LOCUS       sequence-394269-        4812 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  AAV-mediated expression of mNeonGreen under the TRE promoter in 
            mammalian cells.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 4812)
  AUTHORS   Leiwe MN, Fujimoto S, Baba T, Moriyasu D, Saha B, Sakaguchi R, 
            Inagaki S, Imai T
  TITLE     Automated neuronal reconstruction with super-multicolour Tetbow 
            labelling and threshold-based clustering of colour hues.
  JOURNAL   Nat Commun. 2024 Jun 25;15(1):5279. doi: 10.1038/s41467-024-49455-y.
  PUBMED    38918382
REFERENCE   2  (bases 1 to 4812)
  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: "Nat 
            Commun."; date: "2024-06-25"; volume: "15(1):5279. doi"; pages: " 
            10.1038/s41467-024-49455-y"
COMMENT     Sequence Label: pAAV-TRE-mNeonGreen-WPRE
FEATURES             Location/Qualifiers
     source          1..4812
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     repeat_region   19..148
                     /label=AAV2 ITR
     protein_bind    222..240
                     /gene="tetO"
                     /label=tet operator
                     /bound_moiety="tetracycline repressor TetR"
                     /note="bacterial operator O2 for the tetR and tetA genes"
     protein_bind    258..276
                     /gene="tetO"
                     /label=tet operator
                     /bound_moiety="tetracycline repressor TetR"
                     /note="bacterial operator O2 for the tetR and tetA genes"
     protein_bind    293..311
                     /gene="tetO"
                     /label=tet operator
                     /bound_moiety="tetracycline repressor TetR"
                     /note="bacterial operator O2 for the tetR and tetA genes"
     protein_bind    329..347
                     /gene="tetO"
                     /label=tet operator
                     /bound_moiety="tetracycline repressor TetR"
                     /note="bacterial operator O2 for the tetR and tetA genes"
     protein_bind    365..383
                     /gene="tetO"
                     /label=tet operator
                     /bound_moiety="tetracycline repressor TetR"
                     /note="bacterial operator O2 for the tetR and tetA genes"
     protein_bind    400..418
                     /gene="tetO"
                     /label=tet operator
                     /bound_moiety="tetracycline repressor TetR"
                     /note="bacterial operator O2 for the tetR and tetA genes"
     protein_bind    436..454
                     /gene="tetO"
                     /label=tet operator
                     /bound_moiety="tetracycline repressor TetR"
                     /note="bacterial operator O2 for the tetR and tetA genes"
     promoter        466..504
                     /label=minimal CMV promoter
                     /note="human cytomegalovirus (CMV) immediate early 
                     promoter"
     primer_bind     501..525
                     /label=LNCX
                     /note="Human CMV promoter, forward primer"
     regulatory      539..548
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     CDS             545..1255
                     /codon_start=1
                     /product="bright monomeric yellow-green fluorescent protein
                     derived from LanYFP (Shaner et al., 2013)"
                     /label=mNeonGreen
                     /translation="MVSKGEEDNMASLPATHELHIFGSINGVDFDMVGQGTGNPNDGYE
                     ELNLKSTKGDLQFSPWILVPHIGYGFHQYLPYPDGMSPFQAAMVDGSGYQVHRTMQFED
                     GASLTVNYRYTYEGSHIKGEAQVKGTGFPADGPVMTNSLTAADWCRSKKTYPNDKTIIS
                     TFKWSYTTGNGKRYRSTARTTYTFAKPMAANYLKNQPMYVFRKTELKHSKTELNFKEWQ
                     KAFTDVMGMDELYK"
     regulatory      800..809
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     misc_feature    1268..1856
                     /label=WPRE
                     /note="woodchuck hepatitis virus posttranscriptional 
                     regulatory element"
     primer_bind     complement(1321..1341)
                     /label=WPRE-R
                     /note="WPRE, reverse primer"
     CDS             complement(1739..1750)
                     /codon_start=1
                     /product="Factor Xa recognition and cleavage site"
                     /label=Factor Xa site
                     /translation="IEGR"
     primer_bind     complement(1859..1875)
                     /label=KS primer
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(1860..1876)
                     /label=pBluescriptKS
                     /note="For pBluescript vector"
     polyA_signal    1905..2026
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(1916..1935)
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     primer_bind     1970..1989
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     repeat_region   2188..2317
                     /label=AAV2 ITR
     rep_origin      complement(2554..3142)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(2634..2653)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(3313..4104,4105..4173))
                     /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     3936..3955
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(4174..4278)
                     /gene="bla"
                     /label=AmpR promoter
     rep_origin      complement(4304..4759)
                     /direction=LEFT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(4441..4462)
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     4653..4672
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
ORIGIN
        1 tcttcgctat tacgccagct gcgcgctcgc tcgctcactg aggccgcccg ggcaaagccc
       61 gggcgtcggg cgacctttgg tcgcccggcc tcagtgagcg agcgagcgcg cagagaggga
      121 gtggccaact ccatcactag gggttccttg tagttaatga ttaacccgcc atgctactta
      181 tctacgtagc catgctctag gaagatctcg aattggttta ctccctatca gtgatagaga
      241 acgtatgtcg agtttactcc ctatcagtga tagagaacga tgtcgagttt actccctatc
      301 agtgatagag aacgtatgtc gagtttactc cctatcagtg atagagaacg tatgtcgagt
      361 ttactcccta tcagtgatag agaacgtatg tcgagtttat ccctatcagt gatagagaac
      421 gtatgtcgag tttactccct atcagtgata gagaacgtat gtcgaggtag gcgtgtacgg
      481 tgggaggcct atataagcag agctcgttta gtgaaccgtc agatcgcctg gagaattcgc
      541 caccatggtg agcaagggcg aggaggataa catggcctct ctcccagcga cacatgagtt
      601 acacatcttt ggctccatca acggtgtgga ctttgacatg gtgggtcagg gcaccggcaa
      661 tccaaatgat ggttatgagg agttaaacct gaagtccacc aagggtgacc tccagttctc
      721 cccctggatt ctggtccctc atatcgggta tggcttccat cagtacctgc cctaccctga
      781 cgggatgtcg cctttccagg ccgccatggt agatggctcc ggataccaag tccatcgcac
      841 aatgcagttt gaagatggtg cctcccttac tgttaactac cgctacacct acgagggaag
      901 ccacatcaaa ggagaggccc aggtgaaggg gactggtttc cctgctgacg gtcctgtgat
      961 gaccaactcg ctgaccgctg cggactggtg caggtcgaag aagacttacc ccaacgacaa
     1021 aaccatcatc agtaccttta agtggagtta caccactgga aatggcaagc gctaccggag
     1081 cactgcgcgg accacctaca cctttgccaa gccaatggcg gctaactatc tgaagaacca
     1141 gccgatgtac gtgttccgta agacggagct caagcactcc aagaccgagc tcaacttcaa
     1201 ggagtggcaa aaggccttta ccgatgtgat gggcatggac gagctgtaca agtgaaagct
     1261 tatcgataat caacctctgg attacaaaat ttgtgaaaga ttgactggta ttcttaacta
     1321 tgttgctcct tttacgctat gtggatacgc tgctttaatg cctttgtatc atgctattgc
     1381 ttcccgtatg gctttcattt tctcctcctt gtataaatcc tggttgctgt ctctttatga
     1441 ggagttgtgg cccgttgtca ggcaacgtgg cgtggtgtgc actgtgtttg ctgacgcaac
     1501 ccccactggt tggggcattg ccaccacctg tcagctcctt tccgggactt tcgctttccc
     1561 cctccctatt gccacggcgg aactcatcgc cgcctgcctt gcccgctgct ggacaggggc
     1621 tcggctgttg ggcactgaca attccgtggt gttgtcgggg aaatcatcgt cctttccttg
     1681 gctgctcgcc tgtgttgcca cctggattct gcgcgggacg tccttctgct acgtcccttc
     1741 ggccctcaat ccagcggacc ttccttcccg cggcctgctg ccggctctgc ggcctcttcc
     1801 gcgtcttcgc cttcgccctc agacgagtcg gatctccctt tgggccgcct ccccgcatcg
     1861 ataccgtcga cctcgacccg ggcggccgct tcgagcagac atgataagat acattgatga
     1921 gtttggacaa accacaacta gaatgcagtg aaaaaaatgc tttatttgtg aaatttgtga
     1981 tgctattgct ttatttgtaa ccattataag ctgcaataaa caagttaaca acaacaattg
     2041 cattcatttt atgtttcagg ttcaggggga gatgtgggag gttttttaaa gcaagtaaaa
     2101 cctctacaaa tgtggtaaaa tcgataagga tcttcctaga gcatggctac gtagataagt
     2161 agcatggcgg gttaatcatt aactacaagg aacccctagt gatggagttg gccactccct
     2221 ctctgcgcgc tcgctcgctc actgaggccg ggcgaccaaa ggtcgcccga cgcccgggct
     2281 ttgcccgggc ggcctcagtg agcgagcgag cgcgcagctg cattaatgaa tcggccaacg
     2341 cgcggggaga ggcggtttgc gtattgggcg ctcttccgct tcctcgctca ctgactcgct
     2401 gcgctcggtc gttcggctgc ggcgagcggt atcagctcac tcaaaggcgg taatacggtt
     2461 atccacagaa tcaggggata acgcaggaaa gaacatgtga gcaaaaggcc agcaaaaggc
     2521 caggaaccgt aaaaaggccg cgttgctggc gtttttccat aggctccgcc cccctgacga
     2581 gcatcacaaa aatcgacgct caagtcagag gtggcgaaac ccgacaggac tataaagata
     2641 ccaggcgttt ccccctggaa gctccctcgt gcgctctcct gttccgaccc tgccgcttac
     2701 cggatacctg tccgcctttc tcccttcggg aagcgtggcg ctttctcata gctcacgctg
     2761 taggtatctc agttcggtgt aggtcgttcg ctccaagctg ggctgtgtgc acgaaccccc
     2821 cgttcagccc gaccgctgcg ccttatccgg taactatcgt cttgagtcca acccggtaag
     2881 acacgactta tcgccactgg cagcagccac tggtaacagg attagcagag cgaggtatgt
     2941 aggcggtgct acagagttct tgaagtggtg gcctaactac ggctacacta gaagaacagt
     3001 atttggtatc tgcgctctgc tgaagccagt taccttcgga aaaagagttg gtagctcttg
     3061 atccggcaaa caaaccaccg ctggtagcgg tggttttttt gtttgcaagc agcagattac
     3121 gcgcagaaaa aaaggatctc aagaagatcc tttgatcttt tctacggggt ctgacgctca
     3181 gtggaacgaa aactcacgtt aagggatttt ggtcatgaga ttatcaaaaa ggatcttcac
     3241 ctagatcctt ttaaattaaa aatgaagttt taaatcaatc taaagtatat atgagtaaac
     3301 ttggtctgac agttaccaat gcttaatcag tgaggcacct atctcagcga tctgtctatt
     3361 tcgttcatcc atagttgcct gactccccgt cgtgtagata actacgatac gggagggctt
     3421 accatctggc cccagtgctg caatgatacc gcgagaccca cgctcaccgg ctccagattt
     3481 atcagcaata aaccagccag ccggaagggc cgagcgcaga agtggtcctg caactttatc
     3541 cgcctccatc cagtctatta attgttgccg ggaagctaga gtaagtagtt cgccagttaa
     3601 tagtttgcgc aacgttgttg ccattgctac aggcatcgtg gtgtcacgct cgtcgtttgg
     3661 tatggcttca ttcagctccg gttcccaacg atcaaggcga gttacatgat cccccatgtt
     3721 gtgcaaaaaa gcggttagct ccttcggtcc tccgatcgtt gtcagaagta agttggccgc
     3781 agtgttatca ctcatggtta tggcagcact gcataattct cttactgtca tgccatccgt
     3841 aagatgcttt tctgtgactg gtgagtactc aaccaagtca ttctgagaat agtgtatgcg
     3901 gcgaccgagt tgctcttgcc cggcgtcaat acgggataat accgcgccac atagcagaac
     3961 tttaaaagtg ctcatcattg gaaaacgttc ttcggggcga aaactctcaa ggatcttacc
     4021 gctgttgaga tccagttcga tgtaacccac tcgtgcaccc aactgatctt cagcatcttt
     4081 tactttcacc agcgtttctg ggtgagcaaa aacaggaagg caaaatgccg caaaaaaggg
     4141 aataagggcg acacggaaat gttgaatact catactcttc ctttttcaat attattgaag
     4201 catttatcag ggttattgtc tcatgagcgg atacatattt gaatgtattt agaaaaataa
     4261 acaaataggg gttccgcgca catttccccg aaaagtgcca cctaaattgt aagcgttaat
     4321 attttgttaa aattcgcgtt aaatttttgt taaatcagct cattttttaa ccaataggcc
     4381 gaaatcggca aaatccctta taaatcaaaa gaatagaccg agatagggtt gagtgttgtt
     4441 ccagtttgga acaagagtcc actattaaag aacgtggact ccaacgtcaa agggcgaaaa
     4501 accgtctatc agggcgatgg cccactacgt gaaccatcac cctaatcaag ttttttgggg
     4561 tcgaggtgcc gtaaagcact aaatcggaac cctaaaggga gcccccgatt tagagcttga
     4621 cggggaaagc cggcgaacgt ggcgagaaag gaagggaaga aagcgaaagg agcgggcgct
     4681 agggcgctgg caagtgtagc ggtcacgctg cgcgtaacca ccacacccgc cgcgcttaat
     4741 gcgccgctac agggcgcgtc ccattcgcca ttcaggctgc gcaactgttg ggaagggcga
     4801 tcggtgcggg cc
//
