LOCUS       sequence-451679-        7246 bp    DNA     circular SYN 15-APR-2025
DEFINITION  In vitro transcription of cap-independently (synthetic IRES) 
            translated mRNA encoding EGFP..
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 7246)
  TITLE     Enhancing cap-independent translation of mRNA via in vitro 
            transcription priming
  JOURNAL   Unpublished
REFERENCE   2  (bases 1 to 7246)
  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            "Unpublished"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-iSyn-EGFP
FEATURES             Location/Qualifiers
     source          1..7246
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     454..473
                     /label=Kan-R
                     /note="Kanamycin resistance gene, reverse primer"
     primer_bind     538..557
                     /label=pENTR-R
                     /note="pENTR vectors, reverse primer"
     rep_origin      771..1359
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     1260..1279
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     enhancer        1425..1728
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
     intron          2031..3047
                     /label=chimeric intron
                     /note="chimera between introns from chicken beta-actin and 
                     rabbit beta-globin"
     primer_bind     2971..2993
                     /label=pCAGGS-5
                     /note="Chimeric intron in CAG promoter, forward primer"
     primer_bind     3055..3074
                     /label=pCAG-F
                     /note="Rabbit beta-globin intron, for pCAG plasmids, 
                     forward primer"
     primer_bind     3117..3136
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        3117..3135
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     CDS             join(4191..4193,4194..4196,4197..4910)
                     /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(4191..4193,4194..4196,4197..4907)
                     /codon_start=1
                     /product="enhanced GFP"
                     /label=EGFP
                     /translation="MVSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTL
                     KFICTTGKLPVPWPTLVTTLTYGVQCFSRYPDHMKQHDFFKSAMPEGYVQERTIFFKDD
                     GNYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIK
                     VNFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSALSKDPNEKRDHMVLL
                     EFVTAAGITLGMDELYK"
     primer_bind     complement(4236..4257)
                     /label=EGFP-N
                     /note="EGFP, reverse primer"
     primer_bind     complement(4497..4516)
                     /label=EXFP-R
                     /note="For distinguishing EGFP variants, reverse primer"
     primer_bind     4844..4865
                     /label=EGFP-C
                     /note="EGFP, forward primer"
     terminator      5307..5354
                     /label=T7 terminator
                     /note="transcription terminator for bacteriophage T7 RNA 
                     polymerase"
     polyA_signal    5368..5592
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     protein_bind    5635..5651
                     /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     5640..5662
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     primer_bind     5659..5675
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     5659..5675
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     polyA_signal    5724..5845
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(5735..5754)
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     primer_bind     5789..5808
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     CDS             complement(6005..6604)
                     /codon_start=1
                     /gene="pac from Streptomyces alboniger"
                     /product="puromycin N-acetyltransferase"
                     /label=PuroR
                     /note="confers resistance to puromycin"
                     /translation="MTEYKPTVRLATRDDVPRAVRTLAAAFADYPATRHTVDPDRHIER
                     VTELQELFLTRVGLDIGKVWVADDGAAVAVWTTPESVEAGAVFAEIGPRMAELSGSRLA
                     AQQQMEGLLAPHRPKEPAWFLATVGVSPDHQGKGLGSAVVLPGVEAAERAGVPAFLETS
                     APRNLPFYERLGFTVTADVEVPEGPRTWCMTRKPGA"
     primer_bind     complement(6088..6108)
                     /label=Puro-F
                     /note="Puromycin resistance gene, forward primer"
     primer_bind     6585..6604
                     /label=Puro-R
                     /note="Puromycin resistance gene, reverse primer. Also 
                     called puro-variant-R"
     promoter        complement(6615..6943)
                     /label=SV40 promoter
                     /note="SV40 enhancer and early promoter"
     rep_origin      6629..6764
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     primer_bind     complement(6683..6702)
                     /label=SV40pro-F
                     /note="SV40 promoter/origin, forward primer"
     CDS             complement(join(6977..7246,1..546))
                     /codon_start=1
                     /product="aminoglycoside phosphotransferase"
                     /label=KanR
                     /note="confers resistance to kanamycin in bacteria or G418 
                     (Geneticin(R)) in eukaryotes"
                     /translation="MSHIQRETSCSRPRLNSNMDADLYGYKWARDNVGQSGATIYRLYG
                     KPDAPELFLKHGKGSVANDVTDEMVRLNWLTEFMPLPTIKHFIRTPDDAWLLTTAIPGK
                     TAFQVLEEYPDSGENIVDALAVFLRRLHSIPVCNCPFNSDRVFRLAQAQSRMNNGLVDA
                     SDFDDERNGWPVEQVWKEMHKLLPFSPDSVVTHGDFSLDNLIFDEGKLIGCIDVGRVGI
                     ADRYQDLAILWNCLGEFSPSLQKRLFQKYGIDNPDMNKLQFHLMLDEFF"
     CDS             complement(join(6977..7246,1..546))
                     /codon_start=1
                     /gene="aph(3')-Ia"
                     /product="aminoglycoside phosphotransferase"
                     /label=KanR
                     /note="confers resistance to kanamycin in bacteria or G418 
                     (Geneticin(R)) in eukaryotes"
                     /translation="MSHIQRETSCSRPRLNSNMDADLYGYKWARDNVGQSGATIYRLYG
                     KPDAPELFLKHGKGSVANDVTDEMVRLNWLTEFMPLPTIKHFIRTPDDAWLLTTAIPGK
                     TAFQVLEEYPDSGENIVDALAVFLRRLHSIPVCNCPFNSDRVFRLAQAQSRMNNGLVDA
                     SDFDDERNGWPVEQVWKEMHKLLPFSPDSVVTHGDFSLDNLIFDEGKLIGCIDVGRVGI
                     ADRYQDLAILWNCLGEFSPSLQKRLFQKYGIDNPDMNKLQFHLMLDEFF"
ORIGIN
        1 catttctttc cagacttgtt caacaggcca gccattacgc tcgtcatcaa aatcactcgc
       61 atcaaccaaa ccgttattca ttcgtgattg cgcctgagcg aggcgaaata cgcgatcgct
      121 gttaaaagga caattacaaa caggaatcga atgcaaccgg cgcaggaaca ctgccagcgc
      181 atcaacaata ttttcacctg aatcaggata ttcttctaat acctggaatg ctgttttccc
      241 ggggatcgca gtggtgagta accatgcatc atcaggagta cggataaaat gcttgatggt
      301 cggaagaggc ataaattccg tcagccagtt tagtctgacc atctcatctg taacatcatt
      361 ggcaacgcta cctttgccat gtttcagaaa caactctggc gcatcgggct tcccatacaa
      421 tcgatagatt gtcgcacctg attgcccgac attatcgcga gcccatttat acccatataa
      481 atcagcatcc atgttggaat ttaatcgcgg cctggagcaa gacgtttccc gttgaatatg
      541 gctcataaca ccccttgtat tactgtttat gtaagcagac agttttattg ttcatgatga
      601 tatattttta tcttgtgcaa tgtaacatca gagattttga gacacaacgt ggctttgttg
      661 aataaatcga acttttgctg agttgaagga tcagtcatga ccaaaatccc ttaacgtgag
      721 ttttcgttcc actgagcgtc agaccccgta gaaaagatca aaggatcttc ttgagatcct
      781 ttttttctgc gcgtaatctg ctgcttgcaa acaaaaaaac caccgctacc agcggtggtt
      841 tgtttgccgg atcaagagct accaactctt tttccgaagg taactggctt cagcagagcg
      901 cagataccaa atactgttct tctagtgtag ccgtagttag gccaccactt caagaactct
      961 gtagcaccgc ctacatacct cgctctgcta atcctgttac cagtggctgc tgccagtggc
     1021 gataagtcgt gtcttaccgg gttggactca agacgatagt taccggataa ggcgcagcgg
     1081 tcgggctgaa cggggggttc gtgcacacag cccagcttgg agcgaacgac ctacaccgaa
     1141 ctgagatacc tacagcgtga gctatgagaa agcgccacgc ttcccgaagg gagaaaggcg
     1201 gacaggtatc cggtaagcgg cagggtcgga acaggagagc gcacgaggga gcttccaggg
     1261 ggaaacgcct ggtatcttta tagtcctgtc gggtttcgcc acctctgact tgagcgtcga
     1321 tttttgtgat gctcgtcagg ggggcggagc ctatggaaac ctgcaggtta ttaatagtaa
     1381 tcaattacgg ggtcattagt tcatagccca tatatggagt tccgcgttac ataacttacg
     1441 gtaaatggcc cgcctggctg accgcccaac gacccccgcc cattgacgtc aataatgacg
     1501 tatgttccca tagtaacgcc aatagggact ttccattgac gtcaatgggt ggagtattta
     1561 cggtaaactg cccacttggc agtacatcaa gtgtatcata tgccaagtac gccccctatt
     1621 gacgtcaatg acggtaaatg gcccgcctgg cattatgccc agtacatgac cttatgggac
     1681 tttcctactt ggcagtacat ctacgtatta gtcatcgcta ttaccatggt cgaggtgagc
     1741 cccacgttct gcttcactct ccccatctcc cccccctccc cacccccaat tttgtattta
     1801 tttatttttt aattattttg tgcagcgatg ggggcggggg gggggggggg gggggcgccg
     1861 gggggggcgg ggcggggggg gggggggggg gggggggggg gggggggcgg ggcggggcga
     1921 ggcggagagg tgcggcggca gccaatcaga gcggcgcgct ccgaaagttt ccttttatgg
     1981 cgaggcggcg gcggcggcgg ccctataaaa agcgaagcgc gcggcgggcg ggagtcgctg
     2041 cgcgctgcct tcgccccgtg ccccgctccg ccgccgcctc gcgccgcccg ccccggctct
     2101 gactgaccgc gttactccca caggtgagcg ggcgggacgg cccttctcct ccgggctgta
     2161 attagcgctt ggtttaatga cggcttgttt cttttctgtg gctgcgtgaa agccttgagg
     2221 ggctccggga gggccctttg tgcgggggga gcggctcggg gggtgcgtgc gtgtgtgtgt
     2281 gcgtggggag cgccgcgtgc ggctccgcgc tgcccggcgg ctgtgagcgc tgcgggcgcg
     2341 gcgcggggct ttgtgcgctc cgcagtgtgc gcgaggggag cgcggccggg ggcggtgccc
     2401 cgcggtgcgg ggggggctgc gaggggaaca aaggctgcgt gcggggtgtg tgcgtggggg
     2461 ggtgagcagg gggtgtgggc gcgtcggtcg ggctgcaacc ccccctgcac ccccctcccc
     2521 gagttgctga gcacggcccg gcttcgggtg cggggctccg tacggggcgt ggcgcggggc
     2581 tcgccgtgcc gggcgggggg tggcggcagg tgggggtgcc gggcggggcg gggccgcctc
     2641 gggccgggga gggctcgggg gaggggcgcg gcggcccccg gagcgccggc ggctgtcgag
     2701 gcgcggcgag ccgcagccat tgccttttat ggtaatcgtg cgagagggcg cagggacttc
     2761 ctttgtccca aatctgtgcg gagccgaaat ctgggaggcg ccgccgcacc ccctctagcg
     2821 ggcgcggggc gaagcggtgc ggcgccggca ggaaggaaat gggcggggag ggccttcgtg
     2881 cgtcgccgcg ccgccgtccc cttctccctc tccagcctcg gggctgtccg cggggggacg
     2941 gctgccttcg ggggggacgg ggcagggcgg ggttcggctt ctggcgtgtg accggcggct
     3001 ctagagcctc tgctaaccat gttcatgcct tcttcttttt cctacagctc ctgggcaacg
     3061 tgctggttat tgtgctgtct catcattttg gcaaagcggc cgcggccagt gaattgtaat
     3121 acgactcact ataggggtta acaaaaaaaa aaaaaaaaaa aaaaaatcac cgaccgatct
     3181 atctgaaccg gtgggaattc tagagaaaat ttcgtctgga ttagttactt atcgtgtaaa
     3241 atctgataaa tggaattggt tctacataaa tgcctaacga ctatcccttt ggggagtagg
     3301 gtcaagtgac tcgaaacgat agacaacttg ctttaacaag ttggagatat agtctgctct
     3361 gcatggtgac atgcagctgg atataattcc ggggtaagat taacgacctt atctgaacat
     3421 aacgctaccg tttaatattg cgtcatattc ggcgaccatt tgtgtggtaa aaaaaaaaaa
     3481 ccaaaaaaaa aaaacaaaaa aaaaaaataa ttgactaaga tatcttaaaa cagcggatgg
     3541 gtaccccacc atccgaccca ctgggtgtag tactctggta cttcgtacct ttgtacgcct
     3601 gttcttccca ttgtaccctt cctgaacttc caacccaagt aacgttagaa gctcaacatt
     3661 tagtacaaca ggaagcacca catccagtgg tgtttagtac aagcacttct gtttccccgg
     3721 agcgaggtat aggctgtacc cactgccaaa aacctttaac cgttatccgc caaccaacta
     3781 cgtaaaagct agtagtatta tgtttttaac taggcgttcg atcaggtgga tttcccctcc
     3841 actagtttgg tcgatgaggc taggaattcc ccacgggtga ccgtgtccta gcctgcgtgg
     3901 cggccaaccc agcccactca ctatttgttt tcgcgcccag ttgcaaaaag tgtcggggct
     3961 gggacgcctt tttatagaca tggtgtgaag actcgcatgt gcttggttgt gattcctccg
     4021 gcccctgaat gcggctaacc ttaaccctgg agccttgtgt cacaaaccag tgatgataag
     4081 gtcgtaatga gcaattccgg gacgggaccg actactttgg gtgtccgtgt ttcttatttt
     4141 tcttattatt gtcttatggt cacagcatat atataacata tactgtgatc atggtgagca
     4201 agggcgagga gctgttcacc ggggtggtgc ccatcctggt cgagctggac ggcgacgtaa
     4261 acggccacaa gttcagcgtg tccggcgagg gcgagggcga tgccacctac ggcaagctga
     4321 ccctgaagtt catctgcacc accggcaagc tgcccgtgcc ctggcccacc ctcgtgacca
     4381 ccctgaccta cggcgtgcag tgcttcagcc gctaccccga ccacatgaag cagcacgact
     4441 tcttcaagtc cgccatgccc gaaggctacg tccaggagcg caccatcttc ttcaaggacg
     4501 acggcaacta caagacccgc gccgaggtga agttcgaggg cgacaccctg gtgaaccgca
     4561 tcgagctgaa gggcatcgac ttcaaggagg acggcaacat cctggggcac aagctggagt
     4621 acaactacaa cagccacaac gtctatatca tggccgacaa gcagaagaac ggcatcaagg
     4681 tgaacttcaa gatccgccac aacatcgagg acggcagcgt gcagctcgcc gaccactacc
     4741 agcagaacac ccccatcggc gacggccccg tgctgctgcc cgacaaccac tacctgagca
     4801 cccagtccgc cctgagcaaa gaccccaacg agaagcgcga tcacatggtc ctgctggagt
     4861 tcgtgaccgc cgccgggatc actctcggca tggacgagct gtacaagtaa taacaattgg
     4921 ctggagcctc ggtggccatg cttcttgccc cttgggcctc cccccagccc ctcctcccct
     4981 tcctgcaccc gtacccccgt ggtctttgaa taaagtctga accacacaaa tggtcgccga
     5041 ctcagtagat gttttcttgg gttaattgag gcctgagtat aaggtgactt atacttgtaa
     5101 tctatctaaa cggggaacct ctctagtaga caatcccgtg ctaaattgta ggactgccct
     5161 ttaataaata cttctatatt taaagaggta tttatgaaaa gcggaattta tcagattaaa
     5221 aatactttct ctagagtcga cctgcagacg cgttcagata gatcggtcgg tgaaaaaaaa
     5281 aaaaaaaaaa aaaaaaaagc ggccgcctag cataacccct tggggcctct aaacgggtct
     5341 tgaggggttt tttggctagc ggccggcctg tgccttctag ttgccagcca tctgttgttt
     5401 gcccctcccc cgtgccttcc ttgaccctgg aaggtgccac tcccactgtc ctttcctaat
     5461 aaaatgagga aattgcatcg cattgtctga gtaggtgtca ttctattctg gggggtgggg
     5521 tggggcagga cagcaagggg gaggattggg aagacaatag caggcatgct ggggatgcgg
     5581 tgggctctat ggcggcgcgc cactttatgc ttccggctcg tatgttgtgt ggaattgtga
     5641 gcggataaca atttcacaca ggaaacagct atgaccatga ttacgccaag ctctagctag
     5701 aggtcgacgg tatacagaca tgataagata cattgatgag tttggacaaa ccacaactag
     5761 aatgcagtga aaaaaatgct ttatttgtga aatttgtgat gctattgctt tatttgtaac
     5821 cattataagc tgcaataaac aagttggggt gggcgaagaa ctccagcatg agatccccgc
     5881 gctggaggat catccagccg gcgtcccgga aaacgattcc gaagcccaac ctttcataga
     5941 aggcggcggt ggaatcgaaa tctcgtgatg gcaggttggg cgtcgcttgg tcggtcattt
     6001 cgaatcaggc accgggcttg cgggtcatgc accaggtgcg cggtccttcg ggcacctcga
     6061 cgtcggcggt gacggtgaag ccgagccgct cgtagaaggg gaggttgcgg ggcgcggagg
     6121 tttccaggaa ggcgggcacc ccggcgcgct cggccgcctc cactccgggg agcacgacgg
     6181 cgctgcccag acccttgccc tggtggtcgg gcgacacgcc gacggtggcc aggaaccacg
     6241 cgggctcctt gggccggtgc ggcgccagga ggccttccat ctgttgctgc gcggccagcc
     6301 gggaaccgct caactcggcc atgcgcgggc cgatctcggc gaacaccgcc cccgcttcga
     6361 cgctctccgg cgtggtccag accgccaccg cggcgccgtc gtccgcgacc cacaccttgc
     6421 cgatgtcgag cccgacgcgc gtgaggaaga gttcttgcag ctcggtgacc cgctcgatgt
     6481 ggcggtccgg atcgacggtg tggcgcgtgg cggggtagtc ggcgaacgcg gcggcgaggg
     6541 tgcgtacggc cctggggacg tcgtcgcggg tggcgaggcg caccgtgggc ttgtactcgg
     6601 tcatgcggga gctttttgca aaagcctagg cctccaaaaa agcctcctca ctacttctgg
     6661 aatagctcag aggcagaggc ggcctcggcc tctgcataaa taaaaaaaat tagtcagcca
     6721 tggggcggag aatgggcgga actgggcgga gttaggggcg ggatgggcgg agttaggggc
     6781 gggactatgg ttgctgacta attgagatgc atgctttgca tacttctgcc tgctggggag
     6841 cctggggact ttccacacct ggttgctgac taattgagat gcatgctttg catacttctg
     6901 cctgctgggg agcctgggga ctttccacac cctaactgac acagagctcg tgttacaacc
     6961 aattaaccaa ttctgattag aaaaactcat cgagcatcaa atgaaactgc aatttattca
     7021 tatcaggatt atcaatacca tatttttgaa aaagccgttt ctgtaatgaa ggagaaaact
     7081 caccgaggca gttccatagg atggcaagat cctggtatcg gtctgcgatt ccgactcgtc
     7141 caacatcaat acaacctatt aatttcccct cgtcaaaaat aaggttatca agtgagaaat
     7201 caccatgagt gacgactgaa tccggtgaga atggcaaaag cttatg
//
