LOCUS       sequence-252266-        6433 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Expresses an eGFP mRNA with the PPP1R8 G-quadruplex (G4) region in 
            the 3' UTR.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 6433)
  AUTHORS   Xiao MS, Wilusz JE
  TITLE     An improved method for circular RNA purification using RNase R that 
            efficiently removes linear RNAs containing G-quadruplexes or 
            structured 3' ends.
  JOURNAL   Nucleic Acids Res. 2019 Jul 3. pii: 5527976. doi: 
            10.1093/nar/gkz576.
  PUBMED    31269210
REFERENCE   2  (bases 1 to 6433)
  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"; journalName: "Nucleic 
            Acids Res. 2019 Jul 3. pii: 5527976. doi: 10.1093/nar/gkz576."
COMMENT     Sequence Label: pcDNA3.1(+) eGFP PPP1R8 G4
FEATURES             Location/Qualifiers
     source          1..6433
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     promoter        complement(45..149)
                     /gene="bla"
                     /label=AmpR promoter
     primer_bind     complement(224..243)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     enhancer        415..794
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
     promoter        795..998
                     /label=CMV promoter
                     /note="human cytomegalovirus (CMV) immediate early 
                     promoter"
     primer_bind     949..969
                     /label=CMV-F
                     /note="Human CMV immediate early promoter, forward primer"
     primer_bind     1043..1062
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        1043..1061
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     regulatory      1123..1132
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     CDS             join(1129..1131,1132..1134,1135..1848)
                     /codon_start=1
                     /product="enhanced GFP with monomerizing A206K mutation 
                     (Zacharias et al., 2002)"
                     /label=mEGFP
                     /note="mammalian codon-optimized"
                     /translation="MVSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTL
                     KFICTTGKLPVPWPTLVTTLTYGVQCFSRYPDHMKQHDFFKSAMPEGYVQERTIFFKDD
                     GNYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIK
                     VNFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSKLSKDPNEKRDHMVLL
                     EFVTAAGITLGMDELYK"
     primer_bind     complement(1174..1195)
                     /label=EGFP-N
                     /note="EGFP, reverse primer"
     primer_bind     complement(1435..1454)
                     /label=EXFP-R
                     /note="For distinguishing EGFP variants, reverse primer"
     primer_bind     1782..1803
                     /label=EGFP-C
                     /note="EGFP, forward primer"
     primer_bind     complement(2204..2221)
                     /label=BGH-rev
                     /note="Bovine growth hormone terminator, reverse primer. 
                     Also called BGH reverse"
     polyA_signal    2210..2434
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     rep_origin      2480..2908
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(2567..2586)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     2777..2798
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     complement(2917..2937)
                     /label=pBABE 3'
                     /note="SV40 enhancer, reverse primer for pBABE vectors"
     promoter        2922..3251
                     /label=SV40 promoter
                     /note="SV40 enhancer and early promoter"
     rep_origin      3102..3237
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     primer_bind     3164..3183
                     /label=SV40pro-F
                     /note="SV40 promoter/origin, forward primer"
     CDS             3318..4112
                     /codon_start=1
                     /gene="aph(3')-II (or nptII)"
                     /product="aminoglycoside phosphotransferase from Tn5"
                     /label=NeoR/KanR
                     /note="confers resistance to neomycin, kanamycin, and G418 
                     (Geneticin(R))"
                     /translation="MIEQDGLHAGSPAAWVERLFGYDWAQQTIGCSDAAVFRLSAQGRP
                     VLFVKTDLSGALNELQDEAARLSWLATTGVPCAAVLDVVTEAGRDWLLLGEVPGQDLLS
                     SHLAPAEKVSIMADAMRRLHTLDPATCPFDHQAKHRIERARTRMEAGLVDQDDLDEEHQ
                     GLAPAELFARLKARMPDGEDLVVTHGDACLPNIMVENGRFSGFIDCGRLGVADRYQDIA
                     LATRDIAEELGGEWADRFLVLYGIAAPDSQRIAFYRLLDEFF"
     primer_bind     complement(3372..3391)
                     /label=Neo-R
                     /note="Neomycin resistance gene, reverse primer"
     primer_bind     3982..4001
                     /label=Neo-F
                     /note="Neomycin resistance gene, forward primer"
     polyA_signal    4286..4407
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(4323..4342)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     4377..4396
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     primer_bind     complement(4456..4472)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(4456..4472)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(4469..4491)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    4480..4496
                     /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(4504..4510,4511..4528,4529..4534))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    4549..4570
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(4687..4704)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(4858..5446)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(4938..4957)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(5617..6408,6409..6433,1..44))
                     /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     6240..6259
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
ORIGIN
        1 gcaaaaaagg gaataagggc gacacggaaa tgttgaatac tcatactctt cctttttcaa
       61 tattattgaa gcatttatca gggttattgt ctcatgagcg gatacatatt tgaatgtatt
      121 tagaaaaata aacaaatagg ggttccgcgc acatttcccc gaaaagtgcc acctgacgtc
      181 gacggatcgg gagatctccc gatcccctat ggtgcactct cagtacaatc tgctctgatg
      241 ccgcatagtt aagccagtat ctgctccctg cttgtgtgtt ggaggtcgct gagtagtgcg
      301 cgagcaaaat ttaagctaca acaaggcaag gcttgaccga caattgcatg aagaatctgc
      361 ttagggttag gcgttttgcg ctgcttcgcg atgtacgggc cagatatacg cgttgacatt
      421 gattattgac tagttattaa tagtaatcaa ttacggggtc attagttcat agcccatata
      481 tggagttccg cgttacataa cttacggtaa atggcccgcc tggctgaccg cccaacgacc
      541 cccgcccatt gacgtcaata atgacgtatg ttcccatagt aacgccaata gggactttcc
      601 attgacgtca atgggtggag tatttacggt aaactgccca cttggcagta catcaagtgt
      661 atcatatgcc aagtacgccc cctattgacg tcaatgacgg taaatggccc gcctggcatt
      721 atgcccagta catgacctta tgggactttc ctacttggca gtacatctac gtattagtca
      781 tcgctattac catggtgatg cggttttggc agtacatcaa tgggcgtgga tagcggtttg
      841 actcacgggg atttccaagt ctccacccca ttgacgtcaa tgggagtttg ttttggcacc
      901 aaaatcaacg ggactttcca aaatgtcgta acaactccgc cccattgacg caaatgggcg
      961 gtaggcgtgt acggtgggag gtctatataa gcagagctct ctggctaact agagaaccca
     1021 ctgcttactg gcttatcgaa attaatacga ctcactatag ggagacccaa gctggctagc
     1081 gtttaaactt aagcttggca atccggtact gttggtaaat aagccaccat ggtgagcaag
     1141 ggcgaggagc tgttcaccgg ggtggtgccc atcctggtcg agctggacgg cgacgtaaac
     1201 ggccacaagt tcagcgtgtc cggcgagggc gagggcgatg ccacctacgg caagctgacc
     1261 ctgaagttca tctgcaccac cggcaagctg cccgtgccct ggcccaccct cgtgaccacc
     1321 ctgacctacg gcgtgcagtg cttcagccgc taccccgacc acatgaagca gcacgacttc
     1381 ttcaagtccg ccatgcccga aggctacgtc caggagcgca ccatcttctt caaggacgac
     1441 ggcaactaca agacccgcgc cgaggtgaag ttcgagggcg acaccctggt gaaccgcatc
     1501 gagctgaagg gcatcgactt caaggaggac ggcaacatcc tggggcacaa gctggagtac
     1561 aactacaaca gccacaacgt ctatatcatg gccgacaagc agaagaacgg catcaaggtg
     1621 aacttcaaga tccgccacaa catcgaggac ggcagcgtgc agctcgccga ccactaccag
     1681 cagaacaccc ccatcggcga cggccccgtg ctgctgcccg acaaccacta cctgagcacc
     1741 cagtccaagc tgagcaaaga ccccaacgag aagcgcgatc acatggtcct gctggagttc
     1801 gtgaccgccg ccgggatcac tctcggcatg gacgagctgt acaagtaagg ccgcgactct
     1861 agatgtggac ttgactcctg ttgtgccgtc agcagtgaac atgaaccctg caccaaaccc
     1921 tgcagtctat aaccctgaag ctgtaaatga acccaagaag aagaaatatg caaaagaggc
     1981 ttggccaggc aagaagccca caccttcctt gctgatttga tatttttggt catggagaag
     2041 ggtgggattg ggtgggaatg gggtggaagg gtgatgggga gctaatgaac tagggagaaa
     2101 aactttccat gtgtgcggta tcgtctttca gaatgtctcc tggcatccta accatgtaat
     2161 atgacaattg ggggtggggg gcccgtttaa acccgctgat cagcctcgac tgtgccttct
     2221 agttgccagc catctgttgt ttgcccctcc cccgtgcctt ccttgaccct ggaaggtgcc
     2281 actcccactg tcctttccta ataaaatgag gaaattgcat cgcattgtct gagtaggtgt
     2341 cattctattc tggggggtgg ggtggggcag gacagcaagg gggaggattg ggaagacaat
     2401 agcaggcatg ctggggatgc ggtgggctct atggcttctg aggcggaaag aaccagctgg
     2461 ggctctaggg ggtatcccca cgcgccctgt agcggcgcat taagcgcggc gggtgtggtg
     2521 gttacgcgca gcgtgaccgc tacacttgcc agcgccctag cgcccgctcc tttcgctttc
     2581 ttcccttcct ttctcgccac gttcgccggc tttccccgtc aagctctaaa tcgggggctc
     2641 cctttagggt tccgatttag tgctttacgg cacctcgacc ccaaaaaact tgattagggt
     2701 gatggttcac gtagtgggcc atcgccctga tagacggttt ttcgcccttt gacgttggag
     2761 tccacgttct ttaatagtgg actcttgttc caaactggaa caacactcaa ccctatctcg
     2821 gtctattctt ttgatttata agggattttg ccgatttcgg cctattggtt aaaaaatgag
     2881 ctgatttaac aaaaatttaa cgcgaattaa ttctgtggaa tgtgtgtcag ttagggtgtg
     2941 gaaagtcccc aggctcccca gcaggcagaa gtatgcaaag catgcatctc aattagtcag
     3001 caaccaggtg tggaaagtcc ccaggctccc cagcaggcag aagtatgcaa agcatgcatc
     3061 tcaattagtc agcaaccata gtcccgcccc taactccgcc catcccgccc ctaactccgc
     3121 ccagttccgc ccattctccg ccccatggct gactaatttt ttttatttat gcagaggccg
     3181 aggccgcctc tgcctctgag ctattccaga agtagtgagg aggctttttt ggaggcctag
     3241 gcttttgcaa aaagctcccg ggagcttgta tatccatttt cggatctgat caagagacag
     3301 gatgaggatc gtttcgcatg attgaacaag atggattgca cgcaggttct ccggccgctt
     3361 gggtggagag gctattcggc tatgactggg cacaacagac aatcggctgc tctgatgccg
     3421 ccgtgttccg gctgtcagcg caggggcgcc cggttctttt tgtcaagacc gacctgtccg
     3481 gtgccctgaa tgaactgcag gacgaggcag cgcggctatc gtggctggcc acgacgggcg
     3541 ttccttgcgc agctgtgctc gacgttgtca ctgaagcggg aagggactgg ctgctattgg
     3601 gcgaagtgcc ggggcaggat ctcctgtcat ctcaccttgc tcctgccgag aaagtatcca
     3661 tcatggctga tgcaatgcgg cggctgcata cgcttgatcc ggctacctgc ccattcgacc
     3721 accaagcgaa acatcgcatc gagcgagcac gtactcggat ggaagccggt cttgtcgatc
     3781 aggatgatct ggacgaagag catcaggggc tcgcgccagc cgaactgttc gccaggctca
     3841 aggcgcgcat gcccgacggc gaggatctcg tcgtgaccca tggcgatgcc tgcttgccga
     3901 atatcatggt ggaaaatggc cgcttttctg gattcatcga ctgtggccgg ctgggtgtgg
     3961 cggaccgcta tcaggacata gcgttggcta cccgtgatat tgctgaagag cttggcggcg
     4021 aatgggctga ccgcttcctc gtgctttacg gtatcgccgc tcccgattcg cagcgcatcg
     4081 ccttctatcg ccttcttgac gagttcttct gagcgggact ctggggttcg aaatgaccga
     4141 ccaagcgacg cccaacctgc catcacgaga tttcgattcc accgccgcct tctatgaaag
     4201 gttgggcttc ggaatcgttt tccgggacgc cggctggatg atcctccagc gcggggatct
     4261 catgctggag ttcttcgccc accccaactt gtttattgca gcttataatg gttacaaata
     4321 aagcaatagc atcacaaatt tcacaaataa agcatttttt tcactgcatt ctagttgtgg
     4381 tttgtccaaa ctcatcaatg tatcttatca tgtctgtata ccgtcgacct ctagctagag
     4441 cttggcgtaa tcatggtcat agctgtttcc tgtgtgaaat tgttatccgc tcacaattcc
     4501 acacaacata cgagccggaa gcataaagtg taaagcctgg ggtgcctaat gagtgagcta
     4561 actcacatta attgcgttgc gctcactgcc cgctttccag tcgggaaacc tgtcgtgcca
     4621 gctgcattaa tgaatcggcc aacgcgcggg gagaggcggt ttgcgtattg ggcgctcttc
     4681 cgcttcctcg ctcactgact cgctgcgctc ggtcgttcgg ctgcggcgag cggtatcagc
     4741 tcactcaaag gcggtaatac ggttatccac agaatcaggg gataacgcag gaaagaacat
     4801 gtgagcaaaa ggccagcaaa aggccaggaa ccgtaaaaag gccgcgttgc tggcgttttt
     4861 ccataggctc cgcccccctg acgagcatca caaaaatcga cgctcaagtc agaggtggcg
     4921 aaacccgaca ggactataaa gataccaggc gtttccccct ggaagctccc tcgtgcgctc
     4981 tcctgttccg accctgccgc ttaccggata cctgtccgcc tttctccctt cgggaagcgt
     5041 ggcgctttct catagctcac gctgtaggta tctcagttcg gtgtaggtcg ttcgctccaa
     5101 gctgggctgt gtgcacgaac cccccgttca gcccgaccgc tgcgccttat ccggtaacta
     5161 tcgtcttgag tccaacccgg taagacacga cttatcgcca ctggcagcag ccactggtaa
     5221 caggattagc agagcgaggt atgtaggcgg tgctacagag ttcttgaagt ggtggcctaa
     5281 ctacggctac actagaagaa cagtatttgg tatctgcgct ctgctgaagc cagttacctt
     5341 cggaaaaaga gttggtagct cttgatccgg caaacaaacc accgctggta gcggtggttt
     5401 ttttgtttgc aagcagcaga ttacgcgcag aaaaaaagga tctcaagaag atcctttgat
     5461 cttttctacg gggtctgacg ctcagtggaa cgaaaactca cgttaaggga ttttggtcat
     5521 gagattatca aaaaggatct tcacctagat ccttttaaat taaaaatgaa gttttaaatc
     5581 aatctaaagt atatatgagt aaacttggtc tgacagttac caatgcttaa tcagtgaggc
     5641 acctatctca gcgatctgtc tatttcgttc atccatagtt gcctgactcc ccgtcgtgta
     5701 gataactacg atacgggagg gcttaccatc tggccccagt gctgcaatga taccgcgaga
     5761 cccacgctca ccggctccag atttatcagc aataaaccag ccagccggaa gggccgagcg
     5821 cagaagtggt cctgcaactt tatccgcctc catccagtct attaattgtt gccgggaagc
     5881 tagagtaagt agttcgccag ttaatagttt gcgcaacgtt gttgccattg ctacaggcat
     5941 cgtggtgtca cgctcgtcgt ttggtatggc ttcattcagc tccggttccc aacgatcaag
     6001 gcgagttaca tgatccccca tgttgtgcaa aaaagcggtt agctccttcg gtcctccgat
     6061 cgttgtcaga agtaagttgg ccgcagtgtt atcactcatg gttatggcag cactgcataa
     6121 ttctcttact gtcatgccat ccgtaagatg cttttctgtg actggtgagt actcaaccaa
     6181 gtcattctga gaatagtgta tgcggcgacc gagttgctct tgcccggcgt caatacggga
     6241 taataccgcg ccacatagca gaactttaaa agtgctcatc attggaaaac gttcttcggg
     6301 gcgaaaactc tcaaggatct taccgctgtt gagatccagt tcgatgtaac ccactcgtgc
     6361 acccaactga tcttcagcat cttttacttt caccagcgtt tctgggtgag caaaaacagg
     6421 aaggcaaaat gcc
//
