LOCUS       sequence-323955-        5649 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Expresses mWasabi under the control of CAG promoter, basic vector, 
            multiclonig site at C terminal.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 5649)
  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 5649)
  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-mWasabi-G
FEATURES             Location/Qualifiers
     source          1..5649
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     enhancer        35..414
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
     promoter        416..693
                     /label=chicken beta-actin promoter
     intron          694..1710
                     /label=chimeric intron
                     /note="chimera between introns from chicken beta-actin and 
                     rabbit beta-globin"
     primer_bind     1634..1656
                     /label=pCAGGS-5
                     /note="Chimeric intron in CAG promoter, forward primer"
     primer_bind     1718..1737
                     /label=pCAG-F
                     /note="Rabbit beta-globin intron, for pCAG plasmids, 
                     forward primer"
     regulatory      1774..1783
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     CDS             1780..2487
                     /codon_start=1
                     /product="green variant of teal fluorescent protein mTFP1 
                     (Ai et al., 2008)"
                     /label=mWasabi
                     /translation="MVSKGEETTMGVIKPDMKIKLKMEGNVNGHAFVIEGEGEGKPYDG
                     TNTINLEVKEGAPLPFSYDILTTAFSYGNRAFTKYPDDIPNYFKQSFPEGYSWERTMTF
                     EDKGIVKVKSDISMEEDSFIYEIHLKGENFPPNGPVMQKETTGWDASTERMYVRDGVLK
                     GDVKMKLLLEGGGHHRVDFKTIYRAKKAVKLPDYHFVDHRIEILNHDKDYNKVTVYEIA
                     VARNSTDGMDELYK"
     misc_feature    2545..2601
                     /label=MCS
                     /note="multiple cloning site"
     primer_bind     complement(2701..2720)
                     /label=Bglob-pA-R
                     /note="Rabbit beta-globin polyA region, reverse primer"
     polyA_signal    2766..2821
                     /label=beta-globin poly(A) signal
                     /note="rabbit beta-globin polyadenylation signal (Gil and 
                     Proudfoot, 1987)"
     primer_bind     complement(2820..2839)
                     /label=rbglobpA-R
                     /note="Rabbit beta-globin polyA, reverse primer. Also 
                     called rb-glob-pA-term-R"
     primer_bind     complement(3182..3198)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(3182..3198)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(3195..3217)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    3206..3222
                     /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(3230..3236,3237..3254,3255..3260))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    3275..3296
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     promoter        3355..3550
                     /label=SV40 promoter
                     /note="SV40 early promoter"
     rep_origin      3401..3536
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     polyA_signal    3556..3690
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(3593..3612)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     3647..3666
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     primer_bind     complement(3758..3775)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(3929..4517)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(4009..4028)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(4688..5479,5480..5548))
                     /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     5311..5330
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(join(5549..5649,1..4))
                     /gene="bla"
                     /label=AmpR promoter
ORIGIN
        1 cgcgcacatt tccccgaaaa gtgccacctg ggtcgacatt gattattgac tagttattaa
       61 tagtaatcaa ttacggggtc attagttcat agcccatata tggagttccg cgttacataa
      121 cttacggtaa atggcccgcc tggctgaccg cccaacgacc cccgcccatt gacgtcaata
      181 atgacgtatg ttcccatagt aacgccaata gggactttcc attgacgtca atgggtggag
      241 tatttacggt aaactgccca cttggcagta catcaagtgt atcatatgcc aagtacgccc
      301 cctattgacg tcaatgacgg taaatggccc gcctggcatt atgcccagta catgacctta
      361 tgggactttc ctacttggca gtacatctac gtattagtca tcgctattac catggtcgag
      421 gtgagcccca cgttctgctt cactctcccc atctcccccc cctccccacc cccaattttg
      481 tatttattta ttttttaatt attttgtgca gcgatggggg cggggggggg gggggggcgc
      541 gcgccaggcg gggcggggcg gggcgagggg cggggcgggg cgaggcggag aggtgcggcg
      601 gcagccaatc agagcggcgc gctccgaaag tttcctttta tggcgaggcg gcggcggcgg
      661 cggccctata aaaagcgaag cgcgcggcgg gcgggagtcg ctgcgcgctg ccttcgcccc
      721 gtgccccgct ccgccgccgc ctcgcgccgc ccgccccggc tctgactgac cgcgttactc
      781 ccacaggtga gcgggcggga cggcccttct cctccgggct gtaattagcg cttggtttaa
      841 tgacggcttg tttcttttct gtggctgcgt gaaagccttg aggggctccg ggagggccct
      901 ttgtgcgggg ggagcggctc ggggggtgcg tgcgtgtgtg tgtgcgtggg gagcgccgcg
      961 tgcggctccg cgctgcccgg cggctgtgag cgctgcgggc gcggcgcggg gctttgtgcg
     1021 ctccgcagtg tgcgcgaggg gagcgcggcc gggggcggtg ccccgcggtg cggggggggc
     1081 tgcgagggga acaaaggctg cgtgcggggt gtgtgcgtgg gggggtgagc agggggtgtg
     1141 ggcgcgtcgg tcgggctgca accccccctg cacccccctc cccgagttgc tgagcacggc
     1201 ccggcttcgg gtgcggggct ccgtacgggg cgtggcgcgg ggctcgccgt gccgggcggg
     1261 gggtggcggc aggtgggggt gccgggcggg gcggggccgc ctcgggccgg ggagggctcg
     1321 ggggaggggc gcggcggccc ccggagcgcc ggcggctgtc gaggcgcggc gagccgcagc
     1381 cattgccttt tatggtaatc gtgcgagagg gcgcagggac ttcctttgtc ccaaatctgt
     1441 gcggagccga aatctgggag gcgccgccgc accccctcta gcgggcgcgg ggcgaagcgg
     1501 tgcggcgccg gcaggaagga aatgggcggg gagggccttc gtgcgtcgcc gcgccgccgt
     1561 ccccttctcc ctctccagcc tcggggctgt ccgcgggggg acggctgcct tcggggggga
     1621 cggggcaggg cggggttcgg cttctggcgt gtgaccggcg gctctagagc ctctgctaac
     1681 catgttcatg ccttcttctt tttcctacag ctcctgggca acgtgctggt tattgtgctg
     1741 tctcatcatt ttggcaaagc tagcctaccg gtcgccacca tggtgagcaa gggcgaggag
     1801 accacaatgg gcgtaatcaa gcccgacatg aagatcaagc tgaagatgga gggcaacgtg
     1861 aatggccacg ccttcgtgat cgagggcgag ggcgagggca agccctacga cggcaccaac
     1921 accatcaacc tggaggtgaa ggagggagcc cccctgccct tctcctacga cattctgacc
     1981 accgcgttca gttacggcaa cagggccttc accaagtacc ccgacgacat ccccaactac
     2041 ttcaagcagt ccttccccga gggctactct tgggagcgca ccatgacctt cgaggacaag
     2101 ggcatcgtga aggtgaagtc cgacatctcc atggaggagg actccttcat ctacgagata
     2161 cacctcaagg gcgagaactt cccccccaac ggccccgtga tgcagaagga gaccaccggc
     2221 tgggacgcct ccaccgagag gatgtacgtg cgcgacggcg tgctgaaggg cgacgtcaag
     2281 atgaagctgc tgctggaggg cggcggccac caccgcgttg acttcaagac catctacagg
     2341 gccaagaagg cggtgaagct gcccgactat cactttgtgg accaccgcat cgagatcctg
     2401 aaccacgaca aggactacaa caaggtgacc gtttacgaga tcgccgtggc ccgcaactcc
     2461 accgacggca tggacgagct gtacaagtcc ggactcaccg gtggaggcgg ttcaggcgga
     2521 ggtggctctg gcggtggcgg atcgagatct cgagctcaag cttcgaattc tgcagtcgac
     2581 ggtaccgcgg gcccgggatc caccggatct agataactga tcaactcctc aggtgcaggc
     2641 tgcctatcag aaggtggtgg ctggtgtggc caatgccctg gctcacaaat accactgaga
     2701 tctttttccc tctgccaaaa attatgggga catcatgaag ccccttgagc atctgacttc
     2761 tggctaataa aggaaattta ttttcattgc aatagtgtgt tggaattttt tgtgtctctc
     2821 actcggaagg acatatggga gggcaaatca tttaaaacat cagaatgagt atttggttta
     2881 gagtttggca acatatgccc atatgctggc tgccatgaac aaaggttggc tataaagagg
     2941 tcatcagtat atgaaacagc cccctgctgt ccattcctta ttccatagaa aagccttgac
     3001 ttgaggttag atttttttta tattttgttt tgtgttattt ttttctttaa catccctaaa
     3061 attttcctta catgttttac tagccagatt tttcctcctc tcctgactac tcccagtcat
     3121 agctgtccct cttctcttat ggagatccct cgacctgcag cccaagcttg gcgtaatcat
     3181 ggtcatagct gtttcctgtg tgaaattgtt atccgctcac aattccacac aacatacgag
     3241 ccggaagcat aaagtgtaaa gcctggggtg cctaatgagt gagctaactc acattaattg
     3301 cgttgcgctc actgcccgct ttccagtcgg gaaacctgtc gtgccagcgg atccgcatct
     3361 caattagtca gcaaccatag tcccgcccct aactccgccc atcccgcccc taactccgcc
     3421 cagttccgcc cattctccgc cccatggctg actaattttt tttatttatg cagaggccga
     3481 ggccgcctcg gcctctgagc tattccagaa gtagtgagga ggcttttttg gaggcctagg
     3541 cttttgcaaa aagctaactt gtttattgca gcttataatg gttacaaata aagcaatagc
     3601 atcacaaatt tcacaaataa agcatttttt tcactgcatt ctagttgtgg tttgtccaaa
     3661 ctcatcaatg tatcttatca tgtctggatc cgctgcatta atgaatcggc caacgcgcgg
     3721 ggagaggcgg tttgcgtatt gggcgctctt ccgcttcctc gctcactgac tcgctgcgct
     3781 cggtcgttcg gctgcggcga gcggtatcag ctcactcaaa ggcggtaata cggttatcca
     3841 cagaatcagg ggataacgca ggaaagaaca tgtgagcaaa aggccagcaa aaggccagga
     3901 accgtaaaaa ggccgcgttg ctggcgtttt tccataggct ccgcccccct gacgagcatc
     3961 acaaaaatcg acgctcaagt cagaggtggc gaaacccgac aggactataa agataccagg
     4021 cgtttccccc tggaagctcc ctcgtgcgct ctcctgttcc gaccctgccg cttaccggat
     4081 acctgtccgc ctttctccct tcgggaagcg tggcgctttc tcatagctca cgctgtaggt
     4141 atctcagttc ggtgtaggtc gttcgctcca agctgggctg tgtgcacgaa ccccccgttc
     4201 agcccgaccg ctgcgcctta tccggtaact atcgtcttga gtccaacccg gtaagacacg
     4261 acttatcgcc actggcagca gccactggta acaggattag cagagcgagg tatgtaggcg
     4321 gtgctacaga gttcttgaag tggtggccta actacggcta cactagaaga acagtatttg
     4381 gtatctgcgc tctgctgaag ccagttacct tcggaaaaag agttggtagc tcttgatccg
     4441 gcaaacaaac caccgctggt agcggtggtt tttttgtttg caagcagcag attacgcgca
     4501 gaaaaaaagg atctcaagaa gatcctttga tcttttctac ggggtctgac gctcagtgga
     4561 acgaaaactc acgttaaggg attttggtca tgagattatc aaaaaggatc ttcacctaga
     4621 tccttttaaa ttaaaaatga agttttaaat caatctaaag tatatatgag taaacttggt
     4681 ctgacagtta ccaatgctta atcagtgagg cacctatctc agcgatctgt ctatttcgtt
     4741 catccatagt tgcctgactc cccgtcgtgt agataactac gatacgggag ggcttaccat
     4801 ctggccccag tgctgcaatg ataccgcgag acccacgctc accggctcca gatttatcag
     4861 caataaacca gccagccgga agggccgagc gcagaagtgg tcctgcaact ttatccgcct
     4921 ccatccagtc tattaattgt tgccgggaag ctagagtaag tagttcgcca gttaatagtt
     4981 tgcgcaacgt tgttgccatt gctacaggca tcgtggtgtc acgctcgtcg tttggtatgg
     5041 cttcattcag ctccggttcc caacgatcaa ggcgagttac atgatccccc atgttgtgca
     5101 aaaaagcggt tagctccttc ggtcctccga tcgttgtcag aagtaagttg gccgcagtgt
     5161 tatcactcat ggttatggca gcactgcata attctcttac tgtcatgcca tccgtaagat
     5221 gcttttctgt gactggtgag tactcaacca agtcattctg agaatagtgt atgcggcgac
     5281 cgagttgctc ttgcccggcg tcaatacggg ataataccgc gccacatagc agaactttaa
     5341 aagtgctcat cattggaaaa cgttcttcgg ggcgaaaact ctcaaggatc ttaccgctgt
     5401 tgagatccag ttcgatgtaa cccactcgtg cacccaactg atcttcagca tcttttactt
     5461 tcaccagcgt ttctgggtga gcaaaaacag gaaggcaaaa tgccgcaaaa aagggaataa
     5521 gggcgacacg gaaatgttga atactcatac tcttcctttt tcaatattat tgaagcattt
     5581 atcagggtta ttgtctcatg agcggataca tatttgaatg tatttagaaa aataaacaaa
     5641 taggggttc
//
