LOCUS       sequence-252271-        6118 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Expresses eGFP.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 6118)
  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 6118)
  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
FEATURES             Location/Qualifiers
     source          1..6118
                     /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(1889..1906)
                     /label=BGH-rev
                     /note="Bovine growth hormone terminator, reverse primer. 
                     Also called BGH reverse"
     polyA_signal    1895..2119
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     rep_origin      2165..2593
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(2252..2271)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     2462..2483
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     complement(2602..2622)
                     /label=pBABE 3'
                     /note="SV40 enhancer, reverse primer for pBABE vectors"
     promoter        2607..2936
                     /label=SV40 promoter
                     /note="SV40 enhancer and early promoter"
     rep_origin      2787..2922
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     primer_bind     2849..2868
                     /label=SV40pro-F
                     /note="SV40 promoter/origin, forward primer"
     CDS             3003..3797
                     /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(3057..3076)
                     /label=Neo-R
                     /note="Neomycin resistance gene, reverse primer"
     primer_bind     3667..3686
                     /label=Neo-F
                     /note="Neomycin resistance gene, forward primer"
     polyA_signal    3971..4092
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(4008..4027)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     4062..4081
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     primer_bind     complement(4141..4157)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(4141..4157)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(4154..4176)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    4165..4181
                     /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(4189..4195,4196..4213,4214..4219))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    4234..4255
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(4372..4389)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(4543..5131)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(4623..4642)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(5302..6093,6094..6118,1..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     5925..5944
                     /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 agagggcccg tttaaacccg ctgatcagcc tcgactgtgc cttctagttg ccagccatct
     1921 gttgtttgcc cctcccccgt gccttccttg accctggaag gtgccactcc cactgtcctt
     1981 tcctaataaa atgaggaaat tgcatcgcat tgtctgagta ggtgtcattc tattctgggg
     2041 ggtggggtgg ggcaggacag caagggggag gattgggaag acaatagcag gcatgctggg
     2101 gatgcggtgg gctctatggc ttctgaggcg gaaagaacca gctggggctc tagggggtat
     2161 ccccacgcgc cctgtagcgg cgcattaagc gcggcgggtg tggtggttac gcgcagcgtg
     2221 accgctacac ttgccagcgc cctagcgccc gctcctttcg ctttcttccc ttcctttctc
     2281 gccacgttcg ccggctttcc ccgtcaagct ctaaatcggg ggctcccttt agggttccga
     2341 tttagtgctt tacggcacct cgaccccaaa aaacttgatt agggtgatgg ttcacgtagt
     2401 gggccatcgc cctgatagac ggtttttcgc cctttgacgt tggagtccac gttctttaat
     2461 agtggactct tgttccaaac tggaacaaca ctcaacccta tctcggtcta ttcttttgat
     2521 ttataaggga ttttgccgat ttcggcctat tggttaaaaa atgagctgat ttaacaaaaa
     2581 tttaacgcga attaattctg tggaatgtgt gtcagttagg gtgtggaaag tccccaggct
     2641 ccccagcagg cagaagtatg caaagcatgc atctcaatta gtcagcaacc aggtgtggaa
     2701 agtccccagg ctccccagca ggcagaagta tgcaaagcat gcatctcaat tagtcagcaa
     2761 ccatagtccc gcccctaact ccgcccatcc cgcccctaac tccgcccagt tccgcccatt
     2821 ctccgcccca tggctgacta atttttttta tttatgcaga ggccgaggcc gcctctgcct
     2881 ctgagctatt ccagaagtag tgaggaggct tttttggagg cctaggcttt tgcaaaaagc
     2941 tcccgggagc ttgtatatcc attttcggat ctgatcaaga gacaggatga ggatcgtttc
     3001 gcatgattga acaagatgga ttgcacgcag gttctccggc cgcttgggtg gagaggctat
     3061 tcggctatga ctgggcacaa cagacaatcg gctgctctga tgccgccgtg ttccggctgt
     3121 cagcgcaggg gcgcccggtt ctttttgtca agaccgacct gtccggtgcc ctgaatgaac
     3181 tgcaggacga ggcagcgcgg ctatcgtggc tggccacgac gggcgttcct tgcgcagctg
     3241 tgctcgacgt tgtcactgaa gcgggaaggg actggctgct attgggcgaa gtgccggggc
     3301 aggatctcct gtcatctcac cttgctcctg ccgagaaagt atccatcatg gctgatgcaa
     3361 tgcggcggct gcatacgctt gatccggcta cctgcccatt cgaccaccaa gcgaaacatc
     3421 gcatcgagcg agcacgtact cggatggaag ccggtcttgt cgatcaggat gatctggacg
     3481 aagagcatca ggggctcgcg ccagccgaac tgttcgccag gctcaaggcg cgcatgcccg
     3541 acggcgagga tctcgtcgtg acccatggcg atgcctgctt gccgaatatc atggtggaaa
     3601 atggccgctt ttctggattc atcgactgtg gccggctggg tgtggcggac cgctatcagg
     3661 acatagcgtt ggctacccgt gatattgctg aagagcttgg cggcgaatgg gctgaccgct
     3721 tcctcgtgct ttacggtatc gccgctcccg attcgcagcg catcgccttc tatcgccttc
     3781 ttgacgagtt cttctgagcg ggactctggg gttcgaaatg accgaccaag cgacgcccaa
     3841 cctgccatca cgagatttcg attccaccgc cgccttctat gaaaggttgg gcttcggaat
     3901 cgttttccgg gacgccggct ggatgatcct ccagcgcggg gatctcatgc tggagttctt
     3961 cgcccacccc aacttgttta ttgcagctta taatggttac aaataaagca atagcatcac
     4021 aaatttcaca aataaagcat ttttttcact gcattctagt tgtggtttgt ccaaactcat
     4081 caatgtatct tatcatgtct gtataccgtc gacctctagc tagagcttgg cgtaatcatg
     4141 gtcatagctg tttcctgtgt gaaattgtta tccgctcaca attccacaca acatacgagc
     4201 cggaagcata aagtgtaaag cctggggtgc ctaatgagtg agctaactca cattaattgc
     4261 gttgcgctca ctgcccgctt tccagtcggg aaacctgtcg tgccagctgc attaatgaat
     4321 cggccaacgc gcggggagag gcggtttgcg tattgggcgc tcttccgctt cctcgctcac
     4381 tgactcgctg cgctcggtcg ttcggctgcg gcgagcggta tcagctcact caaaggcggt
     4441 aatacggtta tccacagaat caggggataa cgcaggaaag aacatgtgag caaaaggcca
     4501 gcaaaaggcc aggaaccgta aaaaggccgc gttgctggcg tttttccata ggctccgccc
     4561 ccctgacgag catcacaaaa atcgacgctc aagtcagagg tggcgaaacc cgacaggact
     4621 ataaagatac caggcgtttc cccctggaag ctccctcgtg cgctctcctg ttccgaccct
     4681 gccgcttacc ggatacctgt ccgcctttct cccttcggga agcgtggcgc tttctcatag
     4741 ctcacgctgt aggtatctca gttcggtgta ggtcgttcgc tccaagctgg gctgtgtgca
     4801 cgaacccccc gttcagcccg accgctgcgc cttatccggt aactatcgtc ttgagtccaa
     4861 cccggtaaga cacgacttat cgccactggc agcagccact ggtaacagga ttagcagagc
     4921 gaggtatgta ggcggtgcta cagagttctt gaagtggtgg cctaactacg gctacactag
     4981 aagaacagta tttggtatct gcgctctgct gaagccagtt accttcggaa aaagagttgg
     5041 tagctcttga tccggcaaac aaaccaccgc tggtagcggt ggtttttttg tttgcaagca
     5101 gcagattacg cgcagaaaaa aaggatctca agaagatcct ttgatctttt ctacggggtc
     5161 tgacgctcag tggaacgaaa actcacgtta agggattttg gtcatgagat tatcaaaaag
     5221 gatcttcacc tagatccttt taaattaaaa atgaagtttt aaatcaatct aaagtatata
     5281 tgagtaaact tggtctgaca gttaccaatg cttaatcagt gaggcaccta tctcagcgat
     5341 ctgtctattt cgttcatcca tagttgcctg actccccgtc gtgtagataa ctacgatacg
     5401 ggagggctta ccatctggcc ccagtgctgc aatgataccg cgagacccac gctcaccggc
     5461 tccagattta tcagcaataa accagccagc cggaagggcc gagcgcagaa gtggtcctgc
     5521 aactttatcc gcctccatcc agtctattaa ttgttgccgg gaagctagag taagtagttc
     5581 gccagttaat agtttgcgca acgttgttgc cattgctaca ggcatcgtgg tgtcacgctc
     5641 gtcgtttggt atggcttcat tcagctccgg ttcccaacga tcaaggcgag ttacatgatc
     5701 ccccatgttg tgcaaaaaag cggttagctc cttcggtcct ccgatcgttg tcagaagtaa
     5761 gttggccgca gtgttatcac tcatggttat ggcagcactg cataattctc ttactgtcat
     5821 gccatccgta agatgctttt ctgtgactgg tgagtactca accaagtcat tctgagaata
     5881 gtgtatgcgg cgaccgagtt gctcttgccc ggcgtcaata cgggataata ccgcgccaca
     5941 tagcagaact ttaaaagtgc tcatcattgg aaaacgttct tcggggcgaa aactctcaag
     6001 gatcttaccg ctgttgagat ccagttcgat gtaacccact cgtgcaccca actgatcttc
     6061 agcatctttt actttcacca gcgtttctgg gtgagcaaaa acaggaaggc aaaatgcc
//
