LOCUS       sequence-252259-        6281 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Expresses an eGFP mRNA with the SH3BP5 G-quadruplex (G4) region in 
            the 3' UTR.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 6281)
  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 6281)
  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 SH3BP5 G4
FEATURES             Location/Qualifiers
     source          1..6281
                     /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(2052..2069)
                     /label=BGH-rev
                     /note="Bovine growth hormone terminator, reverse primer. 
                     Also called BGH reverse"
     polyA_signal    2058..2282
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     rep_origin      2328..2756
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(2415..2434)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     2625..2646
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     complement(2765..2785)
                     /label=pBABE 3'
                     /note="SV40 enhancer, reverse primer for pBABE vectors"
     promoter        2770..3099
                     /label=SV40 promoter
                     /note="SV40 enhancer and early promoter"
     rep_origin      2950..3085
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     primer_bind     3012..3031
                     /label=SV40pro-F
                     /note="SV40 promoter/origin, forward primer"
     CDS             3166..3960
                     /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(3220..3239)
                     /label=Neo-R
                     /note="Neomycin resistance gene, reverse primer"
     primer_bind     3830..3849
                     /label=Neo-F
                     /note="Neomycin resistance gene, forward primer"
     polyA_signal    4134..4255
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(4171..4190)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     4225..4244
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     primer_bind     complement(4304..4320)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(4304..4320)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(4317..4339)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    4328..4344
                     /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(4352..4358,4359..4376,4377..4382))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    4397..4418
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(4535..4552)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(4706..5294)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(4786..4805)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(5465..6256,6257..6281,1..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     6088..6107
                     /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 agaaggctac catgagctgc actttttggg gtgggaaagg tgaatgccag tggggatgcg
     1921 ggggatgagg gtaggaggga cttatagaag gggatttgtg gctgtggggg agaaggttct
     1981 acagcataag ccttatcctg ccagccaagg ggatttattc taagaggggc ccgtttaaac
     2041 ccgctgatca gcctcgactg tgccttctag ttgccagcca tctgttgttt gcccctcccc
     2101 cgtgccttcc ttgaccctgg aaggtgccac tcccactgtc ctttcctaat aaaatgagga
     2161 aattgcatcg cattgtctga gtaggtgtca ttctattctg gggggtgggg tggggcagga
     2221 cagcaagggg gaggattggg aagacaatag caggcatgct ggggatgcgg tgggctctat
     2281 ggcttctgag gcggaaagaa ccagctgggg ctctaggggg tatccccacg cgccctgtag
     2341 cggcgcatta agcgcggcgg gtgtggtggt tacgcgcagc gtgaccgcta cacttgccag
     2401 cgccctagcg cccgctcctt tcgctttctt cccttccttt ctcgccacgt tcgccggctt
     2461 tccccgtcaa gctctaaatc gggggctccc tttagggttc cgatttagtg ctttacggca
     2521 cctcgacccc aaaaaacttg attagggtga tggttcacgt agtgggccat cgccctgata
     2581 gacggttttt cgccctttga cgttggagtc cacgttcttt aatagtggac tcttgttcca
     2641 aactggaaca acactcaacc ctatctcggt ctattctttt gatttataag ggattttgcc
     2701 gatttcggcc tattggttaa aaaatgagct gatttaacaa aaatttaacg cgaattaatt
     2761 ctgtggaatg tgtgtcagtt agggtgtgga aagtccccag gctccccagc aggcagaagt
     2821 atgcaaagca tgcatctcaa ttagtcagca accaggtgtg gaaagtcccc aggctcccca
     2881 gcaggcagaa gtatgcaaag catgcatctc aattagtcag caaccatagt cccgccccta
     2941 actccgccca tcccgcccct aactccgccc agttccgccc attctccgcc ccatggctga
     3001 ctaatttttt ttatttatgc agaggccgag gccgcctctg cctctgagct attccagaag
     3061 tagtgaggag gcttttttgg aggcctaggc ttttgcaaaa agctcccggg agcttgtata
     3121 tccattttcg gatctgatca agagacagga tgaggatcgt ttcgcatgat tgaacaagat
     3181 ggattgcacg caggttctcc ggccgcttgg gtggagaggc tattcggcta tgactgggca
     3241 caacagacaa tcggctgctc tgatgccgcc gtgttccggc tgtcagcgca ggggcgcccg
     3301 gttctttttg tcaagaccga cctgtccggt gccctgaatg aactgcagga cgaggcagcg
     3361 cggctatcgt ggctggccac gacgggcgtt ccttgcgcag ctgtgctcga cgttgtcact
     3421 gaagcgggaa gggactggct gctattgggc gaagtgccgg ggcaggatct cctgtcatct
     3481 caccttgctc ctgccgagaa agtatccatc atggctgatg caatgcggcg gctgcatacg
     3541 cttgatccgg ctacctgccc attcgaccac caagcgaaac atcgcatcga gcgagcacgt
     3601 actcggatgg aagccggtct tgtcgatcag gatgatctgg acgaagagca tcaggggctc
     3661 gcgccagccg aactgttcgc caggctcaag gcgcgcatgc ccgacggcga ggatctcgtc
     3721 gtgacccatg gcgatgcctg cttgccgaat atcatggtgg aaaatggccg cttttctgga
     3781 ttcatcgact gtggccggct gggtgtggcg gaccgctatc aggacatagc gttggctacc
     3841 cgtgatattg ctgaagagct tggcggcgaa tgggctgacc gcttcctcgt gctttacggt
     3901 atcgccgctc ccgattcgca gcgcatcgcc ttctatcgcc ttcttgacga gttcttctga
     3961 gcgggactct ggggttcgaa atgaccgacc aagcgacgcc caacctgcca tcacgagatt
     4021 tcgattccac cgccgccttc tatgaaaggt tgggcttcgg aatcgttttc cgggacgccg
     4081 gctggatgat cctccagcgc ggggatctca tgctggagtt cttcgcccac cccaacttgt
     4141 ttattgcagc ttataatggt tacaaataaa gcaatagcat cacaaatttc acaaataaag
     4201 catttttttc actgcattct agttgtggtt tgtccaaact catcaatgta tcttatcatg
     4261 tctgtatacc gtcgacctct agctagagct tggcgtaatc atggtcatag ctgtttcctg
     4321 tgtgaaattg ttatccgctc acaattccac acaacatacg agccggaagc ataaagtgta
     4381 aagcctgggg tgcctaatga gtgagctaac tcacattaat tgcgttgcgc tcactgcccg
     4441 ctttccagtc gggaaacctg tcgtgccagc tgcattaatg aatcggccaa cgcgcgggga
     4501 gaggcggttt gcgtattggg cgctcttccg cttcctcgct cactgactcg ctgcgctcgg
     4561 tcgttcggct gcggcgagcg gtatcagctc actcaaaggc ggtaatacgg ttatccacag
     4621 aatcagggga taacgcagga aagaacatgt gagcaaaagg ccagcaaaag gccaggaacc
     4681 gtaaaaaggc cgcgttgctg gcgtttttcc ataggctccg cccccctgac gagcatcaca
     4741 aaaatcgacg ctcaagtcag aggtggcgaa acccgacagg actataaaga taccaggcgt
     4801 ttccccctgg aagctccctc gtgcgctctc ctgttccgac cctgccgctt accggatacc
     4861 tgtccgcctt tctcccttcg ggaagcgtgg cgctttctca tagctcacgc tgtaggtatc
     4921 tcagttcggt gtaggtcgtt cgctccaagc tgggctgtgt gcacgaaccc cccgttcagc
     4981 ccgaccgctg cgccttatcc ggtaactatc gtcttgagtc caacccggta agacacgact
     5041 tatcgccact ggcagcagcc actggtaaca ggattagcag agcgaggtat gtaggcggtg
     5101 ctacagagtt cttgaagtgg tggcctaact acggctacac tagaagaaca gtatttggta
     5161 tctgcgctct gctgaagcca gttaccttcg gaaaaagagt tggtagctct tgatccggca
     5221 aacaaaccac cgctggtagc ggtggttttt ttgtttgcaa gcagcagatt acgcgcagaa
     5281 aaaaaggatc tcaagaagat cctttgatct tttctacggg gtctgacgct cagtggaacg
     5341 aaaactcacg ttaagggatt ttggtcatga gattatcaaa aaggatcttc acctagatcc
     5401 ttttaaatta aaaatgaagt tttaaatcaa tctaaagtat atatgagtaa acttggtctg
     5461 acagttacca atgcttaatc agtgaggcac ctatctcagc gatctgtcta tttcgttcat
     5521 ccatagttgc ctgactcccc gtcgtgtaga taactacgat acgggagggc ttaccatctg
     5581 gccccagtgc tgcaatgata ccgcgagacc cacgctcacc ggctccagat ttatcagcaa
     5641 taaaccagcc agccggaagg gccgagcgca gaagtggtcc tgcaacttta tccgcctcca
     5701 tccagtctat taattgttgc cgggaagcta gagtaagtag ttcgccagtt aatagtttgc
     5761 gcaacgttgt tgccattgct acaggcatcg tggtgtcacg ctcgtcgttt ggtatggctt
     5821 cattcagctc cggttcccaa cgatcaaggc gagttacatg atcccccatg ttgtgcaaaa
     5881 aagcggttag ctccttcggt cctccgatcg ttgtcagaag taagttggcc gcagtgttat
     5941 cactcatggt tatggcagca ctgcataatt ctcttactgt catgccatcc gtaagatgct
     6001 tttctgtgac tggtgagtac tcaaccaagt cattctgaga atagtgtatg cggcgaccga
     6061 gttgctcttg cccggcgtca atacgggata ataccgcgcc acatagcaga actttaaaag
     6121 tgctcatcat tggaaaacgt tcttcggggc gaaaactctc aaggatctta ccgctgttga
     6181 gatccagttc gatgtaaccc actcgtgcac ccaactgatc ttcagcatct tttactttca
     6241 ccagcgtttc tgggtgagca aaaacaggaa ggcaaaatgc c
//
