LOCUS       sequence-323965-        5365 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Expresses mWasabi under the control of TetO promoter, basic vector 
            for AAV, pAAV-CMV backbone, multiclonig site at C terminal, pAAV-TRE
            backbone.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 5365)
  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 5365)
  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: pAT-mWasabi-G
FEATURES             Location/Qualifiers
     source          1..5365
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     repeat_region   40..169
                     /label=AAV2 ITR (alternate)
                     /note="Functional equivalent of wild-type AAV2 ITR"
     protein_bind    204..222
                     /gene="tetO"
                     /label=tet operator
                     /bound_moiety="tetracycline repressor TetR"
                     /note="bacterial operator O2 for the tetR and tetA genes"
     protein_bind    241..259
                     /gene="tetO"
                     /label=tet operator
                     /bound_moiety="tetracycline repressor TetR"
                     /note="bacterial operator O2 for the tetR and tetA genes"
     protein_bind    278..296
                     /gene="tetO"
                     /label=tet operator
                     /bound_moiety="tetracycline repressor TetR"
                     /note="bacterial operator O2 for the tetR and tetA genes"
     protein_bind    315..333
                     /gene="tetO"
                     /label=tet operator
                     /bound_moiety="tetracycline repressor TetR"
                     /note="bacterial operator O2 for the tetR and tetA genes"
     protein_bind    352..370
                     /gene="tetO"
                     /label=tet operator
                     /bound_moiety="tetracycline repressor TetR"
                     /note="bacterial operator O2 for the tetR and tetA genes"
     protein_bind    389..407
                     /gene="tetO"
                     /label=tet operator
                     /bound_moiety="tetracycline repressor TetR"
                     /note="bacterial operator O2 for the tetR and tetA genes"
     protein_bind    426..444
                     /gene="tetO"
                     /label=tet operator
                     /bound_moiety="tetracycline repressor TetR"
                     /note="bacterial operator O2 for the tetR and tetA genes"
     protein_bind    463..481
                     /gene="tetO"
                     /label=tet operator
                     /bound_moiety="tetracycline repressor TetR"
                     /note="bacterial operator O2 for the tetR and tetA genes"
     primer_bind     503..527
                     /label=LNCX
                     /note="Human CMV promoter, forward primer"
     regulatory      633..642
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     CDS             639..1346
                     /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    1404..1460
                     /label=MCS
                     /note="multiple cloning site"
     misc_feature    1531..2119
                     /label=WPRE
                     /note="woodchuck hepatitis virus posttranscriptional 
                     regulatory element"
     primer_bind     complement(1584..1604)
                     /label=WPRE-R
                     /note="WPRE, reverse primer"
     CDS             complement(2002..2013)
                     /codon_start=1
                     /product="Factor Xa recognition and cleavage site"
                     /label=Factor Xa site
                     /translation="IEGR"
     polyA_signal    2151..2627
                     /label=hGH poly(A) signal
                     /note="human growth hormone polyadenylation signal"
     primer_bind     complement(2350..2369)
                     /label=hGH-PA-R
                     /note="Human growth hormone terminator, reverse primer"
     repeat_region   2667..2807
                     /label=AAV2 ITR
                     /note="inverted terminal repeat of adeno-associated virus 
                     serotype 2"
     repeat_region   2667..2796
                     /label=AAV2 ITR
     rep_origin      2882..3337
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(2969..2988)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     3179..3200
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     complement(3354..3373)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     3473..3495
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     primer_bind     complement(3533..3551)
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     promoter        3619..3723
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(3724..3792,3793..4584)
                     /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(3942..3961)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      4755..5343
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     5244..5263
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
ORIGIN
        1 tacggttcct ggccttttgc tggccttttg ctcacatgtc ctgcaggcag ctgcgcgctc
       61 gctcgctcac tgaggccgcc cgggcgtcgg gcgacctttg gtcgcccggc ctcagtgagc
      121 gagcgagcgc gcagagaggg agtggccaac tccatcacta ggggttcctg cggccgcacg
      181 cgtatcaagt gccacctgac gtctccctat cagtgataga gaagtcgaca cgtctcgagc
      241 tccctatcag tgatagagaa ggtacgtcta gaacgtctcc ctatcagtga tagagaagtc
      301 gacacgtctc gagctcccta tcagtgatag agaaggtacg tctagaacgt ctccctatca
      361 gtgatagaga agtcgacacg tctcgagctc cctatcagtg atagagaagg tacgtctaga
      421 acgtctccct atcagtgata gagaagtcga cacgtctcga gctccctatc agtgatagag
      481 aagctacccc ctatataagc agagctcgtt tagtgaaccg tcagatcgcc tggagacgcc
      541 atccacgctg ttttgacctc catagaagac accgggaccg atccagcctg attgtgctgt
      601 ctcatcattt tggcaaagct agcctaccgg tcgccaccat ggtgagcaag ggcgaggaga
      661 ccacaatggg cgtaatcaag cccgacatga agatcaagct gaagatggag ggcaacgtga
      721 atggccacgc cttcgtgatc gagggcgagg gcgagggcaa gccctacgac ggcaccaaca
      781 ccatcaacct ggaggtgaag gagggagccc ccctgccctt ctcctacgac attctgacca
      841 ccgcgttcag ttacggcaac agggccttca ccaagtaccc cgacgacatc cccaactact
      901 tcaagcagtc cttccccgag ggctactctt gggagcgcac catgaccttc gaggacaagg
      961 gcatcgtgaa ggtgaagtcc gacatctcca tggaggagga ctccttcatc tacgagatac
     1021 acctcaaggg cgagaacttc ccccccaacg gccccgtgat gcagaaggag accaccggct
     1081 gggacgcctc caccgagagg atgtacgtgc gcgacggcgt gctgaagggc gacgtcaaga
     1141 tgaagctgct gctggagggc ggcggccacc accgcgttga cttcaagacc atctacaggg
     1201 ccaagaaggc ggtgaagctg cccgactatc actttgtgga ccaccgcatc gagatcctga
     1261 accacgacaa ggactacaac aaggtgaccg tttacgagat cgccgtggcc cgcaactcca
     1321 ccgacggcat ggacgagctg tacaagtccg gactcaccgg tggaggcggt tcaggcggag
     1381 gtggctctgg cggtggcgga tcgagatctc gagctcaagc ttcgaattct gcagtcgacg
     1441 gtaccgcggg cccgggatcc accggatcta gataactgat caactcctca ggtgcaggct
     1501 gcctatcaat tcgatatcaa gcttatcgat aatcaacctc tggattacaa aatttgtgaa
     1561 agattgactg gtattcttaa ctatgttgct ccttttacgc tatgtggata cgctgcttta
     1621 atgcctttgt atcatgctat tgcttcccgt atggctttca ttttctcctc cttgtataaa
     1681 tcctggttgc tgtctcttta tgaggagttg tggcccgttg tcaggcaacg tggcgtggtg
     1741 tgcactgtgt ttgctgacgc aacccccact ggttggggca ttgccaccac ctgtcagctc
     1801 ctttccggga ctttcgcttt ccccctccct attgccacgg cggaactcat cgccgcctgc
     1861 cttgcccgct gctggacagg ggctcggctg ttgggcactg acaattccgt ggtgttgtcg
     1921 gggaaatcat cgtcctttcc ttggctgctc gcctatgttg ccacctggat tctgcgcggg
     1981 acgtccttct gctacgtccc ttcggccctc aatccagcgg accttccttc ccgcggcctg
     2041 ctgccggctc tgcggcctct tccgcgtctt cgccttcgcc ctcagacgag tcggatctcc
     2101 ctttgggccg cctccccgca tcgataccga gcgctgctcg agagatctac gggtggcatc
     2161 cctgtgaccc ctccccagtg cctctcctgg ccctggaagt tgccactcca gtgcccacca
     2221 gccttgtcct aataaaatta agttgcatca ttttgtctga ctaggtgtcc ttctataata
     2281 ttatggggtg gaggggggtg gtatggagca aggggcaagt tgggaagaca acctgtaggg
     2341 cctgcggggt ctattgggaa ccaagctgga gtgcagtggc acaatcttgg ctcactgcaa
     2401 tctccgcctc ctgggttcaa gcgattctcc tgcctcagcc tcccgagttg ttgggattcc
     2461 aggcatgcat gaccaggctc agctaatttt tgtttttttg gtagagacgg ggtttcacca
     2521 tattggccag gctggtctcc aactcctaat ctcaggtgat ctacccacct tggcctccca
     2581 aattgctggg attacaggcg tgaaccactg ctcccttccc tgtccttctg attttgtagg
     2641 taaccacgtg cggaccgagc ggccgcagga acccctagtg atggagttgg ccactccctc
     2701 tctgcgcgct cgctcgctca ctgaggccgg gcgaccaaag gtcgcccgac gcccgggctt
     2761 tgcccgggcg gcctcagtga gcgagcgagc gcgcagctgc ctgcaggggc gcctgatgcg
     2821 gtattttctc cttacgcatc tgtgcggtat ttcacaccgc atacgtcaaa gcaaccatag
     2881 tacgcgccct gtagcggcgc attaagcgcg gcgggtgtgg tggttacgcg cagcgtgacc
     2941 gctacacttg ccagcgcctt agcgcccgct cctttcgctt tcttcccttc ctttctcgcc
     3001 acgttcgccg gctttccccg tcaagctcta aatcgggggc tccctttagg gttccgattt
     3061 agtgctttac ggcacctcga ccccaaaaaa cttgatttgg gtgatggttc acgtagtggg
     3121 ccatcgccct gatagacggt ttttcgccct ttgacgttgg agtccacgtt ctttaatagt
     3181 ggactcttgt tccaaactgg aacaacactc aactctatct cgggctattc ttttgattta
     3241 taagggattt tgccgatttc ggtctattgg ttaaaaaatg agctgattta acaaaaattt
     3301 aacgcgaatt ttaacaaaat attaacgttt acaattttat ggtgcactct cagtacaatc
     3361 tgctctgatg ccgcatagtt aagccagccc cgacacccgc caacacccgc tgacgcgccc
     3421 tgacgggctt gtctgctccc ggcatccgct tacagacaag ctgtgaccgt ctccgggagc
     3481 tgcatgtgtc agaggttttc accgtcatca ccgaaacgcg cgagacgaaa gggcctcgtg
     3541 atacgcctat ttttataggt taatgtcatg ataataatgg tttcttagac gtcaggtggc
     3601 acttttcggg gaaatgtgcg cggaacccct atttgtttat ttttctaaat acattcaaat
     3661 atgtatccgc tcatgagaca ataaccctga taaatgcttc aataatattg aaaaaggaag
     3721 agtatgagta ttcaacattt ccgtgtcgcc cttattccct tttttgcggc attttgcctt
     3781 cctgtttttg ctcacccaga aacgctggtg aaagtaaaag atgctgaaga tcagttgggt
     3841 gcacgagtgg gttacatcga actggatctc aacagcggta agatccttga gagttttcgc
     3901 cccgaagaac gttttccaat gatgagcact tttaaagttc tgctatgtgg cgcggtatta
     3961 tcccgtattg acgccgggca agagcaactc ggtcgccgca tacactattc tcagaatgac
     4021 ttggttgagt actcaccagt cacagaaaag catcttacgg atggcatgac agtaagagaa
     4081 ttatgcagtg ctgccataac catgagtgat aacactgcgg ccaacttact tctgacaacg
     4141 atcggaggac cgaaggagct aaccgctttt ttgcacaaca tgggggatca tgtaactcgc
     4201 cttgatcgtt gggaaccgga gctgaatgaa gccataccaa acgacgagcg tgacaccacg
     4261 atgcctgtag caatggcaac aacgttgcgc aaactattaa ctggcgaact acttactcta
     4321 gcttcccggc aacaattaat agactggatg gaggcggata aagttgcagg accacttctg
     4381 cgctcggccc ttccggctgg ctggtttatt gctgataaat ctggagccgg tgagcgtggg
     4441 tctcgcggta tcattgcagc actggggcca gatggtaagc cctcccgtat cgtagttatc
     4501 tacacgacgg ggagtcaggc aactatggat gaacgaaata gacagatcgc tgagataggt
     4561 gcctcactga ttaagcattg gtaactgtca gaccaagttt actcatatat actttagatt
     4621 gatttaaaac ttcattttta atttaaaagg atctaggtga agatcctttt tgataatctc
     4681 atgaccaaaa tcccttaacg tgagttttcg ttccactgag cgtcagaccc cgtagaaaag
     4741 atcaaaggat cttcttgaga tccttttttt ctgcgcgtaa tctgctgctt gcaaacaaaa
     4801 aaaccaccgc taccagcggt ggtttgtttg ccggatcaag agctaccaac tctttttccg
     4861 aaggtaactg gcttcagcag agcgcagata ccaaatactg ttcttctagt gtagccgtag
     4921 ttaggccacc acttcaagaa ctctgtagca ccgcctacat acctcgctct gctaatcctg
     4981 ttaccagtgg ctgctgccag tggcgataag tcgtgtctta ccgggttgga ctcaagacga
     5041 tagttaccgg ataaggcgca gcggtcgggc tgaacggggg gttcgtgcac acagcccagc
     5101 ttggagcgaa cgacctacac cgaactgaga tacctacagc gtgagctatg agaaagcgcc
     5161 acgcttcccg aagggagaaa ggcggacagg tatccggtaa gcggcagggt cggaacagga
     5221 gagcgcacga gggagcttcc agggggaaac gcctggtatc tttatagtcc tgtcgggttt
     5281 cgccacctct gacttgagcg tcgatttttg tgatgctcgt caggggggcg gagcctatgg
     5341 aaaaacgcca gcaacgcggc ctttt
//
