFL
                     KTVKKNKHKFYPAFIHILARLMNAHPEFRMAMKDGELVIWDSVHPCYTVFHEQTETFSS
                     LWSEYHDDFRQFLHIYSQDVACYGENLAYFPKGFIENMFFVSANPWVSFTSFDLNVANM
                     DNFFAPVFTMGKYYTQGDKVLMPLAIQVHHAVCDGFHVGRMLNELQQYCDEWQGGA"
     primer_bind     complement(2180..2199)
                     /label=CAT-R
                     /note="Chloramphenicol resistance gene, reverse primer"
     CDS             3115..3420
                     /codon_start=1
                     /gene="ccdB"
                     /product="CcdB, a bacterial toxin that poisons DNA gyrase"
                     /label=ccdB
                     /note="Plasmids containing the ccdB gene cannot be 
                     propagated in standard E. coli strains."
                     /translation="MQFKVYTYKRESRYRLFVDVQSDIIDTPGRRMVIPLASARLLSDK
                     VSRELYPVVHIGDESWRMMTTDMASVPVSVIGEEVADLSHRENDIKNAINLMFWGI"
     primer_bind     3299..3318
                     /label=ccdB-fwd
                     /note="ccdB gene, forward primer"
     protein_bind    complement(3461..3585)
                     /gene="mutant version of attR"
                     /label=attR2
                     /bound_moiety="LR Clonase(TM)"
                     /note="recombination site for the Gateway(R) LR reaction"
     polyA_signal    3718..3799
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(3755..3774)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     complement(4214..4234)
                     /label=T3
                     /note="T3 promoter, forward primer"
     promoter        complement(4214..4232)
                     /label=T3 promoter
                     /note="promoter for bacteriophage T3 RNA polymerase"
     primer_bind     complement(4253..4269)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(4253..4269)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(4266..4288)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    4277..4293
                     /label=lac operator
                     /bound_moiety="lac repressor encoded by lacI"
                     /note="The lac repressor binds to the lac operator to 
                     inhibit transcription in E. coli. This inhibition can be 
                     relieved by adding lactose or 
                     isopropyl-beta-D-thiogalactopyranoside (IPTG)."
     promoter        complement(join(4301..4307,4308..4325,4326..4331))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    4346..4367
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(4484..4501)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(4655..5243)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(4735..4754)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(5414..6205,6206..6274))
                     /codon_start=1
                     /gene="bla"
                     /product="beta-lactamase"
                     /label=AmpR
                     /note="confers resistance to ampicillin, carbenicillin, and
                     related antibiotics"
                     /translation="MSIQHFRVALIPFFAAFCLPVFAHPETLVKVKDAEDQLGARVGYI
                     ELDLNSGKILESFRPEERFPMMSTFKVLLCGAVLSRIDAGQEQLGRRIHYSQNDLVEYS
                     PVTEKHLTDGMTVRELCSAAITMSDNTAAKLLLTTIGGPKELTAFLHNMGDHVTRLDRW
                     EPELNEAIPNDERDTTMPVAMATTLRKLLTGELLTLASRQQLIDWMEADKVAGPLLRSA
                     LPAGWFIADKSGAGERGSRGIIAALGPDGKPSRIVVIYTTGSQATMDERNRQIAEIGAS
                     LIKHW"
     primer_bind     6037..6056
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(6275..6379)
                     /gene="bla"
                     /label=AmpR promoter
     rep_origin      6406..6861
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(6493..6512)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     6703..6724
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
ORIGIN
        1 caaggcgatt aagttgggta acgccagggt tttcccagtc acgacgttgt aaaacgacgg
       61 ccagtgagcg cgcgtaatac gactcactat agggcgaatt ggagctcgga tccctataca
      121 gttgaagtcg gaagtttaca tacacttaag ttggagtcat taaaactcgt ttttcaacta
      181 ctccacaaat ttcttgttaa caaacaatag ttttggcaag tcagttagga catctacttt
      241 gtgcatgaca caagtcattt ttccaacaat tgtttacaga cagattattt cacttataat
      301 tcactgtatc acaattccag tgggtcagaa gtttacatac actaagttga ctgtgccttt
      361 aaacagcttg gaaaattcca gaaaatgatg tcatggcttt agaagcttcc atgcattagt
      421 tattaatagt aatcaattac ggggtcatta gttcatagcc catatatgga gttccgcgtt
      481 acataactta cggtaaatgg cccgcctggc tgaccgccca acgacccccg cccattgacg
      541 tcaataatga cgtatgttcc catagtaacg ccaataggga ctttccattg acgtcaatgg
      601 gtggagtatt tacggtaaac tgcccacttg gcagtacatc aagtgtatca tatgccaagt
      661 acgcccccta ttgacgtcaa tgacggtaaa tggcccgcct ggcattatgc ccagtacatg
      721 accttatggg actttcctac ttggcagtac atctacgtat tagtcatcgc tattaccatg
      781 gtgatgcggt tttggcagta catcaatggg cgtggatagc ggtttgactc acggggattt
      841 ccaagtctcc accccattga cgtcaatggg agtttgtttt ggcaccaaaa tcaacgggac
      901 tttccaaaat gtcgtaacaa ctccgcccca ttgacgcaaa tgggcggtag gcgtgtacgg
      961 tgggaggtct atataagcag agctctccct atcagtgata gagatctccc tatcagtgat
     1021 agagatcgtc gacgagctcg tttagtgaac cgtcagatcg cctggagacg ccatccacgc
     1081 tgttttgacc tccatagaag acaccgggac cgatccagcc tccgctagcg ctaccggtcg
     1141 ccaccatggt gagcaagggc gaggagctgt tcaccggggt ggtgcccatc ctggtcgagc
     1201 tggacggcga cgtaaacggc cacaagttca gcgtgtccgg cgagggcgag ggcgatgcca
     1261 cctacggcaa gctgaccctg aagttcatct gcaccaccgg caagctgccc gtgccctggc
     1321 ccaccctcgt gaccaccttc ggctacggcc tgcagtgctt cgcccgctac cccgaccaca
     1381 tgaagcagca cgacttcttc aagtccgcca tgcccgaagg ctacgtccag gagcgcacca
     1441 tcttcttcaa ggacgacggc aactacaaga cccgcgccga ggtgaagttc gagggcgaca
     1501 ccctggtgaa ccgcatcgag ctgaagggca tcgacttcaa ggaggacggc aacatcctgg
     1561 ggcacaagct ggagtacaac tacaacagcc acaacgtcta tatcatggcc gacaagcaga
     1621 agaacggcat caaggtgaac ttcaagatcc gccacaacat cgaggacggc agcgtgcagc
     1681 tcgccgacca ctaccagcag aacaccccca tcggcgacgg ccccgtgctg ctgcccgaca
     1741 accactacct gagctaccag tccgccctga gcaaagaccc caacgagaag cgcgatcaca
     1801 tggtcctgct ggagttcgtg accgccgccg ggatcactct cggcatggac gagctgtaca
     1861 agtccggact cagatcatca acaagtttgt acaaaaaagc tgaacgagaa acgtaaaatg
     1921 atataaatat caatatatta aattagattt tgcataaaaa acagactaca taatactgta
     1981 aaacacaaca tatccagtca ctatggcggc cgcattaggc accccaggct ttacacttta
     2041 tgcttccggc tcgtataatg tgtggatttt gagttaggat ccgtcgagat tttcaggagc
     2101 taaggaagct aaaatggaga aaaaaatcac tggatatacc accgttgata tatcccaatg
     2161 gcatcgtaaa gaacattttg aggcatttca gtcagttgct caatgtacct ataaccagac
     2221 cgttcagctg gatattacgg cctttttaaa gaccgtaaag aaaaataagc acaagtttta
     2281 tccggccttt attcacattc ttgcccgcct gatgaatgct catccggaat tccgtatggc
     2341 aatgaaagac ggtgagctgg tgatatggga tagtgttcac ccttgttaca ccgttttcca
     2401 tgagcaaact gaaacgtttt catcgctctg gagtgaatac cacgacgatt tccggcagtt
     2461 tctacacata tattcgcaag atgtggcgtg ttacggtgaa aacctggcct atttccctaa
     2521 agggtttatt gagaatatgt ttttcgtctc agccaatccc tgggtgagtt tcaccagttt
     2581 tgatttaaac gtggccaata tggacaactt cttcgccccc gttttcacca tgggcaaata
     2641 ttatacgcaa ggcgacaagg tgctgatgcc gctggcgatt caggttcatc atgccgtttg
     2701 tgatggcttc catgtcggca gaatgcttaa tgaattacaa cagtactgcg atgagtggca
     2761 gggcggggcg taaagatctg gatccggctt actaaaagcc agataacagt atgcgtattt
     2821 gcgcgctgat ttttgcggta taagaatata tactgatatg tatacccgaa gtatgtcaaa
     2881 aagaggtatg ctatgaagca gcgtattaca gtgacagttg acagcgacag ctatcagttg
     2941 ctcaaggcat atatgatgtc aatatctccg gtctggtaag cacaaccatg cagaatgaag
     3001 cccgtcgtct gcgtgccgaa cgctggaaag cggaaaatca ggaagggatg gctgaggtcg
     3061 cccggtttat tgaaatgaac ggctcttttg ctgacgagaa caggggctgg tgaaatgcag
     3121 tttaaggttt acacctataa aagagagagc cgttatcgtc tgtttgtgga tgtacagagt
     3181 gatattattg acacgcccgg gcgacggatg gtgatccccc tggccagtgc acgtctgctg
     3241 tcagataaag tctcccgtga actttacccg gtggtgcata tcggggatga aagctggcgc
     3301 atgatgacca ccgatatggc cagtgtgccg gtctccgtta tcggggaaga agtggctgat
     3361 ctcagccacc gcgaaaatga catcaaaaac gccattaacc tgatgttctg gggaatataa
     3421 atgtcaggct cccttataca cagccagtct gcaggtcgac catagtgact ggatatgttg
     3481 tgttttacag tattatgtag tctgtttttt atgcaaaatc taatttaata tattgatatt
     3541 tatatcattt tacgtttctc gttcagcttt cttgtacaaa gtggttgatg atccaccgga
     3601 tctagataac tgatcataat cagccatacc acatttgtag aggttttact tgctttaaaa
     3661 aacctcccac acctccccct gaacctgaaa cataaaatga atgcaattgt tgttgttaac
     3721 ttgtttattg cagcttataa tggttacaaa taaagcaata gcatcacaaa tttcacaaat
     3781 aaagcatttt tttcactgca ttcgatatcc atggaattca ctagtgcgcg cggccgctct
     3841 agatggccag atctagcttg tggaaggcta ctcgaaatgt ttgacccaag ttaaacaatt
     3901 taaaggcaat gctaccaaat actaattgag tgtatgtaaa cttctgaccc actgggaatg
     3961 tgatgaaaga aataaaagct gaaatgaatc attctctcta ctattattct gatatttcac
     4021 attcttaaaa taaagtggtg atcctaactg acctaagaca gggaattttt actaggatta
     4081 aatgtcagga attgtgaaaa agtgagttta aatgtatttg gctaaggtgt atgtaaactt
     4141 ccgacttcaa ctgtataggg atcctctagc tagagtcgac ctcgaggggg ggcccggtac
     4201 ccagcttttg ttccctttag tgagggttaa ttgcgcgctt ggcgtaatca tggtcatagc
     4261 tgtttcctgt gtgaaattgt tatccgctca caattccaca caacatacga gccggaagca
     4321 taaagtgtaa agcctggggt gcctaatgag tgagctaact cacattaatt gcgttgcgct
     4381 cactgcccgc tttccagtcg ggaaacctgt cgtgccagct gcattaatga atcggccaac
     4441 gcgcggggag aggcggtttg cgtattgggc gctcttccgc ttcctcgctc actgactcgc
     4501 tgcgctcggt cgttcggctg cggcgagcgg tatcagctca ctcaaaggcg gtaatacggt
     4561 tatccacaga atcaggggat aacgcaggaa agaacatgtg agcaaaaggc cagcaaaagg
     4621 ccaggaaccg taaaaaggcc gcgttgctgg cgtttttcca taggctccgc ccccctgacg
     4681 agcatcacaa aaatcgacgc tcaagtcaga ggtggcgaaa cccgacagga ctataaagat
     4741 accaggcgtt tccccctgga agctccctcg tgcgctctcc tgttccgacc ctgccgctta
     4801 ccggatacct gtccgccttt ctcccttcgg gaaccgtggc gctttctcat agctcacgct
     4861 gtaggtatct cagttcggtg taggtcgttc gctccaagct gggctgtgtg cacgaacccc
     4921 ccgttcagcc cgaccgctgc gccttatccg gtaactatcg tcttgagtcc aacccggtaa
     4981 gacacgactt atcgccactg gcagcagcca ctggtaacag gattagcaga gcgaggtatg
     5041 taggcggtgc tacagagttc ttgaagtggt ggcctaacta cggctacact agaagaacag
     5101 tatttggtat ctgcgctctg ctgaagccag ttaccttcgg aaaaagagtt ggtagctctt
     5161 gatccggcaa acaaaccacc gctggtagcg gtggtttttt tgtttgcaag cagcagatta
     5221 cgcgcagaaa aaaaggatct caagaagatc ctttgatctt ttctacgggg tctgacgctc
     5281 agtggaacga aaactcacgt taagggattt tggtcatgag attatcaaaa aggatcttca
     5341 cctagatcct tttaaattaa aaatgaagtt ttaaatcaat ctaaagtata tatgagtaaa
     5401 cttggtctga cagttaccaa tgcttaatca gtgaggcacc tatctcagcg atctgtctat
     5461 ttcgttcatc catagttgcc tgactccccg tcgtgtagat aactacgata cgggagggct
     5521 taccatctgg ccccagtgct gcaatgatac cgcgagaccc acgctcaccg gctccagatt
     5581 tatcagcaat aaaccagcca gccggaaggg ccgagcgcag aagtggtcct gcaactttat
     5641 ccgcctccat ccagtctatt aattgttgcc gggaagctag agtaagtagt tcgccagtta
     5701 atagtttgcg caacgttgtt gccattgcta caggcatcgt ggtgtcacgc tcgtcgtttg
     5761 gtatggcttc attcagctcc ggttcccaac gatcaaggcg agttacatga tcccccatgt
     5821 tgtgcaaaaa agcggttagc tccttcggtc ctccgatcgt tgtcagaagt aacttggccg
     5881 cagtgttatc actcatggtt atggcagcac tgcataattc tcttactgtc atgccatccg
     5941 taagatgctt ttctgtgact ggtgagtact caaccaagtc attctgagaa tagtgtatgc
     6001 ggcgaccgag ttgctcttgc ccggcgtcaa tacgggataa taccgcgcca catagcagaa
     6061 ctttaaaagt gctcatcatt ggaaaacgtt cttcggggcg aaaactctca aggatcttac
     6121 cgctgttgag atccagttcg atgtaaccca ctcgtgcacc caactgatct tcagcatctt
     6181 ttactttcac cagcgtttct gggtgagcaa aaacaggaag gcaaaatgcc gcaaaaaagg
     6241 gaataagggc gacacggaaa tgttgaatac tcatactctt cctttttcaa tattattgaa
     6301 gcatttatca gggttattgt ctcatgagcg gatacatatt tgaatgtatt tagaaaaata
     6361 aacaaatagg ggttccgcgc acatttcccc gaaaagtgcc acctgacgcg ccctgtagcg
     6421 gcgcattaag cgcggcggga gtggtggtta cgcgcagcgt gaccgctaca cttgccagcg
     6481 ccctagcgcc cgctcctttc gctttcttcc cttcctttct cgccacgttc gccggctttc
     6541 cccgtcaagc tctaaatcgg gggctccctt tagggttccg atttagtgct ttacggcacc
     6601 tcgaccccaa aaaacttgat tagggtgatg gttcacgtag tgggccatcg ccctgataga
     6661 cggtttttcg ccctttgacg ttggagtcca cgttctttaa tagtggactc ttgttccaaa
     6721 ctggaacaac actcaaccct atctcggtct attcttttga tttataaggg attttgccga
     6781 tttcggccta ttggttaaaa aatgagctga tttaacaaaa atttaacgcg aattttaaca
     6841 aaatattaac gcttacaatt tccattcgcc attcaggctg cgcaactgtt gggaagggcg
     6901 atcggtgcgg gcctcttcgc tattacgcca gctggcgaaa gggggatgtg ctg
//
