LOCUS       sequence-323963-        5916 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Expresses mWasabi under the control of CMV promoter, basic vector 
            for AAV, pAAV-CMV backbone, multiclonig site at C terminal.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 5916)
  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 5916)
  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: pAC-mWasabi-G4
FEATURES             Location/Qualifiers
     source          1..5916
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     enhancer        118..421
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
     promoter        422..625
                     /label=CMV promoter
                     /note="human cytomegalovirus (CMV) immediate early 
                     promoter"
     primer_bind     576..596
                     /label=CMV-F
                     /note="Human CMV immediate early promoter, forward primer"
     intron          716..1094
                     /label=chimeric intron
                     /note="fusion between human cytomegalovirus intron A and 
                     human beta-globin intron 2"
     regulatory      1201..1210
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     CDS             1207..1914
                     /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    1972..2028
                     /label=MCS
                     /note="multiple cloning site"
     polyA_signal    2078..2554
                     /label=hGH poly(A) signal
                     /note="human growth hormone polyadenylation signal"
     primer_bind     complement(2277..2296)
                     /label=hGH-PA-R
                     /note="Human growth hormone terminator, reverse primer"
     repeat_region   2567..2707
                     /label=AAV2 ITR
                     /note="inverted terminal repeat of adeno-associated virus 
                     serotype 2"
     repeat_region   2567..2696
                     /label=AAV2 ITR
     primer_bind     complement(2717..2734)
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     complement(2717..2733)
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(2726..2748)
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     rep_origin      2946..3401
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(3033..3052)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     3243..3264
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     complement(3418..3437)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     3537..3559
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     primer_bind     complement(3597..3615)
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     promoter        3683..3787
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(3788..3856,3857..4648)
                     /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     complement(4006..4025)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      4819..5407
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     5308..5327
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     protein_bind    5695..5716
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     promoter        join(5731..5736,5737..5754,5755..5761)
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    5769..5785
                     /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     5774..5796
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     primer_bind     5793..5809
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     5793..5809
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     repeat_region   join(5825..5916,1..49)
                     /label=AAV2 ITR
                     /note="inverted terminal repeat of adeno-associated virus 
                     serotype 2"
     repeat_region   join(5836..5916,1..49)
                     /label=AAV2 ITR
ORIGIN
        1 gagcgagcgc gcagagaggg agtggccaac tccatcacta ggggttccta tcgatatcaa
       61 gctttaatag taatcaatta cggggtcatt agttcatagc ccatatatgg agttccgcgt
      121 tacataactt acggtaaatg gcccgcctgg ctgaccgccc aacgaccccc gcccattgac
      181 gtcaataatg acgtatgttc ccatagtaac gccaataggg actttccatt gacgtcaatg
      241 ggtggagtat ttacggtaaa ctgcccactt ggcagtacat caagtgtatc atatgccaag
      301 tacgccccct attgacgtca atgacggtaa atggcccgcc tggcattatg cccagtacat
      361 gaccttatgg gactttccta cttggcagta catctacgta ttagtcatcg ctattaccat
      421 ggtgatgcgg ttttggcagt acatcaatgg gcgtggatag cggtttgact cacggggatt
      481 tccaagtctc caccccattg acgtcaatgg gagtttgttt tggcaccaaa atcaacggga
      541 ctttccaaaa tgtcgtaaca actccgcccc attgacgcaa atgggcggta ggcgtgtacg
      601 gtgggaggtc tatataagca gagctggttt agtggatatc cttaagggcc cagccggccc
      661 gaatcccggc cgggaacggt gcattggaac gcggattccc cgtgccaaga gtgacgtaag
      721 taccgcctat agagtctata ggcccacaaa aaatgctttc ttcttttaat atactttttt
      781 gtttatctta tttctaatac tttccctaat ctctttcttt cagggcaata atgatacaat
      841 gtatcatgcc tctttgcacc attctaaaga ataacagtga taatttctgg gttaaggcaa
      901 tagcaatatt tctgcatata aatatttctg catataaatt gtaactgatg taagaggttt
      961 catattgcta atagcagcta caatccagct accattctgc ttttatttta tggttgggat
     1021 aaggctggat tattctgagt ccaagctagg cccttttgct aatcatgttc atacctctta
     1081 tcttcctccc acagctcctg ggcaacgtgc tggtctgtgt gctggcccat cactttggca
     1141 aagaattggg attcgcgaat tgtgctgtct catcattttg gcaaagctag cctaccggtc
     1201 gccaccatgg tgagcaaggg cgaggagacc acaatgggcg taatcaagcc cgacatgaag
     1261 atcaagctga agatggaggg caacgtgaat ggccacgcct tcgtgatcga gggcgagggc
     1321 gagggcaagc cctacgacgg caccaacacc atcaacctgg aggtgaagga gggagccccc
     1381 ctgcccttct cctacgacat tctgaccacc gcgttcagtt acggcaacag ggccttcacc
     1441 aagtaccccg acgacatccc caactacttc aagcagtcct tccccgaggg ctactcttgg
     1501 gagcgcacca tgaccttcga ggacaagggc atcgtgaagg tgaagtccga catctccatg
     1561 gaggaggact ccttcatcta cgagatacac ctcaagggcg agaacttccc ccccaacggc
     1621 cccgtgatgc agaaggagac caccggctgg gacgcctcca ccgagaggat gtacgtgcgc
     1681 gacggcgtgc tgaagggcga cgtcaagatg aagctgctgc tggagggcgg cggccaccac
     1741 cgcgttgact tcaagaccat ctacagggcc aagaaggcgg tgaagctgcc cgactatcac
     1801 tttgtggacc accgcatcga gatcctgaac cacgacaagg actacaacaa ggtgaccgtt
     1861 tacgagatcg ccgtggcccg caactccacc gacggcatgg acgagctgta caagtccgga
     1921 ctcaccggtg gaggcggttc aggcggaggt ggctctggcg gtggcggatc gagatctcga
     1981 gctcaagctt cgaattctgc agtcgacggt accgcgggcc cgggatccac cggatctaga
     2041 taactgatca actcctcagg tgcaggctgc ctatcacggg tggcatccct gtgacccctc
     2101 cccagtgcct ctcctggccc tggaagttgc cactccagtg cccaccagcc ttgtcctaat
     2161 aaaattaagt tgcatcattt tgtctgacta ggtgtccttc tataatatta tggggtggag
     2221 gggggtggta tggagcaagg ggcaagttgg gaagacaacc tgtagggcct gcggggtcta
     2281 ttgggaacca agctggagtg cagtggcaca atcttggctc actgcaatct ccgcctcctg
     2341 ggttcaagcg attctcctgc ctcagcctcc cgagttgttg ggattccagg catgcatgac
     2401 caggctcagc taatttttgt ttttttggta gagacggggt ttcaccatat tggccaggct
     2461 ggtctccaac tcctaatctc aggtgatcta cccaccttgg cctcccaaat tgctgggatt
     2521 acaggcgtga accactgctc ccttccctgt ccttatcgat agatctagga acccctagtg
     2581 atggagttgg ccactccctc tctgcgcgct cgctcgctca ctgaggccgg gcgaccaaag
     2641 gtcgcccgac gcccgggctt tgcccgggcg gcctcagtga gcgagcgagc gcgcagctgc
     2701 ctgcaggcag cttggcactg gccgtcgttt tacaacgtcg tgactgggaa aaccctggcg
     2761 ttacccaact taatcgcctt gcagcacatc cccctttcgc cagctggcgt aatagcgaag
     2821 aggcccgcac cgatcgccct tcccaacagt tgcgcagcct gaatggcgaa tggcgcctga
     2881 tgcggtattt tctccttacg catctgtgcg gtatttcaca ccgcatacgt caaagcaacc
     2941 atagtacgcg ccctgtagcg gcgcattaag cgcggcgggt gtggtggtta cgcgcagcgt
     3001 gaccgctaca cttgccagcg ccctagcgcc cgctcctttc gctttcttcc cttcctttct
     3061 cgccacgttc gccggctttc cccgtcaagc tctaaatcgg gggctccctt tagggttccg
     3121 atttagtgct ttacggcacc tcgaccccaa aaaacttgat ttgggtgatg gttcacgtag
     3181 tgggccatcg ccctgataga cggtttttcg ccctttgacg ttggagtcca cgttctttaa
     3241 tagtggactc ttgttccaaa ctggaacaac actcaaccct atctcgggct attcttttga
     3301 tttataaggg attttgccga tttcggccta ttggttaaaa aatgagctga tttaacaaaa
     3361 atttaacgcg aattttaaca aaatattaac gtttacaatt ttatggtgca ctctcagtac
     3421 aatctgctct gatgccgcat agttaagcca gccccgacac ccgccaacac ccgctgacgc
     3481 gccctgacgg gcttgtctgc tcccggcatc cgcttacaga caagctgtga ccgtctccgg
     3541 gagctgcatg tgtcagaggt tttcaccgtc atcaccgaaa cgcgcgagac gaaagggcct
     3601 cgtgatacgc ctatttttat aggttaatgt catgataata atggtttctt agacgtcagg
     3661 tggcactttt cggggaaatg tgcgcggaac ccctatttgt ttatttttct aaatacattc
     3721 aaatatgtat ccgctcatga gacaataacc ctgataaatg cttcaataat attgaaaaag
     3781 gaagagtatg agtattcaac atttccgtgt cgcccttatt cccttttttg cggcattttg
     3841 ccttcctgtt tttgctcacc cagaaacgct ggtgaaagta aaagatgctg aagatcagtt
     3901 gggtgcacga gtgggttaca tcgaactgga tctcaacagc ggtaagatcc ttgagagttt
     3961 tcgccccgaa gaacgttttc caatgatgag cacttttaaa gttctgctat gtggcgcggt
     4021 attatcccgt attgacgccg ggcaagagca actcggtcgc cgcatacact attctcagaa
     4081 tgacttggtt gagtactcac cagtcacaga aaagcatctt acggatggca tgacagtaag
     4141 agaattatgc agtgctgcca taaccatgag tgataacact gcggccaact tacttctgac
     4201 aacgatcgga ggaccgaagg agctaaccgc ttttttgcac aacatggggg atcatgtaac
     4261 tcgccttgat cgttgggaac cggagctgaa tgaagccata ccaaacgacg agcgtgacac
     4321 cacgatgcct gtagcaatgg caacaacgtt gcgcaaacta ttaactggcg aactacttac
     4381 tctagcttcc cggcaacaat taatagactg gatggaggcg gataaagttg caggaccact
     4441 tctgcgctcg gcccttccgg ctggctggtt tattgctgat aaatctggag ccggtgagcg
     4501 tgggtctcgc ggtatcattg cagcactggg gccagatggt aagccctccc gtatcgtagt
     4561 tatctacacg acggggagtc aggcaactat ggatgaacga aatagacaga tcgctgagat
     4621 aggtgcctca ctgattaagc attggtaact gtcagaccaa gtttactcat atatacttta
     4681 gattgattta aaacttcatt tttaatttaa aaggatctag gtgaagatcc tttttgataa
     4741 tctcatgacc aaaatccctt aacgtgagtt ttcgttccac tgagcgtcag accccgtaga
     4801 aaagatcaaa ggatcttctt gagatccttt ttttctgcgc gtaatctgct gcttgcaaac
     4861 aaaaaaacca ccgctaccag cggtggtttg tttgccggat caagagctac caactctttt
     4921 tccgaaggta actggcttca gcagagcgca gataccaaat actgttcttc tagtgtagcc
     4981 gtagttaggc caccacttca agaactctgt agcaccgcct acatacctcg ctctgctaat
     5041 cctgttacca gtggctgctg ccagtggcga taagtcgtgt cttaccgggt tggactcaag
     5101 acgatagtta ccggataagg cgcagcggtc gggctgaacg gggggttcgt gcacacagcc
     5161 cagcttggag cgaacgacct acaccgaact gagataccta cagcgtgagc tatgagaaag
     5221 cgccacgctt cccgaaggga gaaaggcgga caggtatccg gtaagcggca gggtcggaac
     5281 aggagagcgc acgagggagc ttccaggggg aaacgcctgg tatctttata gtcctgtcgg
     5341 gtttcgccac ctctgacttg agcgtcgatt tttgtgatgc tcgtcagggg ggcggagcct
     5401 atggaaaaac gccagcaacg cggccttttt acggttcctg gccttttgct ggccttttgc
     5461 tcacatgttc tttcctgcgt tatcccctga ttctgtggat aaccgtatta ccgcctttga
     5521 gtgagctgat accgctcgcc gcagccgaac gaccgagcgc agcgagtcag tgagcgagga
     5581 agcggaagag cgcccaatac gcaaaccgcc tctccccgcg cgttggccga ttcattaatg
     5641 cagctggcac gacaggtttc ccgactggaa agcgggcagt gagcgcaacg caattaatgt
     5701 gagttagctc actcattagg caccccaggc tttacacttt atgcttccgg ctcgtatgtt
     5761 gtgtggaatt gtgagcggat aacaatttca cacaggaaac agctatgacc atgattacga
     5821 attgcctgca ggcagctgcg cgctcgctcg ctcactgagg ccgcccgggc aaagcccggg
     5881 cgtcgggcga cctttggtcg cccggcctca gtgagc
//
