LOCUS       sequence-323957-        5646 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Expresses mEGFP (monomeric EGFP with the A206K mutation) under the 
            control of CAG promoter, codon-optimized for Homo sapiens, basic 
            vector, multiclonig site at C terminal.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 5646)
  AUTHORS   Tanida I, Furuta Y, Yamaguchi J, Kakuta S, Oliva Trejo JA, Uchiyama 
            Y
  TITLE     Two-color in-resin CLEM of Epon-embedded cells using osmium 
            resistant green and red fluorescent proteins.
  JOURNAL   Sci Rep. 2020 Dec 14;10(1):21871. doi: 10.1038/s41598-020-78879-x.
  PUBMED    33318540
REFERENCE   2  (bases 1 to 5646)
  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: "Sci Rep."; 
            date: "2020-12-14"; volume: "10(1):21871. doi"; pages: " 
            10.1038/s41598-020-78879-x"
COMMENT     Sequence Label: pCAG-mEGFP-HO-G
FEATURES             Location/Qualifiers
     source          1..5646
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     enhancer        10..389
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
     promoter        391..665
                     /label=chicken beta-actin promoter
     intron          666..1682
                     /label=chimeric intron
                     /note="chimera between introns from chicken beta-actin and 
                     rabbit beta-globin"
     primer_bind     1606..1628
                     /label=pCAGGS-5
                     /note="Chimeric intron in CAG promoter, forward primer"
     primer_bind     1690..1709
                     /label=pCAG-F
                     /note="Rabbit beta-globin intron, for pCAG plasmids, 
                     forward primer"
     regulatory      1737..1746
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     CDS             join(1743..1745,1746..1748,1749..2459)
                     /codon_start=1
                     /product="enhanced GFP with monomerizing A206K mutation 
                     (Zacharias et al., 2002)"
                     /label=mEGFP
                     /note="mammalian codon-optimized"
                     /translation="MVSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTL
                     KFICTTGKLPVPWPTLVTTLTYGVQCFSRYPDHMKQHDFFKSAMPEGYVQERTIFFKDD
                     GNYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIK
                     VNFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSKLSKDPNEKRDHMVLL
                     EFVTAAGITLGMDELYK"
     primer_bind     complement(2049..2068)
                     /label=EXFP-R
                     /note="For distinguishing EGFP variants, reverse primer"
     misc_feature    2517..2573
                     /label=MCS
                     /note="multiple cloning site"
     primer_bind     complement(2673..2692)
                     /label=Bglob-pA-R
                     /note="Rabbit beta-globin polyA region, reverse primer"
     polyA_signal    2738..2793
                     /label=beta-globin poly(A) signal
                     /note="rabbit beta-globin polyadenylation signal (Gil and 
                     Proudfoot, 1987)"
     primer_bind     complement(2792..2811)
                     /label=rbglobpA-R
                     /note="Rabbit beta-globin polyA, reverse primer. Also 
                     called rb-glob-pA-term-R"
     primer_bind     complement(3154..3170)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(3154..3170)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(3167..3189)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    3178..3194
                     /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)."
     promoter        complement(join(3202..3208,3209..3226,3227..3232))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    3247..3268
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     promoter        3327..3522
                     /label=SV40 promoter
                     /note="SV40 early promoter"
     rep_origin      3373..3508
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     polyA_signal    3528..3662
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(3565..3584)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     3619..3638
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     primer_bind     complement(3730..3747)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(3901..4489)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(3981..4000)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(4660..5451,5452..5520))
                     /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     5283..5302
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(5521..5625)
                     /gene="bla"
                     /label=AmpR promoter
ORIGIN
        1 acctgggtcg acattgatta ttgactagtt attaatagta atcaattacg gggtcattag
       61 ttcatagccc atatatggag ttccgcgtta cataacttac ggtaaatggc ccgcctggct
      121 gaccgcccaa cgacccccgc ccattgacgt caataatgac gtatgttccc atagtaacgc
      181 caatagggac tttccattga cgtcaatggg tggagtattt acggtaaact gcccacttgg
      241 cagtacatca agtgtatcat atgccaagta cgccccctat tgacgtcaat gacggtaaat
      301 ggcccgcctg gcattatgcc cagtacatga ccttatggga ctttcctact tggcagtaca
      361 tctacgtatt agtcatcgct attaccatgg tcgaggtgag ccccacgttc tgcttcactc
      421 tccccatctc ccccccctcc ccacccccaa ttttgtattt atttattttt taattatttt
      481 gtgcagcgat gggggcgggg gggggggggc gcgcgccagg cggggcgggg cggggcgagg
      541 ggcggggcgg ggcgaggcgg agaggtgcgg cggcagccaa tcagagcggc gcgctccgaa
      601 agtttccttt tatggcgagg cggcggcggc ggcggcccta taaaaagcga agcgcgcggc
      661 gggcgggagt cgctgcgcgc tgccttcgcc ccgtgccccg ctccgccgcc gcctcgcgcc
      721 gcccgccccg gctctgactg accgcgttac tcccacaggt gagcgggcgg gacggccctt
      781 ctcctccggg ctgtaattag cgcttggttt aatgacggct tgtttctttt ctgtggctgc
      841 gtgaaagcct tgaggggctc cgggagggcc ctttgtgcgg ggggagcggc tcggggggtg
      901 cgtgcgtgtg tgtgtgcgtg gggagcgccg cgtgcggctc cgcgctgccc ggcggctgtg
      961 agcgctgcgg gcgcggcgcg gggctttgtg cgctccgcag tgtgcgcgag gggagcgcgg
     1021 ccgggggcgg tgccccgcgg tgcggggggg gctgcgaggg gaacaaaggc tgcgtgcggg
     1081 gtgtgtgcgt gggggggtga gcagggggtg tgggcgcgtc ggtcgggctg caaccccccc
     1141 tgcacccccc tccccgagtt gctgagcacg gcccggcttc gggtgcgggg ctccgtacgg
     1201 ggcgtggcgc ggggctcgcc gtgccgggcg gggggtggcg gcaggtgggg gtgccgggcg
     1261 gggcggggcc gcctcgggcc ggggagggct cgggggaggg gcgcggcggc ccccggagcg
     1321 ccggcggctg tcgaggcgcg gcgagccgca gccattgcct tttatggtaa tcgtgcgaga
     1381 gggcgcaggg acttcctttg tcccaaatct gtgcggagcc gaaatctggg aggcgccgcc
     1441 gcaccccctc tagcgggcgc ggggcgaagc ggtgcggcgc cggcaggaag gaaatgggcg
     1501 gggagggcct tcgtgcgtcg ccgcgccgcc gtccccttct ccctctccag cctcggggct
     1561 gtccgcgggg ggacggctgc cttcgggggg gacggggcag ggcggggttc ggcttctggc
     1621 gtgtgaccgg cggctctaga gcctctgcta accatgttca tgccttcttc tttttcctac
     1681 agctcctggg caacgtgctg gttattgtgc tgtctcatca ttttggcaaa gctagcgcca
     1741 ccatggtgag caagggcgag gagctgttca ccggcgtggt gcccatcctg gtggagctgg
     1801 acggcgacgt gaacggccac aagttcagcg tgagcggcga gggcgagggc gacgcgacct
     1861 acggcaagct gaccctgaag ttcatctgca ccaccggcaa gctgcccgtg ccctggccca
     1921 ccctggtgac caccctgacc tacggcgtgc agtgcttcag ccgctacccc gaccacatga
     1981 agcagcacga cttcttcaag agcgccatgc ccgagggcta cgtgcaggag cgcaccatct
     2041 tcttcaagga cgacggcaac tacaagaccc gcgccgaggt gaagttcgag ggcgacaccc
     2101 tggtgaaccg catcgagctg aagggcatcg acttcaagga ggacggcaac atcctgggcc
     2161 acaagctgga gtacaactac aacagccaca acgtgtacat catggccgac aagcagaaga
     2221 acggcatcaa ggtgaacttc aagatccgcc acaacatcga ggacggcagc gtgcagctgg
     2281 ccgaccacta ccagcagaac acccccatcg gcgacggccc cgtgctgctg cccgacaacc
     2341 actacctgag cacccagagc aagctgagca aggaccccaa cgagaagcgc gaccacatgg
     2401 tgctgctgga gttcgtgacc gccgccggca tcaccctggg catggacgag ctgtacaagt
     2461 ccggactcac cggtggaggc ggttcaggcg gaggtggctc tggcggtggc ggatcgagat
     2521 ctcgagctca agcttcgaat tctgcagtcg acggtaccgc gggcccggga tccaccggat
     2581 ctagataact gatcaactcc tcaggtgcag gctgcctatc agaaggtggt ggctggtgtg
     2641 gccaatgccc tggctcacaa ataccactga gatctttttc cctctgccaa aaattatggg
     2701 gacatcatga agccccttga gcatctgact tctggctaat aaaggaaatt tattttcatt
     2761 gcaatagtgt gttggaattt tttgtgtctc tcactcggaa ggacatatgg gagggcaaat
     2821 catttaaaac atcagaatga gtatttggtt tagagtttgg caacatatgc ccatatgctg
     2881 gctgccatga acaaaggttg gctataaaga ggtcatcagt atatgaaaca gccccctgct
     2941 gtccattcct tattccatag aaaagccttg acttgaggtt agattttttt tatattttgt
     3001 tttgtgttat ttttttcttt aacatcccta aaattttcct tacatgtttt actagccaga
     3061 tttttcctcc tctcctgact actcccagtc atagctgtcc ctcttctctt atggagatcc
     3121 ctcgacctgc agcccaagct tggcgtaatc atggtcatag ctgtttcctg tgtgaaattg
     3181 ttatccgctc acaattccac acaacatacg agccggaagc ataaagtgta aagcctgggg
     3241 tgcctaatga gtgagctaac tcacattaat tgcgttgcgc tcactgcccg ctttccagtc
     3301 gggaaacctg tcgtgccagc ggatccgcat ctcaattagt cagcaaccat agtcccgccc
     3361 ctaactccgc ccatcccgcc cctaactccg cccagttccg cccattctcc gccccatggc
     3421 tgactaattt tttttattta tgcagaggcc gaggccgcct cggcctctga gctattccag
     3481 aagtagtgag gaggcttttt tggaggccta ggcttttgca aaaagctaac ttgtttattg
     3541 cagcttataa tggttacaaa taaagcaata gcatcacaaa tttcacaaat aaagcatttt
     3601 tttcactgca ttctagttgt ggtttgtcca aactcatcaa tgtatcttat catgtctgga
     3661 tccgctgcat taatgaatcg gccaacgcgc ggggagaggc ggtttgcgta ttgggcgctc
     3721 ttccgcttcc tcgctcactg actcgctgcg ctcggtcgtt cggctgcggc gagcggtatc
     3781 agctcactca aaggcggtaa tacggttatc cacagaatca ggggataacg caggaaagaa
     3841 catgtgagca aaaggccagc aaaaggccag gaaccgtaaa aaggccgcgt tgctggcgtt
     3901 tttccatagg ctccgccccc ctgacgagca tcacaaaaat cgacgctcaa gtcagaggtg
     3961 gcgaaacccg acaggactat aaagatacca ggcgtttccc cctggaagct ccctcgtgcg
     4021 ctctcctgtt ccgaccctgc cgcttaccgg atacctgtcc gcctttctcc cttcgggaag
     4081 cgtggcgctt tctcatagct cacgctgtag gtatctcagt tcggtgtagg tcgttcgctc
     4141 caagctgggc tgtgtgcacg aaccccccgt tcagcccgac cgctgcgcct tatccggtaa
     4201 ctatcgtctt gagtccaacc cggtaagaca cgacttatcg ccactggcag cagccactgg
     4261 taacaggatt agcagagcga ggtatgtagg cggtgctaca gagttcttga agtggtggcc
     4321 taactacggc tacactagaa gaacagtatt tggtatctgc gctctgctga agccagttac
     4381 cttcggaaaa agagttggta gctcttgatc cggcaaacaa accaccgctg gtagcggtgg
     4441 tttttttgtt tgcaagcagc agattacgcg cagaaaaaaa ggatctcaag aagatccttt
     4501 gatcttttct acggggtctg acgctcagtg gaacgaaaac tcacgttaag ggattttggt
     4561 catgagatta tcaaaaagga tcttcaccta gatcctttta aattaaaaat gaagttttaa
     4621 atcaatctaa agtatatatg agtaaacttg gtctgacagt taccaatgct taatcagtga
     4681 ggcacctatc tcagcgatct gtctatttcg ttcatccata gttgcctgac tccccgtcgt
     4741 gtagataact acgatacggg agggcttacc atctggcccc agtgctgcaa tgataccgcg
     4801 agacccacgc tcaccggctc cagatttatc agcaataaac cagccagccg gaagggccga
     4861 gcgcagaagt ggtcctgcaa ctttatccgc ctccatccag tctattaatt gttgccggga
     4921 agctagagta agtagttcgc cagttaatag tttgcgcaac gttgttgcca ttgctacagg
     4981 catcgtggtg tcacgctcgt cgtttggtat ggcttcattc agctccggtt cccaacgatc
     5041 aaggcgagtt acatgatccc ccatgttgtg caaaaaagcg gttagctcct tcggtcctcc
     5101 gatcgttgtc agaagtaagt tggccgcagt gttatcactc atggttatgg cagcactgca
     5161 taattctctt actgtcatgc catccgtaag atgcttttct gtgactggtg agtactcaac
     5221 caagtcattc tgagaatagt gtatgcggcg accgagttgc tcttgcccgg cgtcaatacg
     5281 ggataatacc gcgccacata gcagaacttt aaaagtgctc atcattggaa aacgttcttc
     5341 ggggcgaaaa ctctcaagga tcttaccgct gttgagatcc agttcgatgt aacccactcg
     5401 tgcacccaac tgatcttcag catcttttac tttcaccagc gtttctgggt gagcaaaaac
     5461 aggaaggcaa aatgccgcaa aaaagggaat aagggcgaca cggaaatgtt gaatactcat
     5521 actcttcctt tttcaatatt attgaagcat ttatcagggt tattgtctca tgagcggata
     5581 catatttgaa tgtatttaga aaaataaaca aataggggtt ccgcgcacat ttccccgaaa
     5641 agtgcc
//
