LOCUS       sequence-425824-        4239 bp    DNA     circular SYN 15-APR-2025
DEFINITION  Preparation of EGFP mRNA translated through EMCV IRES..
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 4239)
  TITLE     Enhancing cap-independent translation of mRNA via in vitro 
            transcription priming
  JOURNAL   Unpublished
REFERENCE   2  (bases 1 to 4239)
  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            "Unpublished"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: pRSET-IRES-EGFP
FEATURES             Location/Qualifiers
     source          1..4239
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     288..307
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(526..630)
                     /gene="bla"
                     /label=AmpR promoter
     rep_origin      complement(656..1111)
                     /direction=LEFT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(793..814)
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     1005..1024
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     terminator      complement(1208..1255)
                     /label=T7 terminator
                     /note="transcription terminator for bacteriophage T7 RNA 
                     polymerase"
     primer_bind     1251..1269
                     /label=T7 Term
                     /note="T7 terminator, reverse primer"
     CDS             complement(join(1331..2044,2045..2047,2048..2050))
                     /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             complement(join(1334..2044,2045..2047,2048..2050))
                     /codon_start=1
                     /product="enhanced GFP"
                     /label=EGFP
                     /translation="MVSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTL
                     KFICTTGKLPVPWPTLVTTLTYGVQCFSRYPDHMKQHDFFKSAMPEGYVQERTIFFKDD
                     GNYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIK
                     VNFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSALSKDPNEKRDHMVLL
                     EFVTAAGITLGMDELYK"
     primer_bind     complement(1376..1397)
                     /label=EGFP-C
                     /note="EGFP, forward primer"
     primer_bind     1725..1744
                     /label=EXFP-R
                     /note="For distinguishing EGFP variants, reverse primer"
     primer_bind     1984..2005
                     /label=EGFP-N
                     /note="EGFP, reverse primer"
     regulatory      2047..2056
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     misc_feature    join(2071..2079,2080..2082,2083..2657)
                     /label=IRES2
                     /note="internal ribosome entry site (IRES) of the 
                     encephalomyocarditis virus (EMCV)"
     primer_bind     complement(2244..2263)
                     /label=IRES-F
                     /note="IRES internal ribosome entry site, forward primer"
     primer_bind     2473..2490
                     /label=IRES reverse
                     /note="IRES internal ribosome entry site, reverse primer. 
                     Also called pCDH-rev"
     CDS             complement(join(2717..2731,2732..2740))
                     /codon_start=1
                     /product="Xpress(TM) epitope tag, including an enterokinase
                     recognition and cleavage site"
                     /label=Xpress(TM) tag
                     /translation="DLYDDDDK"
     CDS             complement(2744..2776)
                     /codon_start=1
                     /product="leader peptide from bacteriophage T7 gene 10"
                     /label=T7 tag (gene 10 leader)
                     /note="promotes efficient translation in E. coli"
                     /translation="MASMTGGQQMG"
     CDS             complement(2780..2797)
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     RBS             2817..2839
                     /note="efficient ribosome binding site from bacteriophage 
                     T7 gene 10 (Olins and Rangwala, 1989)"
     primer_bind     complement(2870..2889)
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        complement(2871..2889)
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     primer_bind     complement(2974..2991)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(3145..3733)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(3225..3244)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(3904..4239,1..456,457..525))
                     /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"
ORIGIN
        1 ctcgtcgttt ggtatggctt cattcagctc cggttcccaa cgatcaaggc gagttacatg
       61 atcccccatg ttgtgcaaaa aagcggttag ctccttcggt cctccgatcg ttgtcagaag
      121 taagttggcc gcagtgttat cactcatggt tatggcagca ctgcataatt ctcttactgt
      181 catgccatcc gtaagatgct tttctgtgac tggtgagtac tcaaccaagt cattctgaga
      241 atagtgtatg cggcgaccga gttgctcttg cccggcgtca atacgggata ataccgcgcc
      301 acatagcaga actttaaaag tgctcatcat tggaaaacgt tcttcggggc gaaaactctc
      361 aaggatctta ccgctgttga gatccagttc gatgtaaccc actcgtgcac ccaactgatc
      421 ttcagcatct tttactttca ccagcgtttc tgggtgagca aaaacaggaa ggcaaaatgc
      481 cgcaaaaaag ggaataaggg cgacacggaa atgttgaata ctcatactct tcctttttca
      541 atattattga agcatttatc agggttattg tctcatgagc ggatacatat ttgaatgtat
      601 ttagaaaaat aaacaaatag gggttccgcg cacatttccc cgaaaagtgc cacctaaatt
      661 gtaagcgtta atattttgtt aaaattcgcg ttaaattttt gttaaatcag ctcatttttt
      721 aaccaatagg ccgaaatcgg caaaatccct tataaatcaa aagaatagac cgagataggg
      781 ttgagtgttg ttccagtttg gaacaagagt ccactattaa agaacgtgga ctccaacgtc
      841 aaagggcgaa aaaccgtcta tcagggcgat ggcccactac gtgaaccatc accctaatca
      901 agttttttgg ggtcgaggtg ccgtaaagca ctaaatcgga accctaaagg gagcccccga
      961 tttagagctt gacggggaaa gccggcgaac gtggcgagaa aggaagggaa gaaagcgaaa
     1021 ggagcgggcg ctagggcgct ggcaagtgta gcggtcacgc tgcgcgtaac caccacaccc
     1081 gccgcgctta atgcgccgct acagggcgcg tcccattcgc cattcaggct gcgcaactgt
     1141 tgggaagggc gatcggtgcg ggcctcttcg ctattacgcc agatccggat atagttcctc
     1201 ctttcagcaa aaaacccctc aagacccgtt tagaggcccc aaggggttat gctagttatt
     1261 gctcagcggt ggcagcagcc aactcagctt cctttcgggc tttgttagca gccggatcaa
     1321 gcttcgaatt ttacttgtac agctcgtcca tgccgagagt gatcccggcg gcggtcacga
     1381 actccagcag gaccatgtga tcgcgcttct cgttggggtc tttgctcagg gcggactggg
     1441 tgctcaggta gtggttgtcg ggcagcagca cggggccgtc gccgatgggg gtgttctgct
     1501 ggtagtggtc ggcgagctgc acgctgccgt cctcgatgtt gtggcggatc ttgaagttca
     1561 ccttgatgcc gttcttctgc ttgtcggcca tgatatagac gttgtggctg ttgtagttgt
     1621 actccagctt gtgccccagg atgttgccgt cctccttgaa gtcgatgccc ttcagctcga
     1681 tgcggttcac cagggtgtcg ccctcgaact tcacctcggc gcgggtcttg tagttgccgt
     1741 cgtccttgaa gaagatggtg cgctcctgga cgtagccttc gggcatggcg gacttgaaga
     1801 agtcgtgctg cttcatgtgg tcggggtagc ggctgaagca ctgcacgccg taggtcaggg
     1861 tggtcacgag ggtgggccag ggcacgggca gcttgccggt ggtgcagatg aacttcaggg
     1921 tcagcttgcc gtaggtggca tcgccctcgc cctcgccgga cacgctgaac ttgtggccgt
     1981 ttacgtcgcc gtccagctcg accaggatgg gcaccacccc ggtgaacagc tcctcgccct
     2041 tgctcaccat ggtggcgaat tctttgccaa ggttgtggcc atattatcat cgtgtttttc
     2101 aaaggaaaac cacgtccccg tggttcgggg ggcctagacg tttttttaac ctcgactaaa
     2161 cacatgtaaa gcatgtgcac cgaggcccca gatcagatcc catacaatgg ggtaccttct
     2221 gggcatcctt cagccccttg ttgaatacgc ttgaggagag ccatttgact ctttccacaa
     2281 ctatccaact cacaacgtgg cactggggtt gtgccgcctt tgcaggtgta tcttatacac
     2341 gtggcttttg gccgcagagg cacctgtcgc caggtggggg gttccgctgc ctgcaaaggg
     2401 tcgctacaga cgttgtttgt cttcaagaag cttccagagg aactgcttcc ttcacgacat
     2461 tcaacagacc ttgcattcct ttggcgagag gggaaagacc cctaggaatg ctcgtcaaga
     2521 agacagggcc aggtttccgg gccctcacat tgccaaaaga cggcaatatg gtggaaaata
     2581 acatatagac aaacgcacac cggccttatt ccaagcggct tcggccagta acgttagggg
     2641 ggggggaggg agaggggggg ggggcggaat tcgatatcaa gcttatcgag cggccgcatc
     2701 acaccttggc ctccagctta tcgtcatcgt cgtacagatc ccgacccatt tgctgtccac
     2761 cagtcatgct agccatacca tgatgatgat gatgatgaga accccgcata tgtatatctc
     2821 cttcttaaag ttaaacaaaa ttatttctag agggaaaccg ttgtggtctc cctatagtga
     2881 gtcgtattaa tttcgcggga tcgagatcct gcattaatga atcggccaac gcgcggggag
     2941 aggcggtttg cgtattgggc gctcttccgc ttcctcgctc actgactcgc tgcgctcggt
     3001 cgttcggctg cggcgagcgg tatcagctca ctcaaaggcg gtaatacggt tatccacaga
     3061 atcaggggat aacgcaggaa agaacatgtg agcaaaaggc cagcaaaagg ccaggaaccg
     3121 taaaaaggcc gcgttgctgg cgtttttcca taggctccgc ccccctgacg agcatcacaa
     3181 aaatcgacgc tcaagtcaga ggtggcgaaa cccgacagga ctataaagat accaggcgtt
     3241 tccccctgga agctccctcg tgcgctctcc tgttccgacc ctgccgctta ccggatacct
     3301 gtccgccttt ctcccttcgg gaagcgtggc gctttctcat agctcacgct gtaggtatct
     3361 cagttcggtg taggtcgttc gctccaagct gggctgtgtg cacgaacccc ccgttcagcc
     3421 cgaccgctgc gccttatccg gtaactatcg tcttgagtcc aacccggtaa gacacgactt
     3481 atcgccactg gcagcagcca ctggtaacag gattagcaga gcgaggtatg taggcggtgc
     3541 tacagagttc ttgaagtggt ggcctaacta cggctacact agaagaacag tatttggtat
     3601 ctgcgctctg ctgaagccag ttaccttcgg aaaaagagtt ggtagctctt gatccggcaa
     3661 acaaaccacc gctggtagcg gtggtttttt tgtttgcaag cagcagatta cgcgcagaaa
     3721 aaaaggatct caagaagatc ctttgatctt ttctacgggg tctgacgctc agtggaacga
     3781 aaactcacgt taagggattt tggtcatgag attatcaaaa aggatcttca cctagatcct
     3841 tttaaattaa aaatgaagtt ttaaatcaat ctaaagtata tatgagtaaa cttggtctga
     3901 cagttaccaa tgcttaatca gtgaggcacc tatctcagcg atctgtctat ttcgttcatc
     3961 catagttgcc tgactccccg tcgtgtagat aactacgata cgggagggct taccatctgg
     4021 ccccagtgct gcaatgatac cgcgagaccc acgctcaccg gctccagatt tatcagcaat
     4081 aaaccagcca gccggaaggg ccgagcgcag aagtggtcct gcaactttat ccgcctccat
     4141 ccagtctatt aattgttgcc gggaagctag agtaagtagt tcgccagtta atagtttgcg
     4201 caacgttgtt gccattgcta caggcatcgt ggtgtcacg
//
