LOCUS       sequence-489514-        5833 bp    DNA     circular SYN 16-OCT-2025
DEFINITION  Transient expression of 99xGly-EGFP-3xHA.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 5833)
  AUTHORS   Yang J, Xu Y, Ziehr DR, Taylor MS, Valenstein ML, Frenkel EM, Bush 
            JR, Rutter K, Stevanovski I, Shi CY, Kesavan M, Pinto RM, Deveson I,
            Bartel DP, Sabatini DM, Chivukula RR
  TITLE     Polyglycine-mediated aggregation of FAM98B disrupts tRNA processing 
            in GGC repeat disorders.
  JOURNAL   Science. 2025 Jul 17;389(6757):eado2403. doi: 
            10.1126/science.ado2403. Epub 2025 Jul 17.
  PUBMED    40674500
REFERENCE   2  (bases 1 to 5833)
  AUTHORS   .
  TITLE     Direct Submission
  JOURNAL   Exported Oct 16, 2025 from SnapGene Server 8.0.1
            https://www.snapgene.com
COMMENT     SGRef: number: 1; type: "Journal Article"; journalName: "Science."; 
            date: "2025-07-17"; volume: "389(6757):eado2403. doi"; pages: " 
            10.1126/science.ado2403. Epub 2025 Jul 17"
COMMENT     Sequence Label: pRK5R 99xGly
FEATURES             Location/Qualifiers
     source          1..5833
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     rep_origin      complement(1..589)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(81..100)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(760..1551,1552..1620))
                     /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     1383..1402
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(1621..1725)
                     /gene="bla"
                     /label=AmpR promoter
     primer_bind     1793..1811
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     primer_bind     complement(1849..1871)
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     primer_bind     1971..1990
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     rep_origin      complement(2007..2462)
                     /direction=LEFT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(2144..2165)
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     2356..2375
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     2660..2682
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     primer_bind     2674..2691
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     2675..2691
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     enhancer        2711..3090
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
     promoter        3091..3294
                     /label=CMV promoter
                     /note="human cytomegalovirus (CMV) immediate early 
                     promoter"
     primer_bind     3245..3265
                     /label=CMV-F
                     /note="Human CMV immediate early promoter, forward primer"
     primer_bind     3291..3315
                     /label=LNCX
                     /note="Human CMV promoter, forward primer"
     promoter        3525..3543
                     /label=SP6 promoter
                     /note="promoter for bacteriophage SP6 RNA polymerase"
     primer_bind     3525..3542
                     /label=SP6
                     /note="SP6 promoter, forward primer"
     primer_bind     3556..3575
                     /label=pMT2-F
                     /note="Synthetic intron, forward primer"
     regulatory      3642..3651
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     CDS             join(3978..3980,3981..4691)
                     /codon_start=1
                     /product="enhanced GFP"
                     /label=EGFP
                     /translation="VSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTLK
                     FICTTGKLPVPWPTLVTTLTYGVQCFSRYPDHMKQHDFFKSAMPEGYVQERTIFFKDDG
                     NYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIKV
                     NFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSALSKDPNEKRDHMVLLE
                     FVTAAGITLGMDELYK"
     CDS             join(3978..3980,3981..4691)
                     /codon_start=1
                     /product="the original enhanced GFP (Yang et al., 1996)"
                     /label=EGFP
                     /note="mammalian codon-optimized"
                     /translation="VSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTLK
                     FICTTGKLPVPWPTLVTTLTYGVQCFSRYPDHMKQHDFFKSAMPEGYVQERTIFFKDDG
                     NYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIKV
                     NFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSALSKDPNEKRDHMVLLE
                     FVTAAGITLGMDELYK"
     primer_bind     complement(4020..4041)
                     /label=EGFP-N
                     /note="EGFP, reverse primer"
     primer_bind     complement(4281..4300)
                     /label=EXFP-R
                     /note="For distinguishing EGFP variants, reverse primer"
     primer_bind     4628..4649
                     /label=EGFP-C
                     /note="EGFP, forward primer"
     CDS             4704..4730
                     /codon_start=1
                     /product="HA (human influenza hemagglutinin) epitope tag"
                     /label=HA
                     /translation="YPYDVPDYA"
     CDS             4743..4769
                     /codon_start=1
                     /product="HA (human influenza hemagglutinin) epitope tag"
                     /label=HA
                     /translation="YPYDVPDYA"
     CDS             4782..4808
                     /codon_start=1
                     /product="HA (human influenza hemagglutinin) epitope tag"
                     /label=HA
                     /translation="YPYDVPDYA"
     regulatory      4829..4838
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     polyA_signal    4842..4976
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(4879..4898)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     4933..4952
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     promoter        5045..5402
                     /label=SV40 promoter
                     /note="SV40 enhancer and early promoter"
     enhancer        5045..5236
                     /label=SV40 enhancer
                     /note="enhancer for the SV40 early promoter (Herr, 1993)"
     primer_bind     complement(5068..5088)
                     /label=pBABE 3'
                     /note="SV40 enhancer, reverse primer for pBABE vectors"
     rep_origin      5253..5388
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     primer_bind     complement(5432..5448)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(5432..5448)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(5445..5467)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    5456..5472
                     /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(5480..5486,5487..5504,5505..5510))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    5525..5546
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(5663..5680)
                     /label=L4440
                     /note="L4440 vector, forward primer"
ORIGIN
        1 tttccatagg ctccgccccc ctgacgagca tcacaaaaat cgacgctcaa gtcagaggtg
       61 gcgaaacccg acaggactat aaagatacca ggcgtttccc cctggaagct ccctcgtgcg
      121 ctctcctgtt ccgaccctgc cgcttaccgg atacctgtcc gcctttctcc cttcgggaag
      181 cgtggcgctt tctcatagct cacgctgtag gtatctcagt tcggtgtagg tcgttcgctc
      241 caagctgggc tgtgtgcacg aaccccccgt tcagcccgac cgctgcgcct tatccggtaa
      301 ctatcgtctt gagtccaacc cggtaagaca cgacttatcg ccactggcag cagccactgg
      361 taacaggatt agcagagcga ggtatgtagg cggtgctaca gagttcttga agtggtggcc
      421 taactacggc tacactagaa gaacagtatt tggtatctgc gctctgctga agccagttac
      481 cttcggaaaa agagttggta gctcttgatc cggcaaacaa accaccgctg gtagcggtgg
      541 tttttttgtt tgcaagcagc agattacgcg cagaaaaaaa ggatctcaag aagatccttt
      601 gatcttttct acggggtctg acgctcagtg gaacgaaaac tcacgttaag ggattttggt
      661 catgagatta tcaaaaagga tcttcaccta gatcctttta aattaaaaat gaagttttaa
      721 atcaatctaa agtatatatg agtaaacttg gtctgacagt taccaatgct taatcagtga
      781 ggcacctatc tcagcgatct gtctatttcg ttcatccata gttgcctgac tccccgtcgt
      841 gtagataact acgatacggg agggcttacc atctggcccc agtgctgcaa tgataccgcg
      901 agacccacgc tcaccggctc cagatttatc agcaataaac cagccagccg gaagggccga
      961 gcgcagaagt ggtcctgcaa ctttatccgc ctccatccag tctattaatt gttgccggga
     1021 agctagagta agtagttcgc cagttaatag tttgcgcaac gttgttgcca ttgctacagg
     1081 catcgtggtg tcacgctcgt cgtttggtat ggcttcattc agctccggtt cccaacgatc
     1141 aaggcgagtt acatgatccc ccatgttgtg caaaaaagcg gttagctcct tcggtcctcc
     1201 gatcgttgtc agaagtaagt tggccgcagt gttatcactc atggttatgg cagcactgca
     1261 taattctctt actgtcatgc catccgtaag atgcttttct gtgactggtg agtactcaac
     1321 caagtcattc tgagaatagt gtatgcggcg accgagttgc tcttgcccgg cgtcaatacg
     1381 ggataatacc gcgccacata gcagaacttt aaaagtgctc atcattggaa aacgttcttc
     1441 ggggcgaaaa ctctcaagga tcttaccgct gttgagatcc agttcgatgt aacccactcg
     1501 tgcacccaac tgatcttcag catcttttac tttcaccagc gtttctgggt gagcaaaaac
     1561 aggaaggcaa aatgccgcaa aaaagggaat aagggcgaca cggaaatgtt gaatactcat
     1621 actcttcctt tttcaatatt attgaagcat ttatcagggt tattgtctca tgagcggata
     1681 catatttgaa tgtatttaga aaaataaaca aataggggtt ccgcgcacat ttccccgaaa
     1741 agtgccacct gacgtctaag aaaccattat tatcatgaca ttaacctata aaaataggcg
     1801 tatcacgagg ccctttcgtc tcgcgcgttt cggtgatgac ggtgaaaacc tctgacacat
     1861 gcagctcccg gagacggtca cagcttgtct gtaagcggat gccgggagca gacaagcccg
     1921 tcagggcgcg tcagcgggtg ttggcgggtg tcggggctgg cttaactatg cggcatcaga
     1981 gcagattgta ctgagagtgc accataaaat tgtaaacgtt aatattttgt taaaattcgc
     2041 gttaaatttt tgttaaatca gctcattttt taaccaatag gccgaaatcg gcaaaatccc
     2101 ttataaatca aaagaatagc ccgagatagg gttgagtgtt gttccagttt ggaacaagag
     2161 tccactatta aagaacgtgg actccaacgt caaagggcga aaaaccgtct atcagggcga
     2221 tggcccacta cgtgaaccat cacccaaatc aagttttttg gggtcgaggt gccgtaaagc
     2281 actaaatcgg aaccctaaag ggagcccccg atttagagct tgacggggaa agccggcgaa
     2341 cgtggcgaga aaggaaggga agaaagcgaa aggagcgggc gctagggcgc tggcaagtgt
     2401 agcggtcacg ctgcgcgtaa ccaccacacc cgccgcgctt aatgcgccgc tacagggcgc
     2461 gtactatggt tgctttgacg tatgcggtgt gaaataccgc acagatgcgt aaggagaaaa
     2521 taccgcatca ggcgccattc gccattcagg ctgcgcaact gttgggaagg gcgatcggtg
     2581 cgggcctctt cgctattacg ccagctggcg aaagggggat gtgctgcaag gcgattaagt
     2641 tgggtaacgc cagggttttc ccagtcacga cgttgtaaaa cgacggccag taattaattc
     2701 gagctcgccc gacattgatt attgactagt tattaatagt aatcaattac ggggtcatta
     2761 gttcatagcc catatatgga gttccgcgtt acataactta cggtaaatgg cccgcctggc
     2821 tgaccgccca acgacccccg cccattgacg tcaataatga cgtatgttcc catagtaacg
     2881 ccaataggga ctttccattg acgtcaatgg gtggagtatt tacggtaaac tgcccacttg
     2941 gcagtacatc aagtgtatca tatgccaagt acgcccccta ttgacgtcaa tgacggtaaa
     3001 tggcccgcct ggcattatgc ccagtacatg accttatggg actttcctac ttggcagtac
     3061 atctacgtat tagtcatcgc tattaccatg gtgatgcggt tttggcagta catcaatggg
     3121 cgtggatagc ggtttgactc acggggattt ccaagtctcc accccattga cgtcaatggg
     3181 agtttgtttt ggcaccaaaa tcaacgggac tttccaaaat gtcgtaacaa ctccgcccca
     3241 ttgacgcaaa tgggcggtag gcgtgtacgg tgggaggtct atataagcag agctcgttta
     3301 gtgaaccgtc agatcgcctg gagacgccat ccacgctgtt ttgacctcca tagaagacac
     3361 cgggaccgat ccagcctccg cggccgggaa cggtgcattg gaacgcggat tccccgtgcc
     3421 aagagtgacg taagtaccgc ctatagagtc tataggccca cccccttggc ttcgttagaa
     3481 cgcggctaca attaatacat aaccttatgt atcatacaca tacgatttag gtgacactat
     3541 agaataacat ccactttgcc tttctctcca caggtgtcca ctcccaggtc caactgcacc
     3601 tcggttctag ctagcgctac cggtttaatt aattcgaatt cgccaccatg gggttgccgc
     3661 tgccaggggg cgtgcggcag cgcggcggcg gcggcggcgg cggcggcggc ggcggcggcg
     3721 gcggaggcgg cggcggcggc ggcggcggcg gcggcggcgg cggcggcggc ggcggcggcg
     3781 gcggcggcgg cggcggcggc ggcggcggcg gcggcggcgg cggcggcggc ggcggcggcg
     3841 gcggcggtgg cggcggcggc ggcggcggcg gcggcggcgg cggcggcggc ggcggcggcg
     3901 gcggcggcgg cggcggcggc ggcggcggcg gcggcggcgg cggcggcggc ggcggcggcg
     3961 gcggctgggc ctcgacggtg agcaagggcg aggagctgtt caccggggtg gtgcccatcc
     4021 tggtcgagct ggacggcgac gtaaacggcc acaagttcag cgtgtccggc gagggcgagg
     4081 gcgatgccac ctacggcaag ctgaccctga agttcatctg caccaccggc aagctgcccg
     4141 tgccctggcc caccctcgtg accaccctga cctacggcgt gcagtgcttc agccgctacc
     4201 ccgaccacat gaagcagcac gacttcttca agtccgccat gcccgaaggc tacgtccagg
     4261 agcgcaccat cttcttcaag gacgacggca actacaagac ccgcgccgag gtgaagttcg
     4321 agggcgacac cctggtgaac cgcatcgagc tgaagggcat cgacttcaag gaggacggca
     4381 acatcctggg gcacaagctg gagtacaact acaacagcca caacgtctat atcatggccg
     4441 acaagcagaa gaacggcatc aaggtgaact tcaagatccg ccacaacatc gaggacggca
     4501 gcgtgcagct cgccgaccac taccagcaga acacccccat cggcgacggc cccgtgctgc
     4561 tgcccgacaa ccactacctg agcacccagt ccgccctgag caaagacccc aacgagaagc
     4621 gcgatcacat ggtcctgctg gagttcgtga ccgccgccgg gatcactctc ggcatggacg
     4681 agctgtacaa gggagggagc ggctatccct atgacgtgcc tgattacgcc ggcacaggat
     4741 cataccccta tgatgtgcct gactacgctg gcagcgccgg atacccttat gatgtgcctg
     4801 attatgctta gtgaggatcc ccagcttggc cgccatggcc caacttgttt attgcagctt
     4861 ataatggtta caaataaagc aatagcatca caaatttcac aaataaagca tttttttcac
     4921 tgcattctag ttgtggtttg tccaaactca tcaatgtatc ttatcatgtc tggatcggga
     4981 attaattcgg cgcagcacca tggcctgaaa taacctctga aagaggaact tggttaggta
     5041 ccttctgagg cggaaagaac cagctgtgga atgtgtgtca gttagggtgt ggaaagtccc
     5101 caggctcccc agcaggcaga agtatgcaaa gcatgcatct caattagtca gcaaccaggt
     5161 gtggaaagtc cccaggctcc ccagcaggca gaagtatgca aagcatgcat ctcaattagt
     5221 cagcaaccat agtcccgccc ctaactccgc ccatcccgcc cctaactccg cccagttccg
     5281 cccattctcc gccccatggc tgactaattt tttttattta tgcagaggcc gaggccgcct
     5341 cggcctctga gctattccag aagtagtgag gaggcttttt tggaggccta ggcttttgca
     5401 aaaagctgtt aacagcttgg ctcgtaatca tgtcatagct gtttcctgtg tgaaattgtt
     5461 atccgctcac aattccacac aacatacgag ccggaagcat aaagtgtaaa gcctggggtg
     5521 cctaatgagt gagctaactc acattaattg cgttgcgctc actgcccgct ttccagtcgg
     5581 gaaacctgtc gtgccagctg cattaatgaa tcggccaacg cgcggggaga ggcggtttgc
     5641 gtattgggcg ctcttccgct tcctcgctca ctgactcgct gcgctcggtc gttcggctgc
     5701 ggcgagcggt atcagctcac tcaaaggcgg taatacggtt atccacagaa tcaggggata
     5761 acgcaggaaa gaacatgtga gcaaaaggcc agcaaaaggc caggaaccgt aaaaaggccg
     5821 cgttgctggc gtt
//
