LOCUS       sequence-318603-        5645 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 5645)
  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 5645)
  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..5645
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     enhancer        4..383
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
     promoter        385..662
                     /label=chicken beta-actin promoter
     intron          663..1678
                     /label=chimeric intron
                     /note="chimera between introns from chicken beta-actin and 
                     rabbit beta-globin"
     primer_bind     1602..1624
                     /label=pCAGGS-5
                     /note="Chimeric intron in CAG promoter, forward primer"
     primer_bind     1686..1705
                     /label=pCAG-F
                     /note="Rabbit beta-globin intron, for pCAG plasmids, 
                     forward primer"
     regulatory      1742..1751
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     CDS             1748..2455
                     /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    2513..2569
                     /label=MCS
                     /note="multiple cloning site"
     primer_bind     complement(2669..2688)
                     /label=Bglob-pA-R
                     /note="Rabbit beta-globin polyA region, reverse primer"
     polyA_signal    2734..2789
                     /label=beta-globin poly(A) signal
                     /note="rabbit beta-globin polyadenylation signal (Gil and 
                     Proudfoot, 1987)"
     primer_bind     complement(2788..2807)
                     /label=rbglobpA-R
                     /note="Rabbit beta-globin polyA, reverse primer. Also 
                     called rb-glob-pA-term-R"
     primer_bind     complement(3148..3164)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(3148..3164)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(3161..3183)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    3172..3188
                     /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(3196..3202,3203..3220,3221..3226))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    3241..3262
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     promoter        3321..3516
                     /label=SV40 promoter
                     /note="SV40 early promoter"
     rep_origin      3367..3502
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     polyA_signal    3522..3656
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(3559..3578)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     3613..3632
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     primer_bind     complement(3724..3741)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(3895..4483)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(3975..3994)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(4654..5445,5446..5514))
                     /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     5277..5296
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(5515..5619)
                     /gene="bla"
                     /label=AmpR promoter
ORIGIN
        1 gtcgacattg attattgact agttattaat agtaatcaat tacggggtca ttagttcata
       61 gcccatatat ggagttccgc gttacataac ttacggtaaa tggcccgcct ggctgaccgc
      121 ccaacgaccc ccgcccattg acgtcaataa tgacgtatgt tcccatagta acgccaatag
      181 ggactttcca ttgacgtcaa tgggtggagt atttacggta aactgcccac ttggcagtac
      241 atcaagtgta tcatatgcca agtacgcccc ctattgacgt caatgacggt aaatggcccg
      301 cctggcatta tgcccagtac atgaccttat gggactttcc tacttggcag tacatctacg
      361 tattagtcat cgctattacc atggtcgagg tgagccccac gttctgcttc actctcccca
      421 tctccccccc ctccccaccc ccaattttgt atttatttat tttttaatta ttttgtgcag
      481 cgatgggggc gggggggggg ggggggcgcg cgccaggcgg ggcggggcgg ggcgaggggc
      541 ggggcggggc gaggcggaga ggtgcggcgg cagccaatca gagcggcgcg ctccgaaagt
      601 ttccttttat ggcgaggcgg cggcggcggc ggccctataa aaagcgaagc gcgcggcggg
      661 cgggagtcgc tgcgcgctgc cttcgccccg tgccccgctc cgcgccgcct cgcgccgccc
      721 gccccggctc tgactgaccg cgttactccc acaggtgagc gggcgggacg gcccttctcc
      781 tccgggctgt aattagcgct tggtttaatg acggcttgtt tcttttctgt ggctgcgtga
      841 aagccttgag gggctccggg agggcccttt gtgcgggggg agcggctcgg ggggtgcgtg
      901 cgtgtgtgtg tgcgtgggga gcgccgcgtg cggctccgcg ctgcccggcg gctgtgagcg
      961 ctgcgggcgc ggcgcggggc tttgtgcgct ccgcagtgtg cgcgagggga gcgcggccgg
     1021 gggcggtgcc ccgcggtgcg gggggggctg cgaggggaac aaaggctgcg tgcggggtgt
     1081 gtgcgtgggg gggtgagcag ggggtgtggg cgcgtcggtc gggctgcaac cccccctgca
     1141 cccccctccc cgagttgctg agcacggccc ggcttcgggt gcggggctcc gtacggggcg
     1201 tggcgcgggg ctcgccgtgc cgggcggggg gtggcggcag gtgggggtgc cgggcggggc
     1261 ggggccgcct cgggccgggg agggctcggg ggaggggcgc ggcggccccc ggagcgccgg
     1321 cggctgtcga ggcgcggcga gccgcagcca ttgcctttta tggtaatcgt gcgagagggc
     1381 gcagggactt cctttgtccc aaatctgtgc ggagccgaaa tctgggaggc gccgccgcac
     1441 cccctctagc gggcgcgggg cgaagcggtg cggcgccggc aggaaggaaa tgggcgggga
     1501 gggccttcgt gcgtcgccgc gccgccgtcc ccttctccct ctccagcctc ggggctgtcc
     1561 gcggggggac ggctgccttc gggggggacg gggcagggcg gggttcggct tctggcgtgt
     1621 gaccggcggc tctagagcct ctgctaacca tgttcatgcc ttcttctttt tcctacagct
     1681 cctgggcaac gtgctggtta ttgtgctgtc tcatcatttt ggcaaagcta gcctaccggt
     1741 cgccaccatg gtgagcaagg gcgaggagac cacaatgggc gtaatcaagc ccgacatgaa
     1801 gatcaagctg aagatggagg gcaacgtgaa tggccacgcc ttcgtgatcg agggcgaggg
     1861 cgagggcaag ccctacgacg gcaccaacac catcaacctg gaggtgaagg agggagcccc
     1921 cctgcccttc tcctacgaca ttctgaccac cgcgttcagt tacggcaaca gggccttcac
     1981 caagtacccc gacgacatcc ccaactactt caagcagtcc ttccccgagg gctactcttg
     2041 ggagcgcacc atgaccttcg aggacaaggg catcgtgaag gtgaagtccg acatctccat
     2101 ggaggaggac tccttcatct acgagataca cctcaagggc gagaacttcc cccccaacgg
     2161 ccccgtgatg cagaaggaga ccaccggctg ggacgcctcc accgagagga tgtacgtgcg
     2221 cgacggcgtg ctgaagggcg acgtcaagat gaagctgctg ctggagggcg gcggccacca
     2281 ccgcgttgac ttcaagacca tctacagggc caagaaggcg gtgaagctgc ccgactatca
     2341 ctttgtggac caccgcatcg agatcctgaa ccacgacaag gactacaaca aggtgaccgt
     2401 ttacgagatc gccgtggccc gcaactccac cgacggcatg gacgagctgt acaagtccgg
     2461 actcaccggt ggaggcggtt caggcggagg tggctctggc ggtggcggat cgagatctcg
     2521 agctcaagct tcgaattctg cagtcgacgg taccgcgggc ccgggatcca ccggatctag
     2581 ataactgatc aactcctcag gtgcaggctg cctatcagaa ggtggtggct ggtgtggcca
     2641 atgccctggc tcacaaatac cactgagatc tttttccctc tgccaaaaat tatggggaca
     2701 tcatgaagcc ccttgagcat ctgacttctg gctaataaag gaaatttatt ttcattgcaa
     2761 tagtgtgttg gaattttttg tgtctctcac tcggaaggac atatgggagg gcaaatcatt
     2821 taaaacatca gaatgagtat ttggtttaga gtttggcaac atatgccata tgctggctgc
     2881 catgaacaaa ggtggctata aagaggtcat cagtatatga aacagccccc tgctgtccat
     2941 tccttattcc atagaaaagc cttgacttga ggttagattt tttttatatt ttgttttgtg
     3001 ttattttttt ctttaacatc cctaaaattt tccttacatg ttttactagc cagatttttc
     3061 ctcctctcct gactactccc agtcatagct gtccctcttc tcttatgaag atccctcgac
     3121 ctgcagccca agcttggcgt aatcatggtc atagctgttt cctgtgtgaa attgttatcc
     3181 gctcacaatt ccacacaaca tacgagccgg aagcataaag tgtaaagcct ggggtgccta
     3241 atgagtgagc taactcacat taattgcgtt gcgctcactg cccgctttcc agtcgggaaa
     3301 cctgtcgtgc cagcggatcc gcatctcaat tagtcagcaa ccatagtccc gcccctaact
     3361 ccgcccatcc cgcccctaac tccgcccagt tccgcccatt ctccgcccca tggctgacta
     3421 atttttttta tttatgcaga ggccgaggcc gcctcggcct ctgagctatt ccagaagtag
     3481 tgaggaggct tttttggagg cctaggcttt tgcaaaaagc taacttgttt attgcagctt
     3541 ataatggtta caaataaagc aatagcatca caaatttcac aaataaagca tttttttcac
     3601 tgcattctag ttgtggtttg tccaaactca tcaatgtatc ttatcatgtc tggatccgct
     3661 gcattaatga atcggccaac gcgcggggag aggcggtttg cgtattgggc gctcttccgc
     3721 ttcctcgctc actgactcgc tgcgctcggt cgttcggctg cggcgagcgg tatcagctca
     3781 ctcaaaggcg gtaatacggt tatccacaga atcaggggat aacgcaggaa agaacatgtg
     3841 agcaaaaggc cagcaaaagg ccaggaaccg taaaaaggcc gcgttgctgg cgtttttcca
     3901 taggctccgc ccccctgacg agcatcacaa aaatcgacgc tcaagtcaga ggtggcgaaa
     3961 cccgacagga ctataaagat accaggcgtt tccccctgga agctccctcg tgcgctctcc
     4021 tgttccgacc ctgccgctta ccggatacct gtccgccttt ctcccttcgg gaagcgtggc
     4081 gctttctcaa tgctcacgct gtaggtatct cagttcggtg taggtcgttc gctccaagct
     4141 gggctgtgtg cacgaacccc ccgttcagcc cgaccgctgc gccttatccg gtaactatcg
     4201 tcttgagtcc aacccggtaa gacacgactt atcgccactg gcagcagcca ctggtaacag
     4261 gattagcaga gcgaggtatg taggcggtgc tacagagttc ttgaagtggt ggcctaacta
     4321 cggctacact agaaggacag tatttggtat ctgcgctctg ctgaagccag ttaccttcgg
     4381 aaaaagagtt ggtagctctt gatccggcaa acaaaccacc gctggtagcg gtggtttttt
     4441 tgtttgcaag cagcagatta cgcgcagaaa aaaaggatct caagaagatc ctttgatctt
     4501 ttctacgggg tctgacgctc agtggaacga aaactcacgt taagggattt tggtcatgag
     4561 attatcaaaa aggatcttca cctagatcct tttaaattaa aaatgaagtt ttaaatcaat
     4621 ctaaagtata tatgagtaaa cttggtctga cagttaccaa tgcttaatca gtgaggcacc
     4681 tatctcagcg atctgtctat ttcgttcatc catagttgcc tgactccccg tcgtgtagat
     4741 aactacgata cgggagggct taccatctgg ccccagtgct gcaatgatac cgcgagaccc
     4801 acgctcaccg gctccagatt tatcagcaat aaaccagcca gccggaaggg ccgagcgcag
     4861 aagtggtcct gcaactttat ccgcctccat ccagtctatt aattgttgcc gggaagctag
     4921 agtaagtagt tcgccagtta atagtttgcg caacgttgtt gccattgcta caggcatcgt
     4981 ggtgtcacgc tcgtcgtttg gtatggcttc attcagctcc ggttcccaac gatcaaggcg
     5041 agttacatga tcccccatgt tgtgcaaaaa agcggttagc tccttcggtc ctccgatcgt
     5101 tgtcagaagt aagttggccg cagtgttatc actcatggtt atggcagcac tgcataattc
     5161 tcttactgtc atgccatccg taagatgctt ttctgtgact ggtgagtact caaccaagtc
     5221 attctgagaa tagtgtatgc ggcgaccgag ttgctcttgc ccggcgtcaa tacgggataa
     5281 taccgcgcca catagcagaa ctttaaaagt gctcatcatt ggaaaacgtt cttcggggcg
     5341 aaaactctca aggatcttac cgctgttgag atccagttcg atgtaaccca ctcgtgcacc
     5401 caactgatct tcagcatctt ttactttcac cagcgtttct gggtgagcaa aaacaggaag
     5461 gcaaaatgcc gcaaaaaagg gaataagggc gacacggaaa tgttgaatac tcatactctt
     5521 cctttttcaa tattattgaa gcatttatca gggttattgt ctcatgagcg gatacatatt
     5581 tgaatgtatt tagaaaaata aacaaatagg ggttccgcgc acatttcccc gaaaagtgcc
     5641 acctg
//
