LOCUS       sequence-252034-        6734 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 6734)
  AUTHORS   Meyer C, Garzia A, Morozov P, Molina H, Tuschl T
  TITLE     The G3BP1-Family-USP10 Deubiquitinase Complex Rescues Ubiquitinated 
            40S Subunits of Ribosomes Stalled in Translation from Lysosomal 
            Degradation.
  JOURNAL   Mol Cell. 2020 Mar 19;77(6):1193-1205.e5. doi: 
            10.1016/j.molcel.2019.12.024. Epub 2020 Jan 24.
  PUBMED    31981475
REFERENCE   2  (bases 1 to 6734)
  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.2019.12.024"; journalName: "Mol Cell"; date: 
            "2020-03-19- 19"; volume: "77"; issue: "6"; pages: "1193-1205.e5"
COMMENT     Sequence Label: pFRT_TO_FlagHA_G3BP1
FEATURES             Location/Qualifiers
     source          1..6734
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     151..170
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     404..421
                     /label=L4440
                     /note="L4440 vector, forward primer"
     protein_bind    538..559
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     promoter        join(574..579,580..597,598..604)
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    612..628
                     /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)."
     primer_bind     617..639
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     primer_bind     636..652
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     636..652
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     polyA_signal    701..822
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(712..731)
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     primer_bind     766..785
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     CDS             complement(952..1974)
                     /codon_start=1
                     /product="hygromycin B phosphotransferase"
                     /label=HygR
                     /note="confers resistance to hygromycin"
                     /translation="KKPELTATSVEKFLIEKFDSVSDLMQLSEGEESRAFSFDVGGRGY
                     VLRVNSCADGFYKDRYVYRHFASAALPIPEVLDIGEFSESLTYCISRRAQGVTLQDLPE
                     TELPAVLQPVAEAMDAIAAADLSQTSGFGPFGPQGIGQYTTWRDFICAIADPHVYHWQT
                     VMDDTVSASVAQALDELMLWAEDCPEVRHLVHADFGSNNVLTDNGRITAVIDWSEAMFG
                     DSQYEVANIFFWRPWLACMEQQTRYFERRHPELAGSPRLRAYMLRIGLDQLYQSLVDGN
                     FDDAAWAQGRCDAIVRSGAGTVGRTQIARRSAAVWTDGCVEVLADSGNRRPSTRPRAKE
                     "
     CDS             complement(952..1974)
                     /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
                     "
     protein_bind    complement(1982..2029)
                     /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)."
     primer_bind     2161..2180
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     polyA_signal    2313..2537
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     primer_bind     2526..2543
                     /label=BGH-rev
                     /note="Bovine growth hormone terminator, reverse primer. 
                     Also called BGH reverse"
     CDS             complement(2623..2664)
                     /codon_start=1
                     /product="epitope tag from simian virus 5"
                     /label=V5 tag
                     /translation="GKPIPNPLLGLDST"
     protein_bind    2717..2741
                     /gene="mutant version of attB"
                     /label=attB2
                     /bound_moiety="BP Clonase(TM)"
                     /note="recombination site for the Gateway(R) BP reaction"
     protein_bind    complement(4146..4170)
                     /gene="mutant version of attB"
                     /label=attB1
                     /bound_moiety="BP Clonase(TM)"
                     /note="recombination site for the Gateway(R) BP reaction"
     CDS             complement(4186..4212)
                     /codon_start=1
                     /product="HA (human influenza hemagglutinin) epitope tag"
                     /label=HA
                     /translation="YPYDVPDYA"
     CDS             complement(4213..4236)
                     /codon_start=1
                     /product="FLAG(R) epitope tag, followed by an enterokinase 
                     cleavage site"
                     /label=FLAG
                     /translation="DYKDDDDK"
     primer_bind     complement(4336..4360)
                     /label=LNCX
                     /note="Human CMV promoter, forward primer"
     protein_bind    4370..4388
                     /gene="tetO"
                     /label=tet operator
                     /bound_moiety="tetracycline repressor TetR"
                     /note="bacterial operator O2 for the tetR and tetA genes"
     protein_bind    4391..4409
                     /gene="tetO"
                     /label=tet operator
                     /bound_moiety="tetracycline repressor TetR"
                     /note="bacterial operator O2 for the tetR and tetA genes"
     promoter        complement(4411..4614)
                     /label=CMV promoter
                     /note="human cytomegalovirus (CMV) immediate early 
                     promoter"
     primer_bind     complement(4440..4460)
                     /label=CMV-F
                     /note="Human CMV immediate early promoter, forward primer"
     enhancer        4615..4994
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
     primer_bind     5166..5185
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     promoter        5260..5364
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(5365..5433,5434..6225)
                     /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     complement(5583..5602)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      join(6396..6734,1..250)
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
ORIGIN
        1 gcccagcttg gagcgaacga cctacaccga actgagatac ctacagcgtg agctatgaga
       61 aagcgccacg cttcccgaag ggagaaaggc ggacaggtat ccggtaagcg gcagggtcgg
      121 aacaggagag cgcacgaggg agcttccagg gggaaacgcc tggtatcttt atagtcctgt
      181 cgggtttcgc cacctctgac ttgagcgtcg atttttgtga tgctcgtcag gggggcggag
      241 cctatggaaa aacgccagca acgcggcctt tttacggttc ctggcctttt gctggccttt
      301 tgctcacatg ttctttcctg cgttatcccc tgattctgtg gataaccgta ttaccgcctt
      361 tgagtgagct gataccgctc gccgcagccg aacgaccgag cgcagcgagt cagtgagcga
      421 ggaagcggaa gagcgcccaa tacgcaaacc gcctctcccc gcgcgttggc cgattcatta
      481 atgcagctgg cacgacaggt ttcccgactg gaaagcgggc agtgagcgca acgcaattaa
      541 tgtgagttag ctcactcatt aggcacccca ggctttacac tttatgcttc cggctcgtat
      601 gttgtgtgga attgtgagcg gataacaatt tcacacagga aacagctatg accatgatta
      661 cgccaagctc tagctagagg tcgacggtat acagacatga taagatacat tgatgagttt
      721 ggacaaacca caactagaat gcagtgaaaa aaatgcttta tttgtgaaat ttgtgatgct
      781 attgctttat ttgtaaccat tataagctgc aataaacaag ttggggtggg cgaagaactc
      841 cagcatgaga tccccgcgct ggaggatcat ccagccggcg tcccggaaaa cgattccgaa
      901 gcccaacctt tcatagaagg cggcggtgga atcgaaatct cgtagtacgt gctattcctt
      961 tgccctcgga cgagtgctgg ggcgtcggtt tccactatcg gcgagtactt ctacacagcc
     1021 atcggtccag acggccgcgc ttctgcgggc gatttgtgta cgcccgacag tcccggctcc
     1081 ggatcggacg attgcgtcgc atcgaccctg cgcccaagct gcatcatcga aattgccgtc
     1141 aaccaagctc tgatagagtt ggtcaagacc aatgcggagc atatacgccc ggagccgcgg
     1201 cgatcctgca agctccggat gcctccgctc gaagtagcgc gtctgctgct ccatacaagc
     1261 caaccacggc ctccagaaga agatgttggc gacctcgtat tgggaatccc cgaacatcgc
     1321 ctcgctccag tcaatgaccg ctgttatgcg gccattgtcc gtcaggacat tgttggagcc
     1381 gaaatccgcg tgcacgaggt gccggacttc ggggcagtcc tcggcccaaa gcatcagctc
     1441 atcgagagcc tgcgcgacgg acgcactgac ggtgtcgtcc atcacagttt gccagtgata
     1501 cacatgggga tcagcaatcg cgcatatgaa atcacgccat gtagtgtatt gaccgattcc
     1561 ttgcggtccg aatgggccga acccgctcgt ctggctaaga tcggccgcag cgatcgcatc
     1621 catggcctcc gcgaccggct gcagaacagc gggcagttcg gtttcaggca ggtcttgcaa
     1681 cgtgacaccc tgtgcacggc gggagatgca ataggtcagg ctctcgctga attccccaat
     1741 gtcaagcact tccggaatcg ggagcgcggc cgatgcaaag tgccgataaa cataacgatc
     1801 tttgtagaaa ccatcggcgc agctatttac ccgcaggaca tatccacgcc ctcctacatc
     1861 gaagctgaaa gcacgagatt cttcgccctc cgagagctgc atcaggtcgg agacgctgtc
     1921 gaacttttcg atcagaaact tctcgacaga cgtcgcggtg agttcaggct ttttggccaa
     1981 ggaagttcct atactttcta gagaatagga acttcggaat aggaacttct aggtacgtga
     2041 accatcaccc taatcaagtt ttttggggtc gaggtgccgt aaagcactaa atcggaaccc
     2101 taaagggagc ccccgattta gagcttgacg gggaaagccg gcgaacgtgg cgagaaagga
     2161 agggaagaaa gcgaaaggag cgggcgctag ggcgctggca agtgtagcgg tcacgctgcg
     2221 cgtaaccacc acacccgccg cgcttaatgc gccgctacag ggcgcgtggg gataccccct
     2281 agagccccag ctggttcttt ccgcctcaga agccatagag cccaccgcat ccccagcatg
     2341 cctgctattg tcttcccaat cctccccctt gctgtcctgc cccaccccac cccccagaat
     2401 agaatgacac ctactcagac aatgcgatgc aatttcctca ttttattagg aaaggacagt
     2461 gggagtggca ccttccaggg tcaaggaagg cacgggggag gggcaaacaa cagatggctg
     2521 gcaactagaa ggcacagtcg aggctgatca gcgggtttaa acgggccctc tagactcgag
     2581 gcacactgga tcagttatct atgcggccgc ggatcaggta cgcgtagaat cgagaccgag
     2641 gagagggtta gggataggct taccttcgaa ccgcgggccc tctagacttg agcggccgcc
     2701 actgtgctgg atatcaacca ctttgtacaa gaaagctggg tctcactgcc gtggcgcaag
     2761 cccccttccc actccaaatc ctggtttctg caccatgcct ccacggggag ggcctctcat
     2821 tccaccaccc agcccacctc gagggcctcc aggtccccga aggcgattat ctcgtcggtc
     2881 gccttccctg gcagctcgag tcttcttctc ttcgacattc agacggacct cacctctgaa
     2941 catgatgggc ctgttgctaa ggactttctg aacaggctca gaatcatcaa acacaacaaa
     3001 accaaaattg ggtaatttcc caccactgtt aatgcgcaac tccaccacgt ttccataact
     3061 ttgaaagaaa tctttaagct ctgatttgtc cacttcatga ggcaggttgc caatgaagag
     3121 ttggtgactg tcagggtgtc tcaccattct tcggggttca atgtcacctt gctcaccagc
     3181 ctcacggatt ggtctgggtc ccctttgggg aggaatattt attcgttgtt ctcgcactct
     3241 ttgatcccgc tgaggtcttt gtggtggaat ctgagattca ggcttagact ctggacgggg
     3301 ctgtgaagct ggtactttaa caacatgagg tggtatccca gtaactggaa cagctccact
     3361 gggtggaaga ttcttactgg tcacagatgc ccaagaaaat gtcctcaagt cttcctgtac
     3421 tgtctgagct atgtctgcag gtgctggaga agaactcttc tgagcatcct caggggcagt
     3481 ttcttctaat actggctcag gcttttcctc ttggatttca gatacaggtt cttgttctgg
     3541 ttctggttca ggatcaggct ctggttcagc aacaggctcc tctaaatgtt cttccatgtc
     3601 attactgaca actgcctgat catagaaagt tccagaatca tcaggtacca cctcaggtgt
     3661 ttgctgtctt tcttcaggtt cctctacttc ttcttcagac tcctcctgag gctcagtgac
     3721 aaacccacca aagacctcat cttggtatct gaagatatca ttgtgaacat agaatttatt
     3781 tgcaacagac ccctcaggag caaggacaaa cgtttgcatg aatctcctca aagcctggtt
     3841 gttgttagag agaagcccca tcacctggac taccacacca tcatttagcg tggcatgagc
     3901 atcaacatgg cgaatcttgg tgtggcagtt ggtgaagttt tgtgacatca ctttcctgtg
     3961 gatttctttc tgtccgtaga ctgcatctgc tggctttcca tttgaatcca atcccccatg
     4021 gacataagaa gagttctttc cataaaatct atgcagcatg tctggggcct ggttcagcag
     4081 tgtgtaatac tgtctcacaa attcccgccc gaccagcagg ggactaggct tctccatcac
     4141 catgaagcct gcttttttgt acaaacttgt tgatatctgg ttcaaagcgt aatcgggcac
     4201 gtcataaggg tacttgtcat cgtcgtcctt gtagtccatg gtaccaagct taagtttaaa
     4261 cgctagagtc cggaggctgg atcggtcccg gtgtcttcta tggaggtcaa aacagcgtgg
     4321 atggcgtctc caggcgatct gacggttcac taaacgagct cgtcgacgat ctctatcact
     4381 gatagggaga tctctatcac tgatagggag agctctgctt atatagacct cccaccgtac
     4441 acgcctaccg cccatttgcg tcaatggggc ggagttgtta cgacattttg gaaagtcccg
     4501 ttgattttgg tgccaaaaca aactcccatt gacgtcaatg gggtggagac ttggaaatcc
     4561 ccgtgagtca aaccgctatc cacgcccatt gatgtactgc caaaaccgca tcaccatggt
     4621 aatagcgatg actaatacgt agatgtactg ccaagtagga aagtcccata aggtcatgta
     4681 ctgggcataa tgccaggcgg gccatttacc gtcattgacg tcaatagggg gcgtacttgg
     4741 catatgatac acttgatgta ctgccaagtg ggcagtttac cgtaaatact ccacccattg
     4801 acgtcaatgg aaagtcccta ttggcgttac tatgggaaca tacgtcatta ttgacgtcaa
     4861 tgggcggggg tcgttgggcg gtcagccagg cgggccattt accgtaagtt atgtaacgcg
     4921 gaactccata tatgggctat gaactaatga ccccgtaatt gattactatt aataactagt
     4981 caataatcaa tgtcaacgcg tatatctggc ccgtacatcg cgaagcagcg caaaacgcct
     5041 aaccctaagc agattcttca tgcaattgtc ggtcaagcct tgccttgttg tagcttaaat
     5101 tttgctcgcg cactactcag cgacctccaa cacacaagca gggagcagat actggcttaa
     5161 ctatgcggca tcagagcaga ttgtactgag agtgcaccat aggggatcgg gagatctccc
     5221 gatccgtcga cgtcaggtgg cacttttcgg ggaaatgtgc gcggaacccc tatttgttta
     5281 tttttctaaa tacattcaaa tatgtatccg ctcatgagac aataaccctg ataaatgctt
     5341 caataatatt gaaaaaggaa gagtatgagt attcaacatt tccgtgtcgc ccttattccc
     5401 ttttttgcgg cattttgcct tcctgttttt gctcacccag aaacgctggt gaaagtaaaa
     5461 gatgctgaag atcagttggg tgcacgagtg ggttacatcg aactggatct caacagcggt
     5521 aagatccttg agagttttcg ccccgaagaa cgttttccaa tgatgagcac ttttaaagtt
     5581 ctgctatgtg gcgcggtatt atcccgtatt gacgccgggc aagagcaact cggtcgccgc
     5641 atacactatt ctcagaatga cttggttgag tactcaccag tcacagaaaa gcatcttacg
     5701 gatggcatga cagtaagaga attatgcagt gctgccataa ccatgagtga taacactgcg
     5761 gccaacttac ttctgacaac gatcggagga ccgaaggagc taaccgcttt tttgcacaac
     5821 atgggggatc atgtaactcg ccttgatcgt tgggaaccgg agctgaatga agccatacca
     5881 aacgacgagc gtgacaccac gatgcctgta gcaatggcaa caacgttgcg caaactatta
     5941 actggcgaac tacttactct agcttcccgg caacaattaa tagactggat ggaggcggat
     6001 aaagttgcag gaccacttct gcgctcggcc cttccggctg gctggtttat tgctgataaa
     6061 tctggagccg gtgagcgtgg gtctcgcggt atcattgcag cactggggcc agatggtaag
     6121 ccctcccgta tcgtagttat ctacacgacg gggagtcagg caactatgga tgaacgaaat
     6181 agacagatcg ctgagatagg tgcctcactg attaagcatt ggtaactgtc agaccaagtt
     6241 tactcatata tactttagat tgatttaaaa cttcattttt aatttaaaag gatctaggtg
     6301 aagatccttt ttgataatct catgaccaaa atcccttaac gtgagttttc gttccactga
     6361 gcgtcagacc ccgtagaaaa gatcaaagga tcttcttgag atcctttttt tctgcgcgta
     6421 atctgctgct tgcaaacaaa aaaaccaccg ctaccagcgg tggtttgttt gccggatcaa
     6481 gagctaccaa ctctttttcc gaaggtaact ggcttcagca gagcgcagat accaaatact
     6541 gttcttctag tgtagccgta gttaggccac cacttcaaga actctgtagc accgcctaca
     6601 tacctcgctc tgctaatcct gttaccagtg gctgctgcca gtggcgataa gtcgtgtctt
     6661 accgggttgg actcaagacg atagttaccg gataaggcgc agcggtcggg ctgaacgggg
     6721 ggttcgtgca caca
//
