LOCUS       sequence-451690-        5751 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  In vitro transcription of cap-independently (EMCV IRES) translated 
            mRNA encoding EGFP..
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 5751)
  AUTHORS   Golojuch S, Largey B, El-Sagheer AH, Brown T
  TITLE     Enhancing cap-independent translation of linear mRNA.
  JOURNAL   Nat Commun. 2025 Oct 16;16(1):9205. doi: 10.1038/s41467-025-64257-6.
  PUBMED    41102175
REFERENCE   2  (bases 1 to 5751)
  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: "Nat 
            Commun."; date: "2025-10-16"; volume: "16(1):9205. doi"; pages: " 
            10.1038/s41467-025-64257-6"
COMMENT     Sequence Label: pmRNA-iEMCV-EGFP-A120
FEATURES             Location/Qualifiers
     source          1..5751
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     rep_origin      74..662
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     563..582
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     enhancer        873..1252
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
     promoter        1253..1456
                     /label=CMV promoter
                     /note="human cytomegalovirus (CMV) immediate early 
                     promoter"
     primer_bind     1407..1427
                     /label=CMV-F
                     /note="Human CMV immediate early promoter, forward primer"
     primer_bind     1453..1477
                     /label=LNCX
                     /note="Human CMV promoter, forward primer"
     intron          1592..1724
                     /label=chimeric intron
                     /note="chimera between introns from human beta-globin and 
                     immunoglobulin heavy chain genes"
     primer_bind     1707..1726
                     /label=pMT2-F
                     /note="Synthetic intron, forward primer"
     misc_feature    1862..2435
                     /label=IRES
                     /note="internal ribosome entry site (IRES) of the 
                     encephalomyocarditis virus (EMCV)"
     misc_feature    1885..2437
                     /label=IRES
                     /note="internal ribosome entry site (IRES) of the 
                     encephalomyocarditis virus (EMCV)"
     primer_bind     complement(2029..2046)
                     /label=IRES reverse
                     /note="IRES internal ribosome entry site, reverse primer. 
                     Also called pCDH-rev"
     primer_bind     2256..2275
                     /label=IRES-F
                     /note="IRES internal ribosome entry site, forward primer"
     CDS             join(2481..2483,2484..2486,2487..3200)
                     /codon_start=1
                     /product="the original enhanced GFP (Yang et al., 1996)"
                     /label=EGFP
                     /note="mammalian codon-optimized"
                     /translation="MVSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTL
                     KFICTTGKLPVPWPTLVTTLTYGVQCFSRYPDHMKQHDFFKSAMPEGYVQERTIFFKDD
                     GNYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIK
                     VNFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSALSKDPNEKRDHMVLL
                     EFVTAAGITLGMDELYK"
     CDS             join(2481..2483,2484..2486,2487..3197)
                     /codon_start=1
                     /product="enhanced GFP"
                     /label=EGFP
                     /translation="MVSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTL
                     KFICTTGKLPVPWPTLVTTLTYGVQCFSRYPDHMKQHDFFKSAMPEGYVQERTIFFKDD
                     GNYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIK
                     VNFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSALSKDPNEKRDHMVLL
                     EFVTAAGITLGMDELYK"
     primer_bind     complement(2526..2547)
                     /label=EGFP-N
                     /note="EGFP, reverse primer"
     primer_bind     complement(2787..2806)
                     /label=EXFP-R
                     /note="For distinguishing EGFP variants, reverse primer"
     primer_bind     3134..3155
                     /label=EGFP-C
                     /note="EGFP, forward primer"
     polyA_signal    3666..3787
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(3677..3696)
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     primer_bind     3731..3750
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     rep_origin      3968..4423
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(4055..4074)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     4265..4286
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     complement(4490..4509)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     4609..4631
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     primer_bind     complement(4669..4687)
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     promoter        4755..4859
                     /gene="bla"
                     /label=AmpR promoter
     CDS             4860..5654
                     /codon_start=1
                     /product="aminoglycoside phosphotransferase"
                     /label=NeoR/KanR
                     /note="confers resistance to neomycin, kanamycin, and G418 
                     (Geneticin(R))"
                     /translation="MIEQDGLHAGSPAAWVERLFGYDWAQQTIGCSDAAVFRLSAQGRP
                     VLFVKTDLSGALNELQDEAARLSWLATTGVPCAAVLDVVTEAGRDWLLLGEVPGQDLLS
                     SHLAPAEKVSIMADAMRRLHTLDPATCPFDHQAKHRIERARTRMEAGLVDQDDLDEEHQ
                     GLAPAELFARLKASMPDGEDLVVTHGDACLPNIMVENGRFSGFIDCGRLGVADRYQDIA
                     LATRDIAEELGGEWADRFLVLYGIAAPDSQRIAFYRLLDEFF"
     CDS             4860..5654
                     /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
                     GLAPAELFARLKASMPDGEDLVVTHGDACLPNIMVENGRFSGFIDCGRLGVADRYQDIA
                     LATRDIAEELGGEWADRFLVLYGIAAPDSQRIAFYRLLDEFF"
     primer_bind     complement(4914..4933)
                     /label=Neo-R
                     /note="Neomycin resistance gene, reverse primer"
     primer_bind     5524..5543
                     /label=Neo-F
                     /note="Neomycin resistance gene, forward primer"
ORIGIN
        1 tgaccaaaat cccttaacgt gagttttcgt tccactgagc gtcagacccc gtagaaaaga
       61 tcaaaggatc ttcttgagat cctttttttc tgcgcgtaat ctgctgcttg caaacaaaaa
      121 aaccaccgct accagcggtg gtttgtttgc cggatcaaga gctaccaact ctttttccga
      181 aggtaactgg cttcagcaga gcgcagatac caaatactgt tcttctagtg tagccgtagt
      241 taggccacca cttcaagaac tctgtagcac cgcctacata cctcgctctg ctaatcctgt
      301 taccagtggc tgctgccagt ggcgataagt cgtgtcttac cgggttggac tcaagacgat
      361 agttaccgga taaggcgcag cggtcgggct gaacgggggg ttcgtgcaca cagcccagct
      421 tggagcgaac gacctacacc gaactgagat acctacagcg tgagctatga gaaagcgcca
      481 cgcttcccga agggagaaag gcggacaggt atccggtaag cggcagggtc ggaacaggag
      541 agcgcacgag ggagcttcca gggggaaacg cctggtatct ttatagtcct gtcgggtttc
      601 gccacctctg acttgagcgt cgatttttgt gatgctcgtc aggggggcgg agcctatgga
      661 aaaacgccag caacgcggcc tttttacggt tcctggcctt ttgctggcct tttgctcaca
      721 tggctcgaca gatcatcaat attggccatt agccatatta ttcattggtt atatagcata
      781 aatcaatatt ggctattggc cattgcatac gttgtatcta tatcataata tgtacattta
      841 tattggctca tgtccaatat gaccgccatg ttggcattga ttattgacta gttattaata
      901 gtaatcaatt acggggtcat tagttcatag cccatatatg gagttccgcg ttacataact
      961 tacggtaaat ggcccgcctg gctgaccgcc caacgacccc cgcccattga cgtcaataat
     1021 gacgtatgtt cccatagtaa cgccaatagg gactttccat tgacgtcaat gggtggagta
     1081 tttacggtaa actgcccact tggcagtaca tcaagtgtat catatgccaa gtccgccccc
     1141 tattgacgtc aatgacggta aatggcccgc ctggcattat gcccagtaca tgaccttacg
     1201 ggactttcct acttggcagt acatctacgt attagtcatc gctattacca tggtgatgcg
     1261 gttttggcag tacaccaatg ggcgtggata gcggtttgac tcacggggat ttccaagtct
     1321 ccaccccatt gacgtcaatg ggagtttgtt ttggcaccaa aatcaacggg actttccaaa
     1381 atgtcgtaat aaccccgccc cgttgacgca aatgggcggt aggcgtgtac ggtgggaggt
     1441 ctatataagc agagctcgtt tagtgaaccg tcagatcact agacgcttta ttgcggtagt
     1501 ttatcacagt taaattgcta acgcagtcag tgcttctgac acaacagtct cgaacttaag
     1561 ctgcagaagt tggtcgtgag gcactgggca ggtaagtatc aaggttacaa gacaggttta
     1621 aggagaccaa tagaaactgg gcttgtcgag acagagaaga ctcttgcgtt tctgataggc
     1681 acctattggt cttactgaca tccactttgc ctttctctcc acaggtgtcc actcccagtt
     1741 caattacagc tcttaaggct agagtcacct gcaacatgac ttaatacgac tcactataag
     1801 gagagatatc acgcgttcta gagctagcct cgataagctt gatatcgaat tccgcccccc
     1861 ccccctctcc ctcccccccc cctaacgtta ctggccgaag ccgcttggaa taaggccggt
     1921 gtgcgtttgt ctatatgtta ttttccacca tattgccgtc ttttggcaat gtgagggccc
     1981 ggaaacctgg ccctgtcttc ttgacgagca ttcctagggg tctttcccct ctcgccaaag
     2041 gaatgcaagg tctgttgaat gtcgtgaagg aagcagttcc tctggaagct tcttgaagac
     2101 aaacaacgtc tgtagcgacc ctttgcaggc agcggaaccc cccacctggc gacaggtgcc
     2161 tctgcggcca aaagccacgt gtataagata cacctgcaaa ggcggcacaa ccccagtgcc
     2221 acgttgtgag ttggatagtt gtggaaagag tcaaatggct ctcctcaagc gtattcaaca
     2281 aggggctgaa ggatgcccag aaggtacccc attgtatggg atctgatctg gggcctcggt
     2341 gcacatgctt tacatgtgtt tagtcgaggt taaaaaaacg tctaggcccc ccgaaccacg
     2401 gggacgtggt tttcctttga aaaacacgat gataatatga cgatgataat atgacttcga
     2461 aagtttatga tccagaacaa atggtgagca agggcgagga gctgttcacc ggggtggtgc
     2521 ccatcctggt cgagctggac ggcgacgtaa acggccacaa gttcagcgtg tccggcgagg
     2581 gcgagggcga tgccacctac ggcaagctga ccctgaagtt catctgcacc accggcaagc
     2641 tgcccgtgcc ctggcccacc ctcgtgacca ccctgaccta cggcgtgcag tgcttcagcc
     2701 gctaccccga ccacatgaag cagcacgact tcttcaagtc cgccatgccc gaaggctacg
     2761 tccaggagcg caccatcttc ttcaaggacg acggcaacta caagacccgc gccgaggtga
     2821 agttcgaggg cgacaccctg gtgaaccgca tcgagctgaa gggcatcgac ttcaaggagg
     2881 acggcaacat cctggggcac aagctggagt acaactacaa cagccacaac gtctatatca
     2941 tggccgacaa gcagaagaac ggcatcaagg tgaacttcaa gatccgccac aacatcgagg
     3001 acggcagcgt gcagctcgcc gaccactacc agcagaacac ccccatcggc gacggccccg
     3061 tgctgctgcc cgacaaccac tacctgagca cccagtccgc cctgagcaaa gaccccaacg
     3121 agaagcgcga tcacatggtc ctgctggagt tcgtgaccgc cgccgggatc actctcggca
     3181 tggacgagct gtacaagtaa agcggccgca gctcgctttc ttgctgtcca atttctatta
     3241 aaggttcctt tgttccctaa gtccaactac taaactgggg gatattatga agggccttga
     3301 gcatttggat tctgcctaat aaaaaacatt tattttcatt gcgtttagct cgctttcttg
     3361 ctgtccaatt tctattaaag gttcctttgt tccctaagtc caactactaa actgggggat
     3421 attatgaagg gccttgagca tttggattct gcctaataaa aaacatttat tttcattgca
     3481 tttaaatgtt taaacaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa
     3541 aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa
     3601 aaaaaaaaaa aaaaaagaga ccggtacgtt cgtgataaac aaacggccgc ttcgagcaga
     3661 catgataaga tacattgatg agtttggaca aaccacaact agaatgcagt gaaaaaaatg
     3721 ctttatttgt gaaatttgtg atgctattgc tttatttgta accattataa gctgcaataa
     3781 acaagttaac aacaacaatt gcattcattt tatgtttcag gttcaggggg agatgtggga
     3841 ggttttttaa agcaagtaaa acctctacaa atgtggtaaa atcgataagg atcagggctg
     3901 gcgtaatagc gaagaggccc gcaccgatcg cccttcccaa cagttgcgca gcctgaatgg
     3961 cgaatggacg cgccctgtag cggcgcatta agcgcggcgg gtgtggtggt tacgcgcagc
     4021 gtgaccgcta cacttgccag cgccctagcg cccgctcctt tcgctttctt cccttccttt
     4081 ctcgccacgt tcgccggctt tccccgtcaa gctctaaatc gggggctccc tttagggttc
     4141 cgatttagtg ctttacggca cctcgacccc aaaaaacttg attagggtga tggttcacgt
     4201 agtgggccat cgccctgata gacggttttt cgccctttga cgttggagtc cacgttcttt
     4261 aatagtggac tcttgttcca aactggaaca acactcaacc ctatctcggt ctattctttt
     4321 gatttataag ggattttgcc gatttcggcc tattggttaa aaaatgagct gatttaacaa
     4381 aaatttaacg cgaattttaa caaaatatta acgcttacaa tttcctgatg cggtattttc
     4441 tccttacgca tctgtgcggt atttcacacc gcatatggtg cactctcagt acaatctgct
     4501 ctgatgccgc atagttaagc cagccccgac acccgccaac acccgctgac gcgccctgac
     4561 gggcttgtct gctcccggca tccgcttaca gacaagctgt gaccgtctcc gggagctgca
     4621 tgtgtcagag gttttcaccg tcatcaccga aacgcgcgag acgaaagggc ctcgtgatac
     4681 gcctattttt ataggttaat gtcatgataa taatggtttc ttagacgtca ggtggcactt
     4741 ttcggggaaa tgtgcgcgga acccctattt gtttattttt ctaaatacat tcaaatatgt
     4801 atccgctcat gagacaataa ccctgataaa tgcttcaata atattgaaaa aggaagagta
     4861 tgattgaaca agatggattg cacgcaggtt ctccggccgc ttgggtggag aggctattcg
     4921 gctatgactg ggcacaacag acaatcggct gctctgatgc cgccgtgttc cggctgtcag
     4981 cgcaggggcg cccggttctt tttgtcaaga ccgacctgtc cggtgccctg aatgaactgc
     5041 aagacgaggc agcgcggcta tcgtggctgg ccacgacggg cgttccttgc gcagctgtgc
     5101 tcgacgttgt cactgaagcg ggaagggact ggctgctatt gggcgaagtg ccggggcagg
     5161 atctcctgtc atctcacctt gctcctgccg agaaagtatc catcatggct gatgcaatgc
     5221 ggcggctgca tacgcttgat ccggctacct gcccattcga ccaccaagcg aaacatcgca
     5281 tcgagcgagc acgtactcgg atggaagccg gtcttgtcga tcaggatgat ctggacgaag
     5341 agcatcaggg gctcgcgcca gccgaactgt tcgccaggct caaggcgagc atgcccgacg
     5401 gcgaggatct cgtcgtgacc catggcgatg cctgcttgcc gaatatcatg gtggaaaatg
     5461 gccgcttttc tggattcatc gactgtggcc ggctgggtgt ggcggaccgc tatcaggaca
     5521 tagcgttggc tacccgtgat attgctgaag agcttggcgg cgaatgggct gaccgcttcc
     5581 tcgtgcttta cggtatcgcc gctcccgatt cgcagcgcat cgccttctat cgccttcttg
     5641 acgagttctt ctgactgtca gaccaagttt actcatatat actttagatt gatttaaaac
     5701 ttcattttta atttaaaagg atctaggtga agatcctttt tgataatctc a
//
