LOCUS       sequence-423200-        6504 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Expresses the split transcription factors in cultured neuron and 
            mouse brain.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 6504)
  AUTHORS   Shen J, Geng L, Li X, Emery C, Kroning K, Shingles G, Lee K, Heyden 
            M, Li P, Wang W
  TITLE     A general method for chemogenetic control of peptide function.
  JOURNAL   Nat Methods. 2023 Jan;20(1):112-122. doi: 
            10.1038/s41592-022-01697-8. Epub 2022 Dec 8.
  PUBMED    36481965
REFERENCE   2  (bases 1 to 6504)
  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"; doi: 
            "10.1038/s41592-022-01697-8"; journalName: "Nat Methods"; date: 
            "2023-01"; volume: "20"; issue: "1"; pages: "112-122"
COMMENT     Sequence Label: 
            pAAV-Synapsin-SspB-EGFP-VP16-P2A-FLAG-TetR-NLS-CapN-SsrA-CapC
FEATURES             Location/Qualifiers
     source          1..6504
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     repeat_region   1..130
                     /label=AAV2 ITR (alternate)
                     /note="Functional equivalent of wild-type AAV2 ITR"
     promoter        181..628
                     /label=hSyn promoter
                     /note="human synapsin I promoter; confers neuron-specific 
                     expression (Kugler et al., 2003)"
     CDS             join(990..992,993..995,996..1706)
                     /codon_start=1
                     /product="enhanced GFP"
                     /label=EGFP
                     /translation="MVSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTL
                     KFICTTGKLPVPWPTLVTTLTYGVQCFSRYPDHMKQHDFFKSAMPEGYVQERTIFFKDD
                     GNYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIK
                     VNFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSALSKDPNEKRDHMVLL
                     EFVTAAGITLGMDELYK"
     CDS             join(990..992,993..995,996..1706)
                     /codon_start=1
                     /product="the original enhanced GFP (Yang et al., 1996)"
                     /label=EGFP
                     /note="mammalian codon-optimized"
                     /translation="MVSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTL
                     KFICTTGKLPVPWPTLVTTLTYGVQCFSRYPDHMKQHDFFKSAMPEGYVQERTIFFKDD
                     GNYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIK
                     VNFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSALSKDPNEKRDHMVLL
                     EFVTAAGITLGMDELYK"
     primer_bind     complement(1035..1056)
                     /label=EGFP-N
                     /note="EGFP, reverse primer"
     primer_bind     complement(1296..1315)
                     /label=EXFP-R
                     /note="For distinguishing EGFP variants, reverse primer"
     primer_bind     1643..1664
                     /label=EGFP-C
                     /note="EGFP, forward primer"
     CDS             1707..1727
                     /codon_start=1
                     /product="nuclear localization signal of SV40 (simian virus
                     40) large T antigen"
                     /label=SV40 NLS
                     /translation="PKKKRKV"
     CDS             1728..1961
                     /codon_start=1
                     /gene="UL48"
                     /product="transcriptional activation domain of herpes 
                     simplex virus protein VP16 (Triezenberg et al., 1988; 
                     Cousens et al., 1989)"
                     /label=VP16 AD
                     /translation="APPTDVSLGDELHLDGEDVAMAHADALDDFDLDMLGDGDSPGPGF
                     TPHDSAPYGALDMADFEFEQMFTDALGIDEYGG"
     CDS             1971..2027
                     /codon_start=1
                     /product="2A peptide from porcine teschovirus-1 
                     polyprotein"
                     /label=P2A
                     /note="Eukaryotic ribosomes fail to insert a peptide bond 
                     between the Gly and Pro residues, yielding separate 
                     polypeptides."
                     /translation="ATNFSLLKQAGDVEENPGP"
     primer_bind     complement(2101..2120)
                     /label=Tet-R
                     /note="Tetracycline resistance gene, reverse primer"
     CDS             2640..2660
                     /codon_start=1
                     /product="nuclear localization signal of SV40 (simian virus
                     40) large T antigen"
                     /label=SV40 NLS
                     /translation="PKKKRKV"
     CDS             3117..3437
                     /codon_start=1
                     /product="F36V mutant of FK506-binding protein FKBP12"
                     /label=FKBP-V
                     /note="binds synthetic ligands that do not bind wild-type 
                     FKBP"
                     /translation="GVQVETISPGDGRTFPKRGQTCVVHYTGMLEDGKKVDSSRDRNKP
                     FKFMLGKQEVIRGWEEGVAQMSVGQRAKLTISPDYAYGATGHPGIIPPHATLVFDVELL
                     KLE"
     CDS             3438..3464
                     /codon_start=1
                     /product="HA (human influenza hemagglutinin) epitope tag"
                     /label=HA
                     /translation="YPYDVPDYA"
     CDS             3465..3491
                     /codon_start=1
                     /product="HA (human influenza hemagglutinin) epitope tag"
                     /label=HA
                     /translation="YPYDVPDYA"
     primer_bind     complement(3525..3542)
                     /label=BGH-rev
                     /note="Bovine growth hormone terminator, reverse primer. 
                     Also called BGH reverse"
     polyA_signal    3531..3738
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     repeat_region   3767..3907
                     /label=AAV2 ITR
                     /note="inverted terminal repeat of adeno-associated virus 
                     serotype 2"
     repeat_region   3767..3896
                     /label=AAV2 ITR
     rep_origin      3982..4437
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(4069..4088)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     4279..4300
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     complement(4454..4473)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     4573..4595
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     primer_bind     complement(4633..4651)
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     promoter        4719..4823
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(4824..4892,4893..5684)
                     /codon_start=1
                     /gene="bla"
                     /product="beta-lactamase"
                     /label=AmpR
                     /note="confers resistance to ampicillin, carbenicillin, and
                     related antibiotics"
                     /translation="MSIQHFRVALIPFFAAFCLPVFAHPETLVKVKDAEDQLGARVGYI
                     ELDLNSGKILESFRPEERFPMMSTFKVLLCGAVLSRIDAGQEQLGRRIHYSQNDLVEYS
                     PVTEKHLTDGMTVRELCSAAITMSDNTAANLLLTTIGGPKELTAFLHNMGDHVTRLDRW
                     EPELNEAIPNDERDTTMPVAMATTLRKLLTGELLTLASRQQLIDWMEVDKVAGPLLRSA
                     LPAGWFIADKSGAGERGSRGIIAALGPDGKPSRIVVIYTTGSQATMDERNRQIAEIGAS
                     LIKHW"
     primer_bind     complement(5042..5061)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      5855..6443
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     6344..6363
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
ORIGIN
        1 cctgcaggca gctgcgcgct cgctcgctca ctgaggccgc ccgggcgtcg ggcgaccttt
       61 ggtcgcccgg cctcagtgag cgagcgagcg cgcagagagg gagtggccaa ctccatcact
      121 aggggttcct gcggccgcac gcgttgtaca gtgtctagac tgcagagggc cctgcgtatg
      181 agtgcaagtg ggttttagga ccaggatgag gcggggtggg ggtgcctacc tgacgaccga
      241 ccccgaccca ctggacaagc acccaacccc cattccccaa attgcgcatc ccctatcaga
      301 gagggggagg ggaaacagga tgcggcgagg cgcgtgcgca ctgccagctt cagcaccgcg
      361 gacagtgcct tcgcccccgc ctggcggcgc gcgccaccgc cgcctcagca ctgaaggcgc
      421 gctgacgtca ctcgccggtc ccccgcaaac tccccttccc ggccaccttg gtcgcgtccg
      481 cgccgccgcc ggcccagccg gaccgcacca cgcgaggcgc gagatagggg ggcacgggcg
      541 cgaccatctg cgctgcggcg ccggcgactc agcgctgcct cagtctgcgg tgggcagcgg
      601 aggagtcgtg tcgtgcctga gagcgcagtc gagaaggatc cgccaccatg agccaactga
      661 ctcctcgaag gccatatttg ctgagggcct tttatgaatg gctcctcgat aatcaactta
      721 ccccacactt ggtggtagat gtaacccttc ctggcgtaca ggtacccatg gaatacgcca
      781 gagacggtca aattgtactt aatattgccc cacgcgccgt tggcaacctt gagctggcaa
      841 acgatgaggt ccgctttaac gcacggtttg gaggcattcc acgacaagta tccgtgcctc
      901 tggccgccgt acttgctata tacgcccgcg agaatggagc cggcaccatg ttcgagccag
      961 aagccgccta tgatggtagt gggggaggaa tggtgagcaa gggcgaggag ctgttcaccg
     1021 gggtggtgcc catcctggtc gagctggacg gcgacgtaaa cggccacaag ttcagcgtgt
     1081 ccggcgaggg cgagggcgat gccacctacg gcaagctgac cctgaagttc atctgcacca
     1141 ccggcaagct gcccgtgccc tggcccaccc tcgtgaccac cctgacctac ggcgtgcagt
     1201 gcttcagccg ctaccccgac cacatgaagc agcacgactt cttcaagtcc gccatgcccg
     1261 aaggctacgt ccaggagcgc accatcttct tcaaggacga cggcaactac aagacccgcg
     1321 ccgaggtgaa gttcgagggc gacaccctgg tgaaccgcat cgagctgaag ggcatcgact
     1381 tcaaggagga cggcaacatc ctggggcaca agctggagta caactacaac agccacaacg
     1441 tctatatcat ggccgacaag cagaagaacg gcatcaaggt gaacttcaag atccgccaca
     1501 acatcgagga cggcagcgtg cagctcgccg accactacca gcagaacacc cccatcggcg
     1561 acggccccgt gctgctgccc gacaaccact acctgagcac ccagtccgcc ctgagcaaag
     1621 accccaacga gaagcgcgat cacatggtcc tgctggagtt cgtgaccgcc gccgggatca
     1681 ctctcggcat ggacgagctg tacaagccca agaagaagag gaaggtggcc cccccgaccg
     1741 atgtcagcct gggggacgaa ttgcacttag acggcgagga cgtggcgatg gcgcatgccg
     1801 acgcgctaga cgatttcgat ctggacatgt tgggggacgg ggattccccg ggtccgggat
     1861 ttacccccca cgactccgcc ccctacggcg ctctggatat ggccgacttc gagtttgagc
     1921 agatgtttac cgatgccctt ggaattgacg agtacggtgg gggaagcgga gctactaact
     1981 tcagcctgct gaagcaggct ggagacgtgg aggagaaccc tggacctatg tctagattag
     2041 ataaaagtaa agtgattaac agcgcattag agctgcttaa tgaggtcgga atcgaaggtt
     2101 taacaacccg taaactcgcc cagaagctag gtgtagagca gcctacattg tattggcatg
     2161 taaaaaataa gcgggctttg ctcgacgcct tagccattga gatgttagat aggcaccata
     2221 ctcacttttg ccctttagaa ggggaaagct ggcaagattt tttacgtaat aacgctaaaa
     2281 gttttagatg tgctttacta agtcatcgcg atggagcaaa agtacattta ggtacacggc
     2341 ctacagaaaa acagtatgaa actctcgaaa atcaattagc ctttttatgc caacaaggtt
     2401 tttcactaga gaatgcatta tatgcactca gcgctgtggg gcattttact ttaggttgcg
     2461 tattggaaga tcaagagcat caagtcgcta aagaagaaag ggaaacacct actactgata
     2521 gtatgccgcc attattacga caagctatcg aattatttga tcaccaaggt gcagagccag
     2581 ccttcttatt cggccttgaa ttgatcatat gcggattaga aaaacaactt aaatgtgaac
     2641 ccaagaagaa gaggaaggtg gctagcggcg tccaagtcga gacaatatct cccggcgacg
     2701 gacgaacgtt tccgaagcgg ggacagactt gcgtcgttca ctacacagga atgctggagg
     2761 atggcaaaaa ggtcgattcc agccgggaca ggaataagcc gttcaaattc atgctgggaa
     2821 aacaggaggt aatacgaggt tgggaggagg gtgtggcgca aatgtctgtc ggtcagcggg
     2881 cgaaactgac catttctcct gattatgctt acggagcgac aggccatccg gggatcattc
     2941 cgccccacgc taccctggta ttcgatgtag aactcttgaa gctcgaaacc agaggggttg
     3001 aagaagtagc cagatattcc cccaacctgg cagcgaatga tgaaaattac ggtactccta
     3061 atttgcggcc ttttggttca ggcggttctg gtactggatc tggttctgga ggttctggag
     3121 tgcaggtgga aaccatctcc ccaggagacg ggcgcacctt ccccaagcgc ggccagacct
     3181 gcgtggtgca ctacaccggg atgcttgaag atggaaagaa agttgattcc tcccgggaca
     3241 gaaacaagcc ctttaagttt atgctaggca agcaggaggt gatccgaggc tgggaagaag
     3301 gggttgccca gatgagtgtg ggtcagagag ccaaactgac tatatctcca gattatgcct
     3361 atggtgccac tgggcaccca ggcatcatcc caccacatgc cactctcgtc ttcgatgtgg
     3421 agcttctaaa actggaatac ccatacgatg tgccagatta cgcctatccc tatgacgtcc
     3481 ctgattacgc ttaagaattc ctcgagctag agctcgctga tcagcctcga ctgtgccttc
     3541 tagttgccag ccatctgttg tttgcccctc ccccgtgcct tccttgaccc tggaaggtgc
     3601 cactcccact gtcctttcct aataaaatga ggaaattgca tcgcattgtc tgagtaggtg
     3661 tcattctatt ctggggggtg gggtggggca ggacagcaag ggggaggatt gggaagacaa
     3721 tagcaggcat gctggggagg taaccacgtg cggaccgagc ggccgcagga acccctagtg
     3781 atggagttgg ccactccctc tctgcgcgct cgctcgctca ctgaggccgg gcgaccaaag
     3841 gtcgcccgac gcccgggctt tgcccgggcg gcctcagtga gcgagcgagc gcgcagctgc
     3901 ctgcaggggc gcctgatgcg gtattttctc cttacgcatc tgtgcggtat ttcacaccgc
     3961 atacgtcaaa gcaaccatag tacgcgccct gtagcggcgc attaagcgcg gcgggtgtgg
     4021 tggttacgcg cagcgtgacc gctacacttg ccagcgcctt agcgcccgct cctttcgctt
     4081 tcttcccttc ctttctcgcc acgttcgccg gctttccccg tcaagctcta aatcgggggc
     4141 tccctttagg gttccgattt agtgctttac ggcacctcga ccccaaaaaa cttgatttgg
     4201 gtgatggttc acgtagtggg ccatcgccct gatagacggt ttttcgccct ttgacgttgg
     4261 agtccacgtt ctttaatagt ggactcttgt tccaaactgg aacaacactc aactctatct
     4321 cgggctattc ttttgattta taagggattt tgccgatttc ggtctattgg ttaaaaaatg
     4381 agctgattta acaaaaattt aacgcgaatt ttaacaaaat attaacgttt acaattttat
     4441 ggtgcactct cagtacaatc tgctctgatg ccgcatagtt aagccagccc cgacacccgc
     4501 caacacccgc tgacgcgccc tgacgggctt gtctgctccc ggcatccgct tacagacaag
     4561 ctgtgaccgt ctccgggagc tgcatgtgtc agaggttttc accgtcatca ccgaaacgcg
     4621 cgagacgaaa gggcctcgtg atacgcctat ttttataggt taatgtcatg ataataatgg
     4681 tttcttagac gtcaggtggc acttttcggg gaaatgtgcg cggaacccct atttgtttat
     4741 ttttctaaat acattcaaat atgtatccgc tcatgagaca ataaccctga taaatgcttc
     4801 aataatattg aaaaaggaag agtatgagta ttcaacattt ccgtgtcgcc cttattccct
     4861 tttttgcggc attttgcctt cctgtttttg ctcacccaga aacgctggtg aaagtaaaag
     4921 atgctgaaga tcagttgggt gcacgagtgg gttacatcga actggatctc aacagcggta
     4981 agatccttga gagttttcgc cccgaagaac gttttccaat gatgagcact tttaaagttc
     5041 tgctatgtgg cgcggtatta tcccgtattg acgccgggca agagcaactc ggtcgccgca
     5101 tacactattc tcagaatgac ttggttgagt actcaccagt cacagaaaag catcttacgg
     5161 atggcatgac agtaagagaa ttatgcagtg ctgccataac catgagtgat aacactgcgg
     5221 ccaacttact tctgacaacg atcggaggac cgaaggagct aaccgctttt ttgcacaaca
     5281 tgggggatca tgtaactcgc cttgatcgtt gggaaccgga gctgaatgaa gccataccaa
     5341 acgacgagcg tgacaccacg atgcctgtag caatggcaac aacgttgcgc aaactattaa
     5401 ctggcgaact acttactcta gcttcccggc aacaattaat agactggatg gaggtggata
     5461 aagttgcagg accacttctg cgctcggccc ttccggctgg ctggtttatt gctgataaat
     5521 ctggagccgg tgagcgtgga agccgcggta tcattgcagc actggggcca gatggtaagc
     5581 cctcccgtat cgtagttatc tacacgacgg ggagtcaggc aactatggat gaacgaaata
     5641 gacagatcgc tgagataggt gcctcactga ttaagcattg gtaactgtca gaccaagttt
     5701 actcatatat actttagatt gatttaaaac ttcattttta atttaaaagg atctaggtga
     5761 agatcctttt tgataatctc atgaccaaaa tcccttaacg tgagttttcg ttccactgag
     5821 cgtcagaccc cgtagaaaag atcaaaggat cttcttgaga tccttttttt ctgcgcgtaa
     5881 tctgctgctt gcaaacaaaa aaaccaccgc taccagcggt ggtttgtttg ccggatcaag
     5941 agctaccaac tctttttccg aaggtaactg gcttcagcag agcgcagata ccaaatactg
     6001 ttcttctagt gtagccgtag ttaggccacc acttcaagaa ctctgtagca ccgcctacat
     6061 acctcgctct gctaatcctg ttaccagtgg ctgctgccag tggcgataag tcgtgtctta
     6121 ccgggttgga ctcaagacga tagttaccgg ataaggcgca gcggtcgggc tgaacggggg
     6181 gttcgtgcac acagcccagc ttggagcgaa cgacctacac cgaactgaga tacctacagc
     6241 gtgagctatg agaaagcgcc acgcttcccg aagggagaaa ggcggacagg tatccggtaa
     6301 gcggcagggt cggaacagga gagcgcacga gggagcttcc agggggaaac gcctggtatc
     6361 tttatagtcc tgtcgggttt cgccacctct gacttgagcg tcgatttttg tgatgctcgt
     6421 caggggggcg gagcctatgg aaaaacgcca gcaacgcggc ctttttacgg ttcctggcct
     6481 tttgctggcc ttttgctcac atgt
//
