LOCUS       sequence-323959-        5608 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Expresses mWasabi fused with an ER-targeting sequence of 
            calreticulin and the ER retrieval sequence, KDEL, under the control 
            of CAG promoter.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 5608)
  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 5608)
  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-ER
FEATURES             Location/Qualifiers
     source          1..5608
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     enhancer        20..399
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
     promoter        401..676
                     /label=chicken beta-actin promoter
     intron          677..1693
                     /label=chimeric intron
                     /note="chimera between introns from chicken beta-actin and 
                     rabbit beta-globin"
     primer_bind     1617..1639
                     /label=pCAGGS-5
                     /note="Chimeric intron in CAG promoter, forward primer"
     primer_bind     1701..1720
                     /label=pCAG-F
                     /note="Rabbit beta-globin intron, for pCAG plasmids, 
                     forward primer"
     CDS             1829..2536
                     /codon_start=1
                     /product="green variant of teal fluorescent protein mTFP1 
                     (Ai et al., 2008)"
                     /label=mWasabi
                     /translation="MVSKGEETTMGVIKPDMKIKLKMEGNVNGHAFVIEGEGEGKPYDG
                     TNTINLEVKEGAPLPFSYDILTTAFSYGNRAFTKYPDDIPNYFKQSFPEGYSWERTMTF
                     EDKGIVKVKSDISMEEDSFIYEIHLKGENFPPNGPVMQKETTGWDASTERMYVRDGVLK
                     GDVKMKLLLEGGGHHRVDFKTIYRAKKAVKLPDYHFVDHRIEILNHDKDYNKVTVYEIA
                     VARNSTDGMDELYK"
     primer_bind     complement(2645..2664)
                     /label=Bglob-pA-R
                     /note="Rabbit beta-globin polyA region, reverse primer"
     polyA_signal    2710..2765
                     /label=beta-globin poly(A) signal
                     /note="rabbit beta-globin polyadenylation signal (Gil and 
                     Proudfoot, 1987)"
     primer_bind     complement(2764..2783)
                     /label=rbglobpA-R
                     /note="Rabbit beta-globin polyA, reverse primer. Also 
                     called rb-glob-pA-term-R"
     primer_bind     complement(3126..3142)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(3126..3142)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(3139..3161)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    3150..3166
                     /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(3174..3180,3181..3198,3199..3204))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    3219..3240
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     promoter        3299..3494
                     /label=SV40 promoter
                     /note="SV40 early promoter"
     rep_origin      3345..3480
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     polyA_signal    3500..3634
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(3537..3556)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     3591..3610
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     primer_bind     complement(3702..3719)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(3873..4461)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(3953..3972)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(4632..5423,5424..5492))
                     /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     5255..5274
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(5493..5597)
                     /gene="bla"
                     /label=AmpR promoter
ORIGIN
        1 gaaaagtgcc acctgggtcg acattgatta ttgactagtt attaatagta atcaattacg
       61 gggtcattag ttcatagccc atatatggag ttccgcgtta cataacttac ggtaaatggc
      121 ccgcctggct gaccgcccaa cgacccccgc ccattgacgt caataatgac gtatgttccc
      181 atagtaacgc caatagggac tttccattga cgtcaatggg tggagtattt acggtaaact
      241 gcccacttgg cagtacatca agtgtatcat atgccaagta cgccccctat tgacgtcaat
      301 gacggtaaat ggcccgcctg gcattatgcc cagtacatga ccttatggga ctttcctact
      361 tggcagtaca tctacgtatt agtcatcgct attaccatgg tcgaggtgag ccccacgttc
      421 tgcttcactc tccccatctc ccccccctcc ccacccccaa ttttgtattt atttattttt
      481 taattatttt gtgcagcgat gggggcgggg gggggggggg cgcgcgccag gcggggcggg
      541 gcggggcgag gggcggggcg gggcgaggcg gagaggtgcg gcggcagcca atcagagcgg
      601 cgcgctccga aagtttcctt ttatggcgag gcggcggcgg cggcggccct ataaaaagcg
      661 aagcgcgcgg cgggcgggag tcgctgcgcg ctgccttcgc cccgtgcccc gctccgccgc
      721 cgcctcgcgc cgcccgcccc ggctctgact gaccgcgtta ctcccacagg tgagcgggcg
      781 ggacggccct tctcctccgg gctgtaatta gcgcttggtt taatgacggc ttgtttcttt
      841 tctgtggctg cgtgaaagcc ttgaggggct ccgggagggc cctttgtgcg gggggagcgg
      901 ctcggggggt gcgtgcgtgt gtgtgtgcgt ggggagcgcc gcgtgcggct ccgcgctgcc
      961 cggcggctgt gagcgctgcg ggcgcggcgc ggggctttgt gcgctccgca gtgtgcgcga
     1021 ggggagcgcg gccgggggcg gtgccccgcg gtgcgggggg ggctgcgagg ggaacaaagg
     1081 ctgcgtgcgg ggtgtgtgcg tgggggggtg agcagggggt gtgggcgcgt cggtcgggct
     1141 gcaacccccc ctgcaccccc ctccccgagt tgctgagcac ggcccggctt cgggtgcggg
     1201 gctccgtacg gggcgtggcg cggggctcgc cgtgccgggc ggggggtggc ggcaggtggg
     1261 ggtgccgggc ggggcggggc cgcctcgggc cggggagggc tcgggggagg ggcgcggcgg
     1321 cccccggagc gccggcggct gtcgaggcgc ggcgagccgc agccattgcc ttttatggta
     1381 atcgtgcgag agggcgcagg gacttccttt gtcccaaatc tgtgcggagc cgaaatctgg
     1441 gaggcgccgc cgcaccccct ctagcgggcg cggggcgaag cggtgcggcg ccggcaggaa
     1501 ggaaatgggc ggggagggcc ttcgtgcgtc gccgcgccgc cgtccccttc tccctctcca
     1561 gcctcggggc tgtccgcggg gggacggctg ccttcggggg ggacggggca gggcggggtt
     1621 cggcttctgg cgtgtgaccg gcggctctag agcctctgct aaccatgttc atgccttctt
     1681 ctttttccta cagctcctgg gcaacgtgct ggttattgtg ctgtctcatc attttggcaa
     1741 agctagccta ccggtcgcca ccatgctgct atccgtgccg ttgctgctcg gcctcctcgg
     1801 cctggccgtc gccgaccggt cgcacaccat ggtgagcaag ggcgaggaga ccacaatggg
     1861 cgtaatcaag cccgacatga agatcaagct gaagatggag ggcaacgtga atggccacgc
     1921 cttcgtgatc gagggcgagg gcgagggcaa gccctacgac ggcaccaaca ccatcaacct
     1981 ggaggtgaag gagggagccc ccctgccctt ctcctacgac attctgacca ccgcgttcag
     2041 ttacggcaac agggccttca ccaagtaccc cgacgacatc cccaactact tcaagcagtc
     2101 cttccccgag ggctactctt gggagcgcac catgaccttc gaggacaagg gcatcgtgaa
     2161 ggtgaagtcc gacatctcca tggaggagga ctccttcatc tacgagatac acctcaaggg
     2221 cgagaacttc ccccccaacg gccccgtgat gcagaaggag accaccggct gggacgcctc
     2281 caccgagagg atgtacgtgc gcgacggcgt gctgaagggc gacgtcaaga tgaagctgct
     2341 gctggagggc ggcggccacc accgcgttga cttcaagacc atctacaggg ccaagaaggc
     2401 ggtgaagctg cccgactatc actttgtgga ccaccgcatc gagatcctga accacgacaa
     2461 ggactacaac aaggtgaccg tttacgagat cgccgtggcc cgcaactcca ccgacggcat
     2521 ggacgagctg tacaagagat cgtacaagaa ggacgagctg taataaaact cctcaggtgc
     2581 aggctgccta tcagaaggtg gtggctggtg tggccaatgc cctggctcac aaataccact
     2641 gagatctttt tccctctgcc aaaaattatg gggacatcat gaagcccctt gagcatctga
     2701 cttctggcta ataaaggaaa tttattttca ttgcaatagt gtgttggaat tttttgtgtc
     2761 tctcactcgg aaggacatat gggagggcaa atcatttaaa acatcagaat gagtatttgg
     2821 tttagagttt ggcaacatat gcccatatgc tggctgccat gaacaaaggt tggctataaa
     2881 gaggtcatca gtatatgaaa cagccccctg ctgtccattc cttattccat agaaaagcct
     2941 tgacttgagg ttagattttt tttatatttt gttttgtgtt atttttttct ttaacatccc
     3001 taaaattttc cttacatgtt ttactagcca gatttttcct cctctcctga ctactcccag
     3061 tcatagctgt ccctcttctc ttatggagat ccctcgacct gcagcccaag cttggcgtaa
     3121 tcatggtcat agctgtttcc tgtgtgaaat tgttatccgc tcacaattcc acacaacata
     3181 cgagccggaa gcataaagtg taaagcctgg ggtgcctaat gagtgagcta actcacatta
     3241 attgcgttgc gctcactgcc cgctttccag tcgggaaacc tgtcgtgcca gcggatccgc
     3301 atctcaatta gtcagcaacc atagtcccgc ccctaactcc gcccatcccg cccctaactc
     3361 cgcccagttc cgcccattct ccgccccatg gctgactaat tttttttatt tatgcagagg
     3421 ccgaggccgc ctcggcctct gagctattcc agaagtagtg aggaggcttt tttggaggcc
     3481 taggcttttg caaaaagcta acttgtttat tgcagcttat aatggttaca aataaagcaa
     3541 tagcatcaca aatttcacaa ataaagcatt tttttcactg cattctagtt gtggtttgtc
     3601 caaactcatc aatgtatctt atcatgtctg gatccgctgc attaatgaat cggccaacgc
     3661 gcggggagag gcggtttgcg tattgggcgc tcttccgctt cctcgctcac tgactcgctg
     3721 cgctcggtcg ttcggctgcg gcgagcggta tcagctcact caaaggcggt aatacggtta
     3781 tccacagaat caggggataa cgcaggaaag aacatgtgag caaaaggcca gcaaaaggcc
     3841 aggaaccgta aaaaggccgc gttgctggcg tttttccata ggctccgccc ccctgacgag
     3901 catcacaaaa atcgacgctc aagtcagagg tggcgaaacc cgacaggact ataaagatac
     3961 caggcgtttc cccctggaag ctccctcgtg cgctctcctg ttccgaccct gccgcttacc
     4021 ggatacctgt ccgcctttct cccttcggga agcgtggcgc tttctcatag ctcacgctgt
     4081 aggtatctca gttcggtgta ggtcgttcgc tccaagctgg gctgtgtgca cgaacccccc
     4141 gttcagcccg accgctgcgc cttatccggt aactatcgtc ttgagtccaa cccggtaaga
     4201 cacgacttat cgccactggc agcagccact ggtaacagga ttagcagagc gaggtatgta
     4261 ggcggtgcta cagagttctt gaagtggtgg cctaactacg gctacactag aagaacagta
     4321 tttggtatct gcgctctgct gaagccagtt accttcggaa aaagagttgg tagctcttga
     4381 tccggcaaac aaaccaccgc tggtagcggt ggtttttttg tttgcaagca gcagattacg
     4441 cgcagaaaaa aaggatctca agaagatcct ttgatctttt ctacggggtc tgacgctcag
     4501 tggaacgaaa actcacgtta agggattttg gtcatgagat tatcaaaaag gatcttcacc
     4561 tagatccttt taaattaaaa atgaagtttt aaatcaatct aaagtatata tgagtaaact
     4621 tggtctgaca gttaccaatg cttaatcagt gaggcaccta tctcagcgat ctgtctattt
     4681 cgttcatcca tagttgcctg actccccgtc gtgtagataa ctacgatacg ggagggctta
     4741 ccatctggcc ccagtgctgc aatgataccg cgagacccac gctcaccggc tccagattta
     4801 tcagcaataa accagccagc cggaagggcc gagcgcagaa gtggtcctgc aactttatcc
     4861 gcctccatcc agtctattaa ttgttgccgg gaagctagag taagtagttc gccagttaat
     4921 agtttgcgca acgttgttgc cattgctaca ggcatcgtgg tgtcacgctc gtcgtttggt
     4981 atggcttcat tcagctccgg ttcccaacga tcaaggcgag ttacatgatc ccccatgttg
     5041 tgcaaaaaag cggttagctc cttcggtcct ccgatcgttg tcagaagtaa gttggccgca
     5101 gtgttatcac tcatggttat ggcagcactg cataattctc ttactgtcat gccatccgta
     5161 agatgctttt ctgtgactgg tgagtactca accaagtcat tctgagaata gtgtatgcgg
     5221 cgaccgagtt gctcttgccc ggcgtcaata cgggataata ccgcgccaca tagcagaact
     5281 ttaaaagtgc tcatcattgg aaaacgttct tcggggcgaa aactctcaag gatcttaccg
     5341 ctgttgagat ccagttcgat gtaacccact cgtgcaccca actgatcttc agcatctttt
     5401 actttcacca gcgtttctgg gtgagcaaaa acaggaaggc aaaatgccgc aaaaaaggga
     5461 ataagggcga cacggaaatg ttgaatactc atactcttcc tttttcaata ttattgaagc
     5521 atttatcagg gttattgtct catgagcgga tacatatttg aatgtattta gaaaaataaa
     5581 caaatagggg ttccgcgcac atttcccc
//
