LOCUS       sequence-449542-        5189 bp    DNA     circular SYN 15-APR-2025
DEFINITION  In vitro transcription of cap-dependently translated mRNA encoding 
            EGFP..
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 5189)
  TITLE     Enhancing cap-independent translation of mRNA via in vitro 
            transcription priming
  JOURNAL   Unpublished
REFERENCE   2  (bases 1 to 5189)
  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            "Unpublished"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-EGFP-A120
FEATURES             Location/Qualifiers
     source          1..5189
                     /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    1823..1903
                     /label=MCS
                     /note="multiple cloning site"
     regulatory      1905..1914
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     CDS             join(1911..1913,1914..1916,1917..2630)
                     /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(1911..1913,1914..1916,1917..2627)
                     /codon_start=1
                     /product="enhanced GFP"
                     /label=EGFP
                     /translation="MVSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTL
                     KFICTTGKLPVPWPTLVTTLTYGVQCFSRYPDHMKQHDFFKSAMPEGYVQERTIFFKDD
                     GNYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIK
                     VNFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSALSKDPNEKRDHMVLL
                     EFVTAAGITLGMDELYK"
     primer_bind     complement(1956..1977)
                     /label=EGFP-N
                     /note="EGFP, reverse primer"
     primer_bind     complement(2217..2236)
                     /label=EXFP-R
                     /note="For distinguishing EGFP variants, reverse primer"
     primer_bind     2564..2585
                     /label=EGFP-C
                     /note="EGFP, forward primer"
     polyA_signal    3104..3225
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(3115..3134)
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     primer_bind     3169..3188
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     rep_origin      3406..3861
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(3493..3512)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     3703..3724
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     complement(3928..3947)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     4047..4069
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     primer_bind     complement(4107..4125)
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     promoter        4193..4297
                     /gene="bla"
                     /label=AmpR promoter
     CDS             4298..5092
                     /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             4298..5092
                     /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(4352..4371)
                     /label=Neo-R
                     /note="Neomycin resistance gene, reverse primer"
     primer_bind     4962..4981
                     /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 gagctagcgc taccggactc agatctcgag ctcaagcttc
     1861 gaattctgca gtcgacggta ccgcgggccc gggatccacc ggtcgccacc atggtgagca
     1921 agggcgagga gctgttcacc ggggtggtgc ccatcctggt cgagctggac ggcgacgtaa
     1981 acggccacaa gttcagcgtg tccggcgagg gcgagggcga tgccacctac ggcaagctga
     2041 ccctgaagtt catctgcacc accggcaagc tgcccgtgcc ctggcccacc ctcgtgacca
     2101 ccctgaccta cggcgtgcag tgcttcagcc gctaccccga ccacatgaag cagcacgact
     2161 tcttcaagtc cgccatgccc gaaggctacg tccaggagcg caccatcttc ttcaaggacg
     2221 acggcaacta caagacccgc gccgaggtga agttcgaggg cgacaccctg gtgaaccgca
     2281 tcgagctgaa gggcatcgac ttcaaggagg acggcaacat cctggggcac aagctggagt
     2341 acaactacaa cagccacaac gtctatatca tggccgacaa gcagaagaac ggcatcaagg
     2401 tgaacttcaa gatccgccac aacatcgagg acggcagcgt gcagctcgcc gaccactacc
     2461 agcagaacac ccccatcggc gacggccccg tgctgctgcc cgacaaccac tacctgagca
     2521 cccagtccgc cctgagcaaa gaccccaacg agaagcgcga tcacatggtc ctgctggagt
     2581 tcgtgaccgc cgccgggatc actctcggca tggacgagct gtacaagtaa agcggccgca
     2641 gctcgctttc ttgctgtcca atttctatta aaggttcctt tgttccctaa gtccaactac
     2701 taaactgggg gatattatga agggccttga gcatttggat tctgcctaat aaaaaacatt
     2761 tattttcatt gcgtttagct cgctttcttg ctgtccaatt tctattaaag gttcctttgt
     2821 tccctaagtc caactactaa actgggggat attatgaagg gccttgagca tttggattct
     2881 gcctaataaa aaacatttat tttcattgca tttaaatgtt taaacaaaaa aaaaaaaaaa
     2941 aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa
     3001 aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaag caggtgggag
     3061 ggtacgttcg tgataaacaa acggccgctt cgagcagaca tgataagata cattgatgag
     3121 tttggacaaa ccacaactag aatgcagtga aaaaaatgct ttatttgtga aatttgtgat
     3181 gctattgctt tatttgtaac cattataagc tgcaataaac aagttaacaa caacaattgc
     3241 attcatttta tgtttcaggt tcagggggag atgtgggagg ttttttaaag caagtaaaac
     3301 ctctacaaat gtggtaaaat cgataaggat cagggctggc gtaatagcga agaggcccgc
     3361 accgatcgcc cttcccaaca gttgcgcagc ctgaatggcg aatggacgcg ccctgtagcg
     3421 gcgcattaag cgcggcgggt gtggtggtta cgcgcagcgt gaccgctaca cttgccagcg
     3481 ccctagcgcc cgctcctttc gctttcttcc cttcctttct cgccacgttc gccggctttc
     3541 cccgtcaagc tctaaatcgg gggctccctt tagggttccg atttagtgct ttacggcacc
     3601 tcgaccccaa aaaacttgat tagggtgatg gttcacgtag tgggccatcg ccctgataga
     3661 cggtttttcg ccctttgacg ttggagtcca cgttctttaa tagtggactc ttgttccaaa
     3721 ctggaacaac actcaaccct atctcggtct attcttttga tttataaggg attttgccga
     3781 tttcggccta ttggttaaaa aatgagctga tttaacaaaa atttaacgcg aattttaaca
     3841 aaatattaac gcttacaatt tcctgatgcg gtattttctc cttacgcatc tgtgcggtat
     3901 ttcacaccgc atatggtgca ctctcagtac aatctgctct gatgccgcat agttaagcca
     3961 gccccgacac ccgccaacac ccgctgacgc gccctgacgg gcttgtctgc tcccggcatc
     4021 cgcttacaga caagctgtga ccgtctccgg gagctgcatg tgtcagaggt tttcaccgtc
     4081 atcaccgaaa cgcgcgagac gaaagggcct cgtgatacgc ctatttttat aggttaatgt
     4141 catgataata atggtttctt agacgtcagg tggcactttt cggggaaatg tgcgcggaac
     4201 ccctatttgt ttatttttct aaatacattc aaatatgtat ccgctcatga gacaataacc
     4261 ctgataaatg cttcaataat attgaaaaag gaagagtatg attgaacaag atggattgca
     4321 cgcaggttct ccggccgctt gggtggagag gctattcggc tatgactggg cacaacagac
     4381 aatcggctgc tctgatgccg ccgtgttccg gctgtcagcg caggggcgcc cggttctttt
     4441 tgtcaagacc gacctgtccg gtgccctgaa tgaactgcaa gacgaggcag cgcggctatc
     4501 gtggctggcc acgacgggcg ttccttgcgc agctgtgctc gacgttgtca ctgaagcggg
     4561 aagggactgg ctgctattgg gcgaagtgcc ggggcaggat ctcctgtcat ctcaccttgc
     4621 tcctgccgag aaagtatcca tcatggctga tgcaatgcgg cggctgcata cgcttgatcc
     4681 ggctacctgc ccattcgacc accaagcgaa acatcgcatc gagcgagcac gtactcggat
     4741 ggaagccggt cttgtcgatc aggatgatct ggacgaagag catcaggggc tcgcgccagc
     4801 cgaactgttc gccaggctca aggcgagcat gcccgacggc gaggatctcg tcgtgaccca
     4861 tggcgatgcc tgcttgccga atatcatggt ggaaaatggc cgcttttctg gattcatcga
     4921 ctgtggccgg ctgggtgtgg cggaccgcta tcaggacata gcgttggcta cccgtgatat
     4981 tgctgaagag cttggcggcg aatgggctga ccgcttcctc gtgctttacg gtatcgccgc
     5041 tcccgattcg cagcgcatcg ccttctatcg ccttcttgac gagttcttct gactgtcaga
     5101 ccaagtttac tcatatatac tttagattga tttaaaactt catttttaat ttaaaaggat
     5161 ctaggtgaag atcctttttg ataatctca
//
