LOCUS       sequence-366412-        3493 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  This plasmid can be used to prepare EGFP mRNA by In Vitro 
            Transcription.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 3493)
  AUTHORS   Perche F, Clemencon R, Schulze K, Ebensen T, Guzman CA, Pichon C
  TITLE     Neutral Lipopolyplexes for In Vivo Delivery of Conventional and 
            Replicative RNA Vaccine.
  JOURNAL   Mol Ther Nucleic Acids. 2019 Sep 6;17:767-775. doi: 
            10.1016/j.omtn.2019.07.014. Epub 2019 Jul 30.
  PUBMED    31446119
REFERENCE   2  (bases 1 to 3493)
  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: "Mol Ther 
            Nucleic Acids."; date: "2019-09-6"; volume: "17:767-775. doi"; 
            pages: " 10.1016/j.omtn.2019.07.014. Epub 2019 Jul 30"
COMMENT     Sequence Label: pGEM4Z-EGFP
FEATURES             Location/Qualifiers
     source          1..3493
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     complement(242..261)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     361..383
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     primer_bind     complement(421..439)
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     promoter        507..611
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(612..680,681..1472)
                     /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     complement(830..849)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      1643..2231
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     2132..2151
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     2385..2402
                     /label=L4440
                     /note="L4440 vector, forward primer"
     protein_bind    2519..2540
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     promoter        join(2555..2560,2561..2578,2579..2585)
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    2593..2609
                     /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)."
     primer_bind     2598..2620
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     primer_bind     2617..2633
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     2617..2633
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     2652..2671
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        2652..2670
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     regulatory      2719..2728
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     CDS             join(2725..2727,2728..2730,2731..3444)
                     /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(2725..2727,2728..2730,2731..3441)
                     /codon_start=1
                     /product="enhanced GFP"
                     /label=EGFP
                     /translation="MVSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTL
                     KFICTTGKLPVPWPTLVTTLTYGVQCFSRYPDHMKQHDFFKSAMPEGYVQERTIFFKDD
                     GNYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIK
                     VNFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSALSKDPNEKRDHMVLL
                     EFVTAAGITLGMDELYK"
     primer_bind     complement(2770..2791)
                     /label=EGFP-N
                     /note="EGFP, reverse primer"
     primer_bind     complement(3031..3050)
                     /label=EXFP-R
                     /note="For distinguishing EGFP variants, reverse primer"
     primer_bind     3378..3399
                     /label=EGFP-C
                     /note="EGFP, forward primer"
ORIGIN
        1 aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa
       61 aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa
      121 aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaactag tggcgcctga
      181 tgcggtattt tctccttacg catctgtgcg gtatttcaca ccgcatatgg tgcactctca
      241 gtacaatctg ctctgatgcc gcatagttaa gccagccccg acacccgcca acacccgctg
      301 acgcgccctg acgggcttgt ctgctcccgg catccgctta cagacaagct gtgaccgtct
      361 ccgggagctg catgtgtcag aggttttcac cgtcatcacc gaaacgcgcg agacgaaagg
      421 gcctcgtgat acgcctattt ttataggtta atgtcatgat aataatggtt tcttagacgt
      481 caggtggcac ttttcgggga aatgtgcgcg gaacccctat ttgtttattt ttctaaatac
      541 attcaaatat gtatccgctc atgagacaat aaccctgata aatgcttcaa taatattgaa
      601 aaaggaagag tatgagtatt caacatttcc gtgtcgccct tattcccttt tttgcggcat
      661 tttgccttcc tgtttttgct cacccagaaa cgctggtgaa agtaaaagat gctgaagatc
      721 agttgggtgc acgagtgggt tacatcgaac tggatctcaa cagcggtaag atccttgaga
      781 gttttcgccc cgaagaacgt tttccaatga tgagcacttt taaagttctg ctatgtggcg
      841 cggtattatc ccgtattgac gccgggcaag agcaactcgg tcgccgcata cactattctc
      901 agaatgactt ggttgagtac tcaccagtca cagaaaagca tcttacggat ggcatgacag
      961 taagagaatt atgcagtgct gccataacca tgagtgataa cactgcggcc aacttacttc
     1021 tgacaacgat cggaggaccg aaggagctaa ccgctttttt gcacaacatg ggggatcatg
     1081 taactcgcct tgatcgttgg gaaccggagc tgaatgaagc cataccaaac gacgagcgtg
     1141 acaccacgat gcctgtagca atggcaacaa cgttgcgcaa actattaact ggcgaactac
     1201 ttactctagc ttcccggcaa caattaatag actggatgga ggcggataaa gttgcaggac
     1261 cacttctgcg ctcggccctt ccggctggct ggtttattgc tgataaatct ggagccggtg
     1321 agcgtgggtc tcgcggtatc attgcagcac tggggccaga tggtaagccc tcccgtatcg
     1381 tagttatcta cacgacgggg agtcaggcaa ctatggatga acgaaataga cagatcgctg
     1441 agataggtgc ctcactgatt aagcattggt aactgtcaga ccaagtttac tcatatatac
     1501 tttagattga tttaaaactt catttttaat ttaaaaggat ctaggtgaag atcctttttg
     1561 ataatctcat gaccaaaatc ccttaacgtg agttttcgtt ccactgagcg tcagaccccg
     1621 tagaaaagat caaaggatct tcttgagatc ctttttttct gcgcgtaatc tgctgcttgc
     1681 aaacaaaaaa accaccgcta ccagcggtgg tttgtttgcc ggatcaagag ctaccaactc
     1741 tttttccgaa ggtaactggc ttcagcagag cgcagatacc aaatactgtt cttctagtgt
     1801 agccgtagtt aggccaccac ttcaagaact ctgtagcacc gcctacatac ctcgctctgc
     1861 taatcctgtt accagtggct gctgccagtg gcgataagtc gtgtcttacc gggttggact
     1921 caagacgata gttaccggat aaggcgcagc ggtcgggctg aacggggggt tcgtgcacac
     1981 agcccagctt ggagcgaacg acctacaccg aactgagata cctacagcgt gagctatgag
     2041 aaagcgccac gcttcccgaa gggagaaagg cggacaggta tccggtaagc ggcagggtcg
     2101 gaacaggaga gcgcacgagg gagcttccag ggggaaacgc ctggtatctt tatagtcctg
     2161 tcgggtttcg ccacctctga cttgagcgtc gatttttgtg atgctcgtca ggggggcgga
     2221 gcctatggaa aaacgccagc aacgcggcct ttttacggtt cctggccttt tgctggcctt
     2281 ttgctcacat gttctttcct gcgttatccc ctgattctgt ggataaccgt attaccgcct
     2341 ttgagtgagc tgataccgct cgccgcagcc gaacgaccga gcgcagcgag tcagtgagcg
     2401 aggaagcgga agagcgccca atacgcaaac cgcctctccc cgcgcgttgg ccgattcatt
     2461 aatgcagctg gcacgacagg tttcccgact ggaaagcggg cagtgagcgc aacgcaatta
     2521 atgtgagtta gctcactcat taggcacccc aggctttaca ctttatgctt ccggctcgta
     2581 tgttgtgtgg aattgtgagc ggataacaat ttcacacagg aaacagctat gaccatgatt
     2641 acgccaagct ctaatacgac tcactatagg gagacaagct tgcatgcctg caggtcgact
     2701 ctagaggatc caccggtcgc caccatggtg agcaagggcg aggagctgtt caccggggtg
     2761 gtgcccatcc tggtcgagct ggacggcgac gtaaacggcc acaagttcag cgtgtccggc
     2821 gagggcgagg gcgatgccac ctacggcaag ctgaccctga agttcatctg caccaccggc
     2881 aagctgcccg tgccctggcc caccctcgtg accaccctga cctacggcgt gcagtgcttc
     2941 agccgctacc ccgaccacat gaagcagcac gacttcttca agtccgccat gcccgaaggc
     3001 tacgtccagg agcgcaccat cttcttcaag gacgacggca actacaagac ccgcgccgag
     3061 gtgaagttcg agggcgacac cctggtgaac cgcatcgagc tgaagggcat cgacttcaag
     3121 gaggacggca acatcctggg gcacaagctg gagtacaact acaacagcca caacgtctat
     3181 atcatggccg acaagcagaa gaacggcatc aaggtgaact tcaagatccg ccacaacatc
     3241 gaggacggca gcgtgcagct cgccgaccac taccagcaga acacccccat cggcgacggc
     3301 cccgtgctgc tgcccgacaa ccactacctg agcacccagt ccgccctgag caaagacccc
     3361 aacgagaagc gcgatcacat ggtcctgctg gagttcgtga ccgccgccgg gatcactctc
     3421 ggcatggacg agctgtacaa gtaaagcggc cgcggatccc cgggtaccga gctcgaattc
     3481 ttaattaaaa aaa
//
