LOCUS       sequence-451682-        5492 bp    DNA     circular SYN 15-APR-2025
DEFINITION  In vitro transcription of cap-independently (HCV IRES) translated 
            mRNA encoding EGFP..
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 5492)
  TITLE     Enhancing cap-independent translation of mRNA via in vitro 
            transcription priming
  JOURNAL   Unpublished
REFERENCE   2  (bases 1 to 5492)
  AUTHORS   .
  TITLE     Direct Submission
  JOURNAL   Exported Apr 15, 2025 from SnapGene Server 8.0.1
            https://www.snapgene.com
COMMENT     SGRef: number: 1; type: "Journal Article"; journalName: 
            "Unpublished"
COMMENT     Sequence Label: pmRNA-iHCV-EGFP-A120
FEATURES             Location/Qualifiers
     source          1..5492
                     /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"
     CDS             join(2214..2216,2217..2219,2220..2933)
                     /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(2214..2216,2217..2219,2220..2930)
                     /codon_start=1
                     /product="enhanced GFP"
                     /label=EGFP
                     /translation="MVSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTL
                     KFICTTGKLPVPWPTLVTTLTYGVQCFSRYPDHMKQHDFFKSAMPEGYVQERTIFFKDD
                     GNYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIK
                     VNFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSALSKDPNEKRDHMVLL
                     EFVTAAGITLGMDELYK"
     primer_bind     complement(2259..2280)
                     /label=EGFP-N
                     /note="EGFP, reverse primer"
     primer_bind     complement(2520..2539)
                     /label=EXFP-R
                     /note="For distinguishing EGFP variants, reverse primer"
     primer_bind     2867..2888
                     /label=EGFP-C
                     /note="EGFP, forward primer"
     polyA_signal    3407..3528
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(3418..3437)
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     primer_bind     3472..3491
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     rep_origin      3709..4164
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(3796..3815)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     4006..4027
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     complement(4231..4250)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     4350..4372
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     primer_bind     complement(4410..4428)
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     promoter        4496..4600
                     /gene="bla"
                     /label=AmpR promoter
     CDS             4601..5395
                     /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             4601..5395
                     /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(4655..4674)
                     /label=Neo-R
                     /note="Neomycin resistance gene, reverse primer"
     primer_bind     5265..5284
                     /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 gagctagcta gtgtacgtag ccagcccccg attgggggcg
     1861 acactccacc atagatcact cccctgtgag gaactactgt cttcacgcag aaagcgtcta
     1921 gccatggcgt tagtatgaga gtcgtgcagc ctccaggacc ccccctcccg ggagagccat
     1981 agtggtctgc ggaaccggtg agtacaccgg aattgccagg acgaccgggt cctttcttgg
     2041 atcaacccgc tcaatgcctg gagatttggg cgtgcccccg caagactgct agccgagtag
     2101 tgttgggtcg cgaaaggcct tgtggtactg cctgataggg tgcttgcgag tgccccggga
     2161 ggtctcgtag accgtgcacc atgagcacga atcctaaacc tcaaagaaaa accatggtga
     2221 gcaagggcga ggagctgttc accggggtgg tgcccatcct ggtcgagctg gacggcgacg
     2281 taaacggcca caagttcagc gtgtccggcg agggcgaggg cgatgccacc tacggcaagc
     2341 tgaccctgaa gttcatctgc accaccggca agctgcccgt gccctggccc accctcgtga
     2401 ccaccctgac ctacggcgtg cagtgcttca gccgctaccc cgaccacatg aagcagcacg
     2461 acttcttcaa gtccgccatg cccgaaggct acgtccagga gcgcaccatc ttcttcaagg
     2521 acgacggcaa ctacaagacc cgcgccgagg tgaagttcga gggcgacacc ctggtgaacc
     2581 gcatcgagct gaagggcatc gacttcaagg aggacggcaa catcctgggg cacaagctgg
     2641 agtacaacta caacagccac aacgtctata tcatggccga caagcagaag aacggcatca
     2701 aggtgaactt caagatccgc cacaacatcg aggacggcag cgtgcagctc gccgaccact
     2761 accagcagaa cacccccatc ggcgacggcc ccgtgctgct gcccgacaac cactacctga
     2821 gcacccagtc cgccctgagc aaagacccca acgagaagcg cgatcacatg gtcctgctgg
     2881 agttcgtgac cgccgccggg atcactctcg gcatggacga gctgtacaag taaagcggcc
     2941 gcagctcgct ttcttgctgt ccaatttcta ttaaaggttc ctttgttccc taagtccaac
     3001 tactaaactg ggggatatta tgaagggcct tgagcatttg gattctgcct aataaaaaac
     3061 atttattttc attgcgttta gctcgctttc ttgctgtcca atttctatta aaggttcctt
     3121 tgttccctaa gtccaactac taaactgggg gatattatga agggccttga gcatttggat
     3181 tctgcctaat aaaaaacatt tattttcatt gcatttaaat gtttaaacaa aaaaaaaaaa
     3241 aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa
     3301 aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aagcaggtgg
     3361 gagggtacgt tcgtgataaa caaacggccg cttcgagcag acatgataag atacattgat
     3421 gagtttggac aaaccacaac tagaatgcag tgaaaaaaat gctttatttg tgaaatttgt
     3481 gatgctattg ctttatttgt aaccattata agctgcaata aacaagttaa caacaacaat
     3541 tgcattcatt ttatgtttca ggttcagggg gagatgtggg aggtttttta aagcaagtaa
     3601 aacctctaca aatgtggtaa aatcgataag gatcagggct ggcgtaatag cgaagaggcc
     3661 cgcaccgatc gcccttccca acagttgcgc agcctgaatg gcgaatggac gcgccctgta
     3721 gcggcgcatt aagcgcggcg ggtgtggtgg ttacgcgcag cgtgaccgct acacttgcca
     3781 gcgccctagc gcccgctcct ttcgctttct tcccttcctt tctcgccacg ttcgccggct
     3841 ttccccgtca agctctaaat cgggggctcc ctttagggtt ccgatttagt gctttacggc
     3901 acctcgaccc caaaaaactt gattagggtg atggttcacg tagtgggcca tcgccctgat
     3961 agacggtttt tcgccctttg acgttggagt ccacgttctt taatagtgga ctcttgttcc
     4021 aaactggaac aacactcaac cctatctcgg tctattcttt tgatttataa gggattttgc
     4081 cgatttcggc ctattggtta aaaaatgagc tgatttaaca aaaatttaac gcgaatttta
     4141 acaaaatatt aacgcttaca atttcctgat gcggtatttt ctccttacgc atctgtgcgg
     4201 tatttcacac cgcatatggt gcactctcag tacaatctgc tctgatgccg catagttaag
     4261 ccagccccga cacccgccaa cacccgctga cgcgccctga cgggcttgtc tgctcccggc
     4321 atccgcttac agacaagctg tgaccgtctc cgggagctgc atgtgtcaga ggttttcacc
     4381 gtcatcaccg aaacgcgcga gacgaaaggg cctcgtgata cgcctatttt tataggttaa
     4441 tgtcatgata ataatggttt cttagacgtc aggtggcact tttcggggaa atgtgcgcgg
     4501 aacccctatt tgtttatttt tctaaataca ttcaaatatg tatccgctca tgagacaata
     4561 accctgataa atgcttcaat aatattgaaa aaggaagagt atgattgaac aagatggatt
     4621 gcacgcaggt tctccggccg cttgggtgga gaggctattc ggctatgact gggcacaaca
     4681 gacaatcggc tgctctgatg ccgccgtgtt ccggctgtca gcgcaggggc gcccggttct
     4741 ttttgtcaag accgacctgt ccggtgccct gaatgaactg caagacgagg cagcgcggct
     4801 atcgtggctg gccacgacgg gcgttccttg cgcagctgtg ctcgacgttg tcactgaagc
     4861 gggaagggac tggctgctat tgggcgaagt gccggggcag gatctcctgt catctcacct
     4921 tgctcctgcc gagaaagtat ccatcatggc tgatgcaatg cggcggctgc atacgcttga
     4981 tccggctacc tgcccattcg accaccaagc gaaacatcgc atcgagcgag cacgtactcg
     5041 gatggaagcc ggtcttgtcg atcaggatga tctggacgaa gagcatcagg ggctcgcgcc
     5101 agccgaactg ttcgccaggc tcaaggcgag catgcccgac ggcgaggatc tcgtcgtgac
     5161 ccatggcgat gcctgcttgc cgaatatcat ggtggaaaat ggccgctttt ctggattcat
     5221 cgactgtggc cggctgggtg tggcggaccg ctatcaggac atagcgttgg ctacccgtga
     5281 tattgctgaa gagcttggcg gcgaatgggc tgaccgcttc ctcgtgcttt acggtatcgc
     5341 cgctcccgat tcgcagcgca tcgccttcta tcgccttctt gacgagttct tctgactgtc
     5401 agaccaagtt tactcatata tactttagat tgatttaaaa cttcattttt aatttaaaag
     5461 gatctaggtg aagatccttt ttgataatct ca
//
