LOCUS       sequence-273300-        7326 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  DOX-inducible S149A 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-S149A
FEATURES             Location/Qualifiers
     source          1..7326
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     enhancer        131..510
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
     promoter        511..714
                     /label=CMV promoter
                     /note="human cytomegalovirus (CMV) immediate early 
                     promoter"
     primer_bind     665..685
                     /label=CMV-F
                     /note="Human CMV immediate early promoter, forward primer"
     protein_bind    716..734
                     /gene="tetO"
                     /label=tet operator
                     /bound_moiety="tetracycline repressor TetR"
                     /note="bacterial operator O2 for the tetR and tetA genes"
     protein_bind    737..755
                     /gene="tetO"
                     /label=tet operator
                     /bound_moiety="tetracycline repressor TetR"
                     /note="bacterial operator O2 for the tetR and tetA genes"
     regulatory      913..922
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     CDS             join(919..921,922..924,925..1635)
                     /codon_start=1
                     /product="enhanced GFP"
                     /label=EGFP
                     /translation="MVSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTL
                     KFICTTGKLPVPWPTLVTTLTYGVQCFSRYPDHMKQHDFFKSAMPEGYVQERTIFFKDD
                     GNYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIK
                     VNFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSALSKDPNEKRDHMVLL
                     EFVTAAGITLGMDELYK"
     CDS             join(919..921,922..924,925..1635)
                     /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(964..985)
                     /label=EGFP-N
                     /note="EGFP, reverse primer"
     primer_bind     complement(1225..1244)
                     /label=EXFP-R
                     /note="For distinguishing EGFP variants, reverse primer"
     primer_bind     1572..1593
                     /label=EGFP-C
                     /note="EGFP, forward primer"
     primer_bind     complement(3174..3191)
                     /label=BGH-rev
                     /note="Bovine growth hormone terminator, reverse primer. 
                     Also called BGH reverse"
     polyA_signal    3180..3404
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     primer_bind     complement(3537..3556)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     protein_bind    3688..3735
                     /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             3743..4765
                     /codon_start=1
                     /product="hygromycin B phosphotransferase"
                     /label=HygR
                     /note="confers resistance to hygromycin"
                     /translation="KKPELTATSVEKFLIEKFDSVSDLMQLSEGEESRAFSFDVGGRGY
                     VLRVNSCADGFYKDRYVYRHFASAALPIPEVLDIGEFSESLTYCISRRAQGVTLQDLPE
                     TELPAVLQPVAEAMDAIAAADLSQTSGFGPFGPQGIGQYTTWRDFICAIADPHVYHWQT
                     VMDDTVSASVAQALDELMLWAEDCPEVRHLVHADFGSNNVLTDNGRITAVIDWSEAMFG
                     DSQYEVANIFFWRPWLACMEQQTRYFERRHPELAGSPRLRAYMLRIGLDQLYQSLVDGN
                     FDDAAWAQGRCDAIVRSGAGTVGRTQIARRSAAVWTDGCVEVLADSGNRRPSTRPRAKE
                     "
     CDS             3743..4765
                     /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    4895..5016
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(4932..4951)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     4986..5005
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     primer_bind     complement(5065..5081)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(5065..5081)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(5078..5100)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    5089..5105
                     /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(5113..5119,5120..5137,5138..5143))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    5158..5179
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(5296..5313)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(5467..6055)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(5547..5566)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(6226..7017,7018..7086))
                     /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     6849..6868
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(7087..7191)
                     /gene="bla"
                     /label=AmpR promoter
     primer_bind     complement(7266..7285)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
ORIGIN
        1 gtcgctgagt agtgcgcgag caaaatttaa gctacaacaa ggcaaggctt gaccgacaat
       61 tgcatgaaga atctgcttag ggttaggcgt tttgcgctgc ttcgcgatgt acgggccaga
      121 tatacgcgtt gacattgatt attgactagt tattaatagt aatcaattac ggggtcatta
      181 gttcatagcc catatatgga gttccgcgtt acataactta cggtaaatgg cccgcctggc
      241 tgaccgccca acgacccccg cccattgacg tcaataatga cgtatgttcc catagtaacg
      301 ccaataggga ctttccattg acgtcaatgg gtggagtatt tacggtaaac tgcccacttg
      361 gcagtacatc aagtgtatca tatgccaagt acgcccccta ttgacgtcaa tgacggtaaa
      421 tggcccgcct ggcattatgc ccagtacatg accttatggg actttcctac ttggcagtac
      481 atctacgtat tagtcatcgc tattaccatg gtgatgcggt tttggcagta catcaatggg
      541 cgtggatagc ggtttgactc acggggattt ccaagtctcc accccattga cgtcaatggg
      601 agtttgtttt ggcaccaaaa tcaacgggac tttccaaaat gtcgtaacaa ctccgcccca
      661 ttgacgcaaa tgggcggtag gcgtgtacgg tgggaggtct atataagcag agctctccct
      721 atcagtgata gagatctccc tatcagtgat agagatcgtc gacgagctcg tttagtgaac
      781 cgtcagatcg cctggagacg ccatccacgc tgttttgacc tccatagaag acaccgggac
      841 cgatccagcc tccggactct agcgtttaaa cttaagctta gtgaaccgtc agatccgcta
      901 gcgctaccgg tcgccaccat ggtgagcaag ggcgaggagc tgttcaccgg ggtggtgccc
      961 atcctggtcg agctggacgg cgacgtaaac ggccacaagt tcagcgtgtc cggcgagggc
     1021 gagggcgatg ccacctacgg caagctgacc ctgaagttca tctgcaccac cggcaagctg
     1081 cccgtgccct ggcccaccct cgtgaccacc ctgacctacg gcgtgcagtg cttcagccgc
     1141 taccccgacc acatgaagca gcacgacttc ttcaagtccg ccatgcccga aggctacgtc
     1201 caggagcgca ccatcttctt caaggacgac ggcaactaca agacccgcgc cgaggtgaag
     1261 ttcgagggcg acaccctggt gaaccgcatc gagctgaagg gcatcgactt caaggaggac
     1321 ggcaacatcc tggggcacaa gctggagtac aactacaaca gccacaacgt ctatatcatg
     1381 gccgacaagc agaagaacgg catcaaggtg aacttcaaga tccgccacaa catcgaggac
     1441 ggcagcgtgc agctcgccga ccactaccag cagaacaccc ccatcggcga cggccccgtg
     1501 ctgctgcccg acaaccacta cctgagcacc cagtccgccc tgagcaaaga ccccaacgag
     1561 aagcgcgatc acatggtcct gctggagttc gtgaccgccg ccgggatcac tctcggcatg
     1621 gacgagctgt acaagtccgg actcagatct atggtgatgg agaagcctag tcccctgctg
     1681 gtcgggcggg aatttgtgag acagtattac acactgctga accaggcccc agacatgctg
     1741 catagatttt atggaaagaa ctcttcttat gtccatgggg gattggattc aaatggaaag
     1801 ccagcagatg cagtctacgg acagaaagaa atccacagga aagtgatgtc acaaaacttc
     1861 accaactgcc acaccaagat tcgccatgtt gatgctcatg ccacgctaaa tgatggtgtg
     1921 gtagtccagg tgatggggct tctctctaac aacaaccagg ctttgaggag attcatgcaa
     1981 acgtttgtcc ttgctcctga ggggtctgtt gcaaataaat tctatgttca caatgatatc
     2041 ttcagatacc aagatgaggt ctttggtggg tttgtcactg agcctcagga ggaggctgaa
     2101 gaagaagtag aggaacctga agaaagacag caaacacctg aggtggtacc tgatgattct
     2161 ggaactttct atgatcaggc agttgtcagt aatgacatgg aagaacattt agaggagcct
     2221 gttgctgaac cagagcctga tcctgaacca gaaccagaac aagaacctgt atctgaaatc
     2281 caagaggaaa agcctgagcc agtattagaa gaaactgccc ctgaggatgc tcagaagagt
     2341 tcttctccag cacctgcaga catagctcag acagtacagg aagacttgag gacattttct
     2401 tgggcatctg tgaccagtaa gaatcttcca cccagtggag ctgttccagt tactgggata
     2461 ccacctcatg ttgttaaagt accagcttca cagccccgtc cagagtctaa gcctgaatct
     2521 cagattccac cacaaagacc tcagcgggat caaagagtgc gagaacaacg aataaatatt
     2581 cctccccaaa ggggacccag accaatccgt gaggctggtg agcaaggtga cattgaaccc
     2641 cgaagaatgg tgagacaccc tgacagtcac caactcttca ttggcaacct gcctcatgaa
     2701 gtggacaaat cagagcttaa agatttcttt caaagttatg gaaacgtggt ggagttgcgc
     2761 attaacagtg gtgggaaatt acccaatttt ggttttgttg tgtttgatga ttctgagcct
     2821 gttcagaaag tccttagcaa cagacccatc atgttcagag gtgaggtccg tctgaatgtc
     2881 gaagagaaga agactcgagc tgccagggaa ggcgaccgac gagataatcg ccttcgggga
     2941 cctggaggcc ctcgaggtgg gctgggtggt ggaatgagag gccctccccg tggaggcatg
     3001 gtgcagaaac caggatttgg agtgggaagg gggcttgcgc cacggcaggg aattctgcag
     3061 tcgacggtac cgcgggcccg ggatccacta gtccagtgtg gtggaattct gcagatatcc
     3121 agcacagtgg cggccgctcg agtctagagg gcccgtttaa acccgctgat cagcctcgac
     3181 tgtgccttct agttgccagc catctgttgt ttgcccctcc cccgtgcctt ccttgaccct
     3241 ggaaggtgcc actcccactg tcctttccta ataaaatgag gaaattgcat cgcattgtct
     3301 gagtaggtgt cattctattc tggggggtgg ggtggggcag gacagcaagg gggaggattg
     3361 ggaagacaat agcaggcatg ctggggatgc ggtgggctct atggcttctg aggcggaaag
     3421 aaccagctgg ggctctaggg ggtatcccca cgcgccctgt agcggcgcat taagcgcggc
     3481 gggtgtggtg gttacgcgca gcgtgaccgc tacacttgcc agcgccctag cgcccgctcc
     3541 tttcgctttc ttcccttcct ttctcgccac gttcgccggc tttccccgtc aagctctaaa
     3601 tcgggggctc cctttagggt tccgatttag tgctttacgg cacctcgacc ccaaaaaact
     3661 tgattagggt gatggttcac gtacctagaa gttcctattc cgaagttcct attctctaga
     3721 aagtatagga acttccttgg ccaaaaagcc tgaactcacc gcgacgtctg tcgagaagtt
     3781 tctgatcgaa aagttcgaca gcgtctccga cctgatgcag ctctcggagg gcgaagaatc
     3841 tcgtgctttc agcttcgatg taggagggcg tggatatgtc ctgcgggtaa atagctgcgc
     3901 cgatggtttc tacaaagatc gttatgttta tcggcacttt gcatcggccg cgctcccgat
     3961 tccggaagtg cttgacattg gggaattcag cgagagcctg acctattgca tctcccgccg
     4021 tgcacagggt gtcacgttgc aagacctgcc tgaaaccgaa ctgcccgctg ttctgcagcc
     4081 ggtcgcggag gccatggatg cgatcgctgc ggccgatctt agccagacga gcgggttcgg
     4141 cccattcgga ccgcaaggaa tcggtcaata cactacatgg cgtgatttca tatgcgcgat
     4201 tgctgatccc catgtgtatc actggcaaac tgtgatggac gacaccgtca gtgcgtccgt
     4261 cgcgcaggct ctcgatgagc tgatgctttg ggccgaggac tgccccgaag tccggcacct
     4321 cgtgcacgcg gatttcggct ccaacaatgt cctgacggac aatggccgca taacagcggt
     4381 cattgactgg agcgaggcga tgttcgggga ttcccaatac gaggtcgcca acatcttctt
     4441 ctggaggccg tggttggctt gtatggagca gcagacgcgc tacttcgagc ggaggcatcc
     4501 ggagcttgca ggatcgccgc ggctccgggc gtatatgctc cgcattggtc ttgaccaact
     4561 ctatcagagc ttggttgacg gcaatttcga tgatgcagct tgggcgcagg gtcgatgcga
     4621 cgcaatcgtc cgatccggag ccgggactgt cgggcgtaca caaatcgccc gcagaagcgc
     4681 ggccgtctgg accgatggct gtgtagaagt actcgccgat agtggaaacc gacgccccag
     4741 cactcgtccg agggcaaagg aatagcacgt actacgagat ttcgattcca ccgccgcctt
     4801 ctatgaaagg ttgggcttcg gaatcgtttt ccgggacgcc ggctggatga tcctccagcg
     4861 cggggatctc atgctggagt tcttcgccca ccccaacttg tttattgcag cttataatgg
     4921 ttacaaataa agcaatagca tcacaaattt cacaaataaa gcattttttt cactgcattc
     4981 tagttgtggt ttgtccaaac tcatcaatgt atcttatcat gtctgtatac cgtcgacctc
     5041 tagctagagc ttggcgtaat catggtcata gctgtttcct gtgtgaaatt gttatccgct
     5101 cacaattcca cacaacatac gagccggaag cataaagtgt aaagcctggg gtgcctaatg
     5161 agtgagctaa ctcacattaa ttgcgttgcg ctcactgccc gctttccagt cgggaaacct
     5221 gtcgtgccag ctgcattaat gaatcggcca acgcgcgggg agaggcggtt tgcgtattgg
     5281 gcgctcttcc gcttcctcgc tcactgactc gctgcgctcg gtcgttcggc tgcggcgagc
     5341 ggtatcagct cactcaaagg cggtaatacg gttatccaca gaatcagggg ataacgcagg
     5401 aaagaacatg tgagcaaaag gccagcaaaa ggccaggaac cgtaaaaagg ccgcgttgct
     5461 ggcgtttttc cataggctcc gcccccctga cgagcatcac aaaaatcgac gctcaagtca
     5521 gaggtggcga aacccgacag gactataaag ataccaggcg tttccccctg gaagctccct
     5581 cgtgcgctct cctgttccga ccctgccgct taccggatac ctgtccgcct ttctcccttc
     5641 gggaagcgtg gcgctttctc atagctcacg ctgtaggtat ctcagttcgg tgtaggtcgt
     5701 tcgctccaag ctgggctgtg tgcacgaacc ccccgttcag cccgaccgct gcgccttatc
     5761 cggtaactat cgtcttgagt ccaacccggt aagacacgac ttatcgccac tggcagcagc
     5821 cactggtaac aggattagca gagcgaggta tgtaggcggt gctacagagt tcttgaagtg
     5881 gtggcctaac tacggctaca ctagaagaac agtatttggt atctgcgctc tgctgaagcc
     5941 agttaccttc ggaaaaagag ttggtagctc ttgatccggc aaacaaacca ccgctggtag
     6001 cggtggtttt tttgtttgca agcagcagat tacgcgcaga aaaaaaggat ctcaagaaga
     6061 tcctttgatc ttttctacgg ggtctgacgc tcagtggaac gaaaactcac gttaagggat
     6121 tttggtcatg agattatcaa aaaggatctt cacctagatc cttttaaatt aaaaatgaag
     6181 ttttaaatca atctaaagta tatatgagta aacttggtct gacagttacc aatgcttaat
     6241 cagtgaggca cctatctcag cgatctgtct atttcgttca tccatagttg cctgactccc
     6301 cgtcgtgtag ataactacga tacgggaggg cttaccatct ggccccagtg ctgcaatgat
     6361 accgcgagac ccacgctcac cggctccaga tttatcagca ataaaccagc cagccggaag
     6421 ggccgagcgc agaagtggtc ctgcaacttt atccgcctcc atccagtcta ttaattgttg
     6481 ccgggaagct agagtaagta gttcgccagt taatagtttg cgcaacgttg ttgccattgc
     6541 tacaggcatc gtggtgtcac gctcgtcgtt tggtatggct tcattcagct ccggttccca
     6601 acgatcaagg cgagttacat gatcccccat gttgtgcaaa aaagcggtta gctccttcgg
     6661 tcctccgatc gttgtcagaa gtaagttggc cgcagtgtta tcactcatgg ttatggcagc
     6721 actgcataat tctcttactg tcatgccatc cgtaagatgc ttttctgtga ctggtgagta
     6781 ctcaaccaag tcattctgag aatagtgtat gcggcgaccg agttgctctt gcccggcgtc
     6841 aatacgggat aataccgcgc cacatagcag aactttaaaa gtgctcatca ttggaaaacg
     6901 ttcttcgggg cgaaaactct caaggatctt accgctgttg agatccagtt cgatgtaacc
     6961 cactcgtgca cccaactgat cttcagcatc ttttactttc accagcgttt ctgggtgagc
     7021 aaaaacagga aggcaaaatg ccgcaaaaaa gggaataagg gcgacacgga aatgttgaat
     7081 actcatactc ttcctttttc aatattattg aagcatttat cagggttatt gtctcatgag
     7141 cggatacata tttgaatgta tttagaaaaa taaacaaata ggggttccgc gcacatttcc
     7201 ccgaaaagtg ccacctgacg tcgacggatc gggagatctc ccgatcccct atggtgcact
     7261 ctcagtacaa tctgctctga tgccgcatag ttaagccagt atctgctccc tgcttgtgtg
     7321 ttggag
//
