LOCUS       sequence-481889-        5596 bp    DNA     circular SYN 04-AUG-2025
DEFINITION  Transient expression of 15xGly-EGFP-3xHA.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 5596)
  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 17 July 2025, 389(6757). doi: 10.1126/science.ado2403
REFERENCE   2  (bases 1 to 5596)
  AUTHORS   .
  TITLE     Direct Submission
  JOURNAL   Exported Aug 4, 2025 from SnapGene Server 8.0.1
            https://www.snapgene.com
COMMENT     SGRef: number: 1; type: "Journal Article"; journalName: "Science 17 
            July 2025, 389(6757). doi: 10.1126/science.ado2403"
COMMENT     Sequence Label: pRK5R 15xGly
FEATURES             Location/Qualifiers
     source          1..5596
                     /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(3741..3743,3744..4454)
                     /codon_start=1
                     /product="enhanced GFP"
                     /label=EGFP
                     /translation="VSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTLK
                     FICTTGKLPVPWPTLVTTLTYGVQCFSRYPDHMKQHDFFKSAMPEGYVQERTIFFKDDG
                     NYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIKV
                     NFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSALSKDPNEKRDHMVLLE
                     FVTAAGITLGMDELYK"
     CDS             join(3741..3743,3744..4454)
                     /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(3783..3804)
                     /label=EGFP-N
                     /note="EGFP, reverse primer"
     primer_bind     complement(4044..4063)
                     /label=EXFP-R
                     /note="For distinguishing EGFP variants, reverse primer"
     primer_bind     4391..4412
                     /label=EGFP-C
                     /note="EGFP, forward primer"
     CDS             4467..4493
                     /codon_start=1
                     /product="HA (human influenza hemagglutinin) epitope tag"
                     /label=HA
                     /translation="YPYDVPDYA"
     CDS             4506..4532
                     /codon_start=1
                     /product="HA (human influenza hemagglutinin) epitope tag"
                     /label=HA
                     /translation="YPYDVPDYA"
     CDS             4545..4571
                     /codon_start=1
                     /product="HA (human influenza hemagglutinin) epitope tag"
                     /label=HA
                     /translation="YPYDVPDYA"
     regulatory      4592..4601
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     polyA_signal    4605..4739
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(4642..4661)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     4696..4715
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     promoter        4808..5165
                     /label=SV40 promoter
                     /note="SV40 enhancer and early promoter"
     enhancer        4808..4999
                     /label=SV40 enhancer
                     /note="enhancer for the SV40 early promoter (Herr, 1993)"
     primer_bind     complement(4831..4851)
                     /label=pBABE 3'
                     /note="SV40 enhancer, reverse primer for pBABE vectors"
     rep_origin      5016..5151
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     primer_bind     complement(5195..5211)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(5195..5211)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(5208..5230)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    5219..5235
                     /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(5243..5249,5250..5267,5268..5273))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    5288..5309
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(5426..5443)
                     /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 gcggaggctg ggcctcgacg gtgagcaagg gcgaggagct gttcaccggg gtggtgccca
     3781 tcctggtcga gctggacggc gacgtaaacg gccacaagtt cagcgtgtcc ggcgagggcg
     3841 agggcgatgc cacctacggc aagctgaccc tgaagttcat ctgcaccacc ggcaagctgc
     3901 ccgtgccctg gcccaccctc gtgaccaccc tgacctacgg cgtgcagtgc ttcagccgct
     3961 accccgacca catgaagcag cacgacttct tcaagtccgc catgcccgaa ggctacgtcc
     4021 aggagcgcac catcttcttc aaggacgacg gcaactacaa gacccgcgcc gaggtgaagt
     4081 tcgagggcga caccctggtg aaccgcatcg agctgaaggg catcgacttc aaggaggacg
     4141 gcaacatcct ggggcacaag ctggagtaca actacaacag ccacaacgtc tatatcatgg
     4201 ccgacaagca gaagaacggc atcaaggtga acttcaagat ccgccacaac atcgaggacg
     4261 gcagcgtgca gctcgccgac cactaccagc agaacacccc catcggcgac ggccccgtgc
     4321 tgctgcccga caaccactac ctgagcaccc agtccgccct gagcaaagac cccaacgaga
     4381 agcgcgatca catggtcctg ctggagttcg tgaccgccgc cgggatcact ctcggcatgg
     4441 acgagctgta caagggaggg agcggctatc cctatgacgt gcctgattac gccggcacag
     4501 gatcataccc ctatgatgtg cctgactacg ctggcagcgc cggataccct tatgatgtgc
     4561 ctgattatgc ttagtgagga tccccagctt ggccgccatg gcccaacttg tttattgcag
     4621 cttataatgg ttacaaataa agcaatagca tcacaaattt cacaaataaa gcattttttt
     4681 cactgcattc tagttgtggt ttgtccaaac tcatcaatgt atcttatcat gtctggatcg
     4741 ggaattaatt cggcgcagca ccatggcctg aaataacctc tgaaagagga acttggttag
     4801 gtaccttctg aggcggaaag aaccagctgt ggaatgtgtg tcagttaggg tgtggaaagt
     4861 ccccaggctc cccagcaggc agaagtatgc aaagcatgca tctcaattag tcagcaacca
     4921 ggtgtggaaa gtccccaggc tccccagcag gcagaagtat gcaaagcatg catctcaatt
     4981 agtcagcaac catagtcccg cccctaactc cgcccatccc gcccctaact ccgcccagtt
     5041 ccgcccattc tccgccccat ggctgactaa ttttttttat ttatgcagag gccgaggccg
     5101 cctcggcctc tgagctattc cagaagtagt gaggaggctt ttttggaggc ctaggctttt
     5161 gcaaaaagct gttaacagct tggctcgtaa tcatgtcata gctgtttcct gtgtgaaatt
     5221 gttatccgct cacaattcca cacaacatac gagccggaag cataaagtgt aaagcctggg
     5281 gtgcctaatg agtgagctaa ctcacattaa ttgcgttgcg ctcactgccc gctttccagt
     5341 cgggaaacct gtcgtgccag ctgcattaat gaatcggcca acgcgcgggg agaggcggtt
     5401 tgcgtattgg gcgctcttcc gcttcctcgc tcactgactc gctgcgctcg gtcgttcggc
     5461 tgcggcgagc ggtatcagct cactcaaagg cggtaatacg gttatccaca gaatcagggg
     5521 ataacgcagg aaagaacatg tgagcaaaag gccagcaaaa ggccaggaac cgtaaaaagg
     5581 ccgcgttgct ggcgtt
//
