LOCUS       sequence-269869-        7326 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  DOX-inducible WT G3BP1 inserted with GFP tagged on the N-terminus 
            and used with the Flp-In T-Rex System.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 7326)
  AUTHORS   Fischer JW, Busa VF, Shao Y, Leung AKL
  TITLE     Structure-Mediated RNA Decay by UPF1 and G3BP1.
  JOURNAL   Mol Cell. 2020 Apr 2;78(1):70-84.e6. doi: 
            10.1016/j.molcel.2020.01.021. Epub 2020 Feb 3.
  PUBMED    32017897
REFERENCE   2  (bases 1 to 7326)
  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: 
            "10.1016/j.molcel.2020.01.021"; journalName: "Mol Cell"; date: 
            "2020-04-2- 2"; volume: "78"; issue: "1"; pages: "70-84.e6"
COMMENT     Sequence Label: pcDNA5 FRT TO-EGFP-G3BP1-WT
FEATURES             Location/Qualifiers
     source          1..7326
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     enhancer        141..520
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
     promoter        521..724
                     /label=CMV promoter
                     /note="human cytomegalovirus (CMV) immediate early 
                     promoter"
     primer_bind     675..695
                     /label=CMV-F
                     /note="Human CMV immediate early promoter, forward primer"
     protein_bind    726..744
                     /gene="tetO"
                     /label=tet operator
                     /bound_moiety="tetracycline repressor TetR"
                     /note="bacterial operator O2 for the tetR and tetA genes"
     protein_bind    747..765
                     /gene="tetO"
                     /label=tet operator
                     /bound_moiety="tetracycline repressor TetR"
                     /note="bacterial operator O2 for the tetR and tetA genes"
     regulatory      923..932
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     CDS             join(929..931,932..934,935..1645)
                     /codon_start=1
                     /product="enhanced GFP"
                     /label=EGFP
                     /translation="MVSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTL
                     KFICTTGKLPVPWPTLVTTLTYGVQCFSRYPDHMKQHDFFKSAMPEGYVQERTIFFKDD
                     GNYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIK
                     VNFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSALSKDPNEKRDHMVLL
                     EFVTAAGITLGMDELYK"
     CDS             join(929..931,932..934,935..1645)
                     /codon_start=1
                     /product="the original enhanced GFP (Yang et al., 1996)"
                     /label=EGFP
                     /note="mammalian codon-optimized"
                     /translation="MVSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTL
                     KFICTTGKLPVPWPTLVTTLTYGVQCFSRYPDHMKQHDFFKSAMPEGYVQERTIFFKDD
                     GNYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIK
                     VNFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSALSKDPNEKRDHMVLL
                     EFVTAAGITLGMDELYK"
     primer_bind     complement(974..995)
                     /label=EGFP-N
                     /note="EGFP, reverse primer"
     primer_bind     complement(1235..1254)
                     /label=EXFP-R
                     /note="For distinguishing EGFP variants, reverse primer"
     primer_bind     1582..1603
                     /label=EGFP-C
                     /note="EGFP, forward primer"
     primer_bind     complement(3184..3201)
                     /label=BGH-rev
                     /note="Bovine growth hormone terminator, reverse primer. 
                     Also called BGH reverse"
     polyA_signal    3190..3414
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     primer_bind     complement(3547..3566)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     protein_bind    3698..3745
                     /label=FRT
                     /bound_moiety="FLP recombinase from the Saccharomyces 
                     cerevisiae 2u plasmid"
                     /note="FLP-mediated recombination occurs in the 8-bp core 
                     sequence TCTAGAAA (Turan and Bode, 2011)."
     CDS             3753..4775
                     /codon_start=1
                     /product="hygromycin B phosphotransferase"
                     /label=HygR
                     /note="confers resistance to hygromycin"
                     /translation="KKPELTATSVEKFLIEKFDSVSDLMQLSEGEESRAFSFDVGGRGY
                     VLRVNSCADGFYKDRYVYRHFASAALPIPEVLDIGEFSESLTYCISRRAQGVTLQDLPE
                     TELPAVLQPVAEAMDAIAAADLSQTSGFGPFGPQGIGQYTTWRDFICAIADPHVYHWQT
                     VMDDTVSASVAQALDELMLWAEDCPEVRHLVHADFGSNNVLTDNGRITAVIDWSEAMFG
                     DSQYEVANIFFWRPWLACMEQQTRYFERRHPELAGSPRLRAYMLRIGLDQLYQSLVDGN
                     FDDAAWAQGRCDAIVRSGAGTVGRTQIARRSAAVWTDGCVEVLADSGNRRPSTRPRAKE
                     "
     CDS             3753..4775
                     /codon_start=1
                     /gene="aph(4)-Ia"
                     /product="aminoglycoside phosphotransferase from E. coli"
                     /label=HygR
                     /note="confers resistance to hygromycin"
                     /translation="KKPELTATSVEKFLIEKFDSVSDLMQLSEGEESRAFSFDVGGRGY
                     VLRVNSCADGFYKDRYVYRHFASAALPIPEVLDIGEFSESLTYCISRRAQGVTLQDLPE
                     TELPAVLQPVAEAMDAIAAADLSQTSGFGPFGPQGIGQYTTWRDFICAIADPHVYHWQT
                     VMDDTVSASVAQALDELMLWAEDCPEVRHLVHADFGSNNVLTDNGRITAVIDWSEAMFG
                     DSQYEVANIFFWRPWLACMEQQTRYFERRHPELAGSPRLRAYMLRIGLDQLYQSLVDGN
                     FDDAAWAQGRCDAIVRSGAGTVGRTQIARRSAAVWTDGCVEVLADSGNRRPSTRPRAKE
                     "
     polyA_signal    4905..5026
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(4942..4961)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     4996..5015
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     primer_bind     complement(5075..5091)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(5075..5091)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(5088..5110)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    5099..5115
                     /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(5123..5129,5130..5147,5148..5153))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    5168..5189
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(5306..5323)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(5477..6065)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(5557..5576)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(6236..7027,7028..7096))
                     /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     6859..6878
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(7097..7201)
                     /gene="bla"
                     /label=AmpR promoter
     primer_bind     complement(7276..7295)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
ORIGIN
        1 tgtgttggag gtcgctgagt agtgcgcgag caaaatttaa gctacaacaa ggcaaggctt
       61 gaccgacaat tgcatgaaga atctgcttag ggttaggcgt tttgcgctgc ttcgcgatgt
      121 acgggccaga tatacgcgtt gacattgatt attgactagt tattaatagt aatcaattac
      181 ggggtcatta gttcatagcc catatatgga gttccgcgtt acataactta cggtaaatgg
      241 cccgcctggc tgaccgccca acgacccccg cccattgacg tcaataatga cgtatgttcc
      301 catagtaacg ccaataggga ctttccattg acgtcaatgg gtggagtatt tacggtaaac
      361 tgcccacttg gcagtacatc aagtgtatca tatgccaagt acgcccccta ttgacgtcaa
      421 tgacggtaaa tggcccgcct ggcattatgc ccagtacatg accttatggg actttcctac
      481 ttggcagtac atctacgtat tagtcatcgc tattaccatg gtgatgcggt tttggcagta
      541 catcaatggg cgtggatagc ggtttgactc acggggattt ccaagtctcc accccattga
      601 cgtcaatggg agtttgtttt ggcaccaaaa tcaacgggac tttccaaaat gtcgtaacaa
      661 ctccgcccca ttgacgcaaa tgggcggtag gcgtgtacgg tgggaggtct atataagcag
      721 agctctccct atcagtgata gagatctccc tatcagtgat agagatcgtc gacgagctcg
      781 tttagtgaac cgtcagatcg cctggagacg ccatccacgc tgttttgacc tccatagaag
      841 acaccgggac cgatccagcc tccggactct agcgtttaaa cttaagctta gtgaaccgtc
      901 agatccgcta gcgctaccgg tcgccaccat ggtgagcaag ggcgaggagc tgttcaccgg
      961 ggtggtgccc atcctggtcg agctggacgg cgacgtaaac ggccacaagt tcagcgtgtc
     1021 cggcgagggc gagggcgatg ccacctacgg caagctgacc ctgaagttca tctgcaccac
     1081 cggcaagctg cccgtgccct ggcccaccct cgtgaccacc ctgacctacg gcgtgcagtg
     1141 cttcagccgc taccccgacc acatgaagca gcacgacttc ttcaagtccg ccatgcccga
     1201 aggctacgtc caggagcgca ccatcttctt caaggacgac ggcaactaca agacccgcgc
     1261 cgaggtgaag ttcgagggcg acaccctggt gaaccgcatc gagctgaagg gcatcgactt
     1321 caaggaggac ggcaacatcc tggggcacaa gctggagtac aactacaaca gccacaacgt
     1381 ctatatcatg gccgacaagc agaagaacgg catcaaggtg aacttcaaga tccgccacaa
     1441 catcgaggac ggcagcgtgc agctcgccga ccactaccag cagaacaccc ccatcggcga
     1501 cggccccgtg ctgctgcccg acaaccacta cctgagcacc cagtccgccc tgagcaaaga
     1561 ccccaacgag aagcgcgatc acatggtcct gctggagttc gtgaccgccg ccgggatcac
     1621 tctcggcatg gacgagctgt acaagtccgg actcagatct atggtgatgg agaagcctag
     1681 tcccctgctg gtcgggcggg aatttgtgag acagtattac acactgctga accaggcccc
     1741 agacatgctg catagatttt atggaaagaa ctcttcttat gtccatgggg gattggattc
     1801 aaatggaaag ccagcagatg cagtctacgg acagaaagaa atccacagga aagtgatgtc
     1861 acaaaacttc accaactgcc acaccaagat tcgccatgtt gatgctcatg ccacgctaaa
     1921 tgatggtgtg gtagtccagg tgatggggct tctctctaac aacaaccagg ctttgaggag
     1981 attcatgcaa acgtttgtcc ttgctcctga ggggtctgtt gcaaataaat tctatgttca
     2041 caatgatatc ttcagatacc aagatgaggt ctttggtggg tttgtcactg agcctcagga
     2101 ggagtctgaa gaagaagtag aggaacctga agaaagacag caaacacctg aggtggtacc
     2161 tgatgattct ggaactttct atgatcaggc agttgtcagt aatgacatgg aagaacattt
     2221 agaggagcct gttgctgaac cagagcctga tcctgaacca gaaccagaac aagaacctgt
     2281 atctgaaatc caagaggaaa agcctgagcc agtattagaa gaaactgccc ctgaggatgc
     2341 tcagaagagt tcttctccag cacctgcaga catagctcag acagtacagg aagacttgag
     2401 gacattttct tgggcatctg tgaccagtaa gaatcttcca cccagtggag ctgttccagt
     2461 tactgggata ccacctcatg ttgttaaagt accagcttca cagccccgtc cagagtctaa
     2521 gcctgaatct cagattccac cacaaagacc tcagcgggat caaagagtgc gagaacaacg
     2581 aataaatatt cctccccaaa ggggacccag accaatccgt gaggctggtg agcaaggtga
     2641 cattgaaccc cgaagaatgg tgagacaccc tgacagtcac caactcttca ttggcaacct
     2701 gcctcatgaa gtggacaaat cagagcttaa agatttcttt caaagttatg gaaacgtggt
     2761 ggagttgcgc attaacagtg gtgggaaatt acccaatttt ggttttgttg tgtttgatga
     2821 ttctgagcct gttcagaaag tccttagcaa cagacccatc atgttcagag gtgaggtccg
     2881 tctgaatgtc gaagagaaga agactcgagc tgccagggaa ggcgaccgac gagataatcg
     2941 ccttcgggga cctggaggcc ctcgaggtgg gctgggtggt ggaatgagag gccctccccg
     3001 tggaggcatg gtgcagaaac caggatttgg agtgggaagg gggcttgcgc cacggcaggg
     3061 aattctgcag tcgacggtac cgcgggcccg ggatccacta gtccagtgtg gtggaattct
     3121 gcagatatcc agcacagtgg cggccgctcg agtctagagg gcccgtttaa acccgctgat
     3181 cagcctcgac tgtgccttct agttgccagc catctgttgt ttgcccctcc cccgtgcctt
     3241 ccttgaccct ggaaggtgcc actcccactg tcctttccta ataaaatgag gaaattgcat
     3301 cgcattgtct gagtaggtgt cattctattc tggggggtgg ggtggggcag gacagcaagg
     3361 gggaggattg ggaagacaat agcaggcatg ctggggatgc ggtgggctct atggcttctg
     3421 aggcggaaag aaccagctgg ggctctaggg ggtatcccca cgcgccctgt agcggcgcat
     3481 taagcgcggc gggtgtggtg gttacgcgca gcgtgaccgc tacacttgcc agcgccctag
     3541 cgcccgctcc tttcgctttc ttcccttcct ttctcgccac gttcgccggc tttccccgtc
     3601 aagctctaaa tcgggggctc cctttagggt tccgatttag tgctttacgg cacctcgacc
     3661 ccaaaaaact tgattagggt gatggttcac gtacctagaa gttcctattc cgaagttcct
     3721 attctctaga aagtatagga acttccttgg ccaaaaagcc tgaactcacc gcgacgtctg
     3781 tcgagaagtt tctgatcgaa aagttcgaca gcgtctccga cctgatgcag ctctcggagg
     3841 gcgaagaatc tcgtgctttc agcttcgatg taggagggcg tggatatgtc ctgcgggtaa
     3901 atagctgcgc cgatggtttc tacaaagatc gttatgttta tcggcacttt gcatcggccg
     3961 cgctcccgat tccggaagtg cttgacattg gggaattcag cgagagcctg acctattgca
     4021 tctcccgccg tgcacagggt gtcacgttgc aagacctgcc tgaaaccgaa ctgcccgctg
     4081 ttctgcagcc ggtcgcggag gccatggatg cgatcgctgc ggccgatctt agccagacga
     4141 gcgggttcgg cccattcgga ccgcaaggaa tcggtcaata cactacatgg cgtgatttca
     4201 tatgcgcgat tgctgatccc catgtgtatc actggcaaac tgtgatggac gacaccgtca
     4261 gtgcgtccgt cgcgcaggct ctcgatgagc tgatgctttg ggccgaggac tgccccgaag
     4321 tccggcacct cgtgcacgcg gatttcggct ccaacaatgt cctgacggac aatggccgca
     4381 taacagcggt cattgactgg agcgaggcga tgttcgggga ttcccaatac gaggtcgcca
     4441 acatcttctt ctggaggccg tggttggctt gtatggagca gcagacgcgc tacttcgagc
     4501 ggaggcatcc ggagcttgca ggatcgccgc ggctccgggc gtatatgctc cgcattggtc
     4561 ttgaccaact ctatcagagc ttggttgacg gcaatttcga tgatgcagct tgggcgcagg
     4621 gtcgatgcga cgcaatcgtc cgatccggag ccgggactgt cgggcgtaca caaatcgccc
     4681 gcagaagcgc ggccgtctgg accgatggct gtgtagaagt actcgccgat agtggaaacc
     4741 gacgccccag cactcgtccg agggcaaagg aatagcacgt actacgagat ttcgattcca
     4801 ccgccgcctt ctatgaaagg ttgggcttcg gaatcgtttt ccgggacgcc ggctggatga
     4861 tcctccagcg cggggatctc atgctggagt tcttcgccca ccccaacttg tttattgcag
     4921 cttataatgg ttacaaataa agcaatagca tcacaaattt cacaaataaa gcattttttt
     4981 cactgcattc tagttgtggt ttgtccaaac tcatcaatgt atcttatcat gtctgtatac
     5041 cgtcgacctc tagctagagc ttggcgtaat catggtcata gctgtttcct gtgtgaaatt
     5101 gttatccgct cacaattcca cacaacatac gagccggaag cataaagtgt aaagcctggg
     5161 gtgcctaatg agtgagctaa ctcacattaa ttgcgttgcg ctcactgccc gctttccagt
     5221 cgggaaacct gtcgtgccag ctgcattaat gaatcggcca acgcgcgggg agaggcggtt
     5281 tgcgtattgg gcgctcttcc gcttcctcgc tcactgactc gctgcgctcg gtcgttcggc
     5341 tgcggcgagc ggtatcagct cactcaaagg cggtaatacg gttatccaca gaatcagggg
     5401 ataacgcagg aaagaacatg tgagcaaaag gccagcaaaa ggccaggaac cgtaaaaagg
     5461 ccgcgttgct ggcgtttttc cataggctcc gcccccctga cgagcatcac aaaaatcgac
     5521 gctcaagtca gaggtggcga aacccgacag gactataaag ataccaggcg tttccccctg
     5581 gaagctccct cgtgcgctct cctgttccga ccctgccgct taccggatac ctgtccgcct
     5641 ttctcccttc gggaagcgtg gcgctttctc atagctcacg ctgtaggtat ctcagttcgg
     5701 tgtaggtcgt tcgctccaag ctgggctgtg tgcacgaacc ccccgttcag cccgaccgct
     5761 gcgccttatc cggtaactat cgtcttgagt ccaacccggt aagacacgac ttatcgccac
     5821 tggcagcagc cactggtaac aggattagca gagcgaggta tgtaggcggt gctacagagt
     5881 tcttgaagtg gtggcctaac tacggctaca ctagaagaac agtatttggt atctgcgctc
     5941 tgctgaagcc agttaccttc ggaaaaagag ttggtagctc ttgatccggc aaacaaacca
     6001 ccgctggtag cggtggtttt tttgtttgca agcagcagat tacgcgcaga aaaaaaggat
     6061 ctcaagaaga tcctttgatc ttttctacgg ggtctgacgc tcagtggaac gaaaactcac
     6121 gttaagggat tttggtcatg agattatcaa aaaggatctt cacctagatc cttttaaatt
     6181 aaaaatgaag ttttaaatca atctaaagta tatatgagta aacttggtct gacagttacc
     6241 aatgcttaat cagtgaggca cctatctcag cgatctgtct atttcgttca tccatagttg
     6301 cctgactccc cgtcgtgtag ataactacga tacgggaggg cttaccatct ggccccagtg
     6361 ctgcaatgat accgcgagac ccacgctcac cggctccaga tttatcagca ataaaccagc
     6421 cagccggaag ggccgagcgc agaagtggtc ctgcaacttt atccgcctcc atccagtcta
     6481 ttaattgttg ccgggaagct agagtaagta gttcgccagt taatagtttg cgcaacgttg
     6541 ttgccattgc tacaggcatc gtggtgtcac gctcgtcgtt tggtatggct tcattcagct
     6601 ccggttccca acgatcaagg cgagttacat gatcccccat gttgtgcaaa aaagcggtta
     6661 gctccttcgg tcctccgatc gttgtcagaa gtaagttggc cgcagtgtta tcactcatgg
     6721 ttatggcagc actgcataat tctcttactg tcatgccatc cgtaagatgc ttttctgtga
     6781 ctggtgagta ctcaaccaag tcattctgag aatagtgtat gcggcgaccg agttgctctt
     6841 gcccggcgtc aatacgggat aataccgcgc cacatagcag aactttaaaa gtgctcatca
     6901 ttggaaaacg ttcttcgggg cgaaaactct caaggatctt accgctgttg agatccagtt
     6961 cgatgtaacc cactcgtgca cccaactgat cttcagcatc ttttactttc accagcgttt
     7021 ctgggtgagc aaaaacagga aggcaaaatg ccgcaaaaaa gggaataagg gcgacacgga
     7081 aatgttgaat actcatactc ttcctttttc aatattattg aagcatttat cagggttatt
     7141 gtctcatgag cggatacata tttgaatgta tttagaaaaa taaacaaata ggggttccgc
     7201 gcacatttcc ccgaaaagtg ccacctgacg tcgacggatc gggagatctc ccgatcccct
     7261 atggtgcact ctcagtacaa tctgctctga tgccgcatag ttaagccagt atctgctccc
     7321 tgcttg
//
