LOCUS       sequence-415565-        7372 bp    DNA     circular SYN 15-APR-2025
DEFINITION  expresses cFOS-FRB-VP16 and EGFP component in rat hippocampal neuron
            cells.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 7372)
  AUTHORS   Cui M, Lee S, Ban SH, Ryu JR, Shen M, Yang SH, Kim JY, Choi SK, Han 
            J, Kim Y, Han K, Lee D, Sun W, Kwon HB, Lee D
  TITLE     A single-component, light-assisted uncaging switch for 
            endoproteolytic release.
  JOURNAL   Nat Chem Biol. 2024 Mar;20(3):353-364. doi: 
            10.1038/s41589-023-01480-6. Epub 2023 Nov 16.
  PUBMED    37973890
REFERENCE   2  (bases 1 to 7372)
  AUTHORS   .
  TITLE     Direct Submission
  JOURNAL   Exported Apr 15, 2025 from SnapGene Server 8.0.1
            https://www.snapgene.com
COMMENT     SGRef: number: 1; type: "Journal Article"; doi: 
            "10.1038/s41589-023-01480-6"; journalName: "Nat Chem Biol"; date: 
            "2024-03"; volume: "20"; issue: "3"; pages: "353-364"
COMMENT     Sequence Label: pAAV-cFOS-FRB-VP16-P2A-EGFP
FEATURES             Location/Qualifiers
     source          1..7372
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     complement(30..52)
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     primer_bind     152..171
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     385..404
                     /label=pBRrevBam
                     /note="pBR322 vectors, tet region, downstream of BamHI, 
                     reverse primer"
     promoter        694..712
                     /label=SP6 promoter
                     /note="promoter for bacteriophage SP6 RNA polymerase"
     repeat_region   733..862
                     /label=AAV2 ITR
     CDS             903..956
                     /codon_start=1
                     /product="2A peptide from Thosea asigna virus capsid 
                     protein"
                     /label=T2A
                     /note="Eukaryotic ribosomes fail to insert a peptide bond 
                     between the Gly and Pro residues, yielding separate 
                     polypeptides."
                     /translation="EGRGSLLTCGDVEENPGP"
     CDS             complement(1754..1765)
                     /codon_start=1
                     /product="WELQut protease recognition and cleavage site"
                     /label=WELQut site
                     /translation="WELQ"
     intron          1823..2052
                     /label=chimeric intron
                     /note="chimera between introns from adenovirus and 
                     immunoglobulin heavy chain genes"
     primer_bind     2035..2054
                     /label=pMT2-F
                     /note="Synthetic intron, forward primer"
     regulatory      2095..2104
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     CDS             2113..2391
                     /codon_start=1
                     /product="T2098L mutant of FKBP-rapamycin binding domain of
                     human FRAP"
                     /label=FRB*
                     /note="binds synthetic ligands such as AP21967"
                     /translation="ILWHEMWHEGLEEASRLYFGERNVKGMFEVLEPLHAMMERGPQTL
                     KETSFNQAYGRDLMEAQEWCRKYMKSGNVKDLLQAWDLYYHVFRRISK"
     CDS             2548..2781
                     /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             2791..2847
                     /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"
     CDS             join(2857..2859,2860..3573)
                     /codon_start=1
                     /product="the original enhanced GFP (Yang et al., 1996)"
                     /label=EGFP
                     /note="mammalian codon-optimized"
                     /translation="VSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTLK
                     FICTTGKLPVPWPTLVTTLTYGVQCFSRYPDHMKQHDFFKSAMPEGYVQERTIFFKDDG
                     NYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIKV
                     NFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSALSKDPNEKRDHMVLLE
                     FVTAAGITLGMDELYK"
     CDS             join(2857..2859,2860..3570)
                     /codon_start=1
                     /product="enhanced GFP"
                     /label=EGFP
                     /translation="VSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTLK
                     FICTTGKLPVPWPTLVTTLTYGVQCFSRYPDHMKQHDFFKSAMPEGYVQERTIFFKDDG
                     NYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIKV
                     NFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSALSKDPNEKRDHMVLLE
                     FVTAAGITLGMDELYK"
     primer_bind     complement(3160..3179)
                     /label=EXFP-R
                     /note="For distinguishing EGFP variants, reverse primer"
     primer_bind     3507..3528
                     /label=EGFP-C
                     /note="EGFP, forward primer"
     CDS             3587..3613
                     /codon_start=1
                     /product="HA (human influenza hemagglutinin) epitope tag"
                     /label=HA
                     /translation="YPYDVPDYA"
     promoter        complement(3683..3701)
                     /label=SP6 promoter
                     /note="promoter for bacteriophage SP6 RNA polymerase"
     intron          3785..3850
                     /label=small t intron
                     /note="SV40 (simian virus 40) small t antigen intron"
     misc_feature    3903..4491
                     /label=WPRE
                     /note="woodchuck hepatitis virus posttranscriptional 
                     regulatory element"
     primer_bind     complement(3956..3976)
                     /label=WPRE-R
                     /note="WPRE, reverse primer"
     CDS             complement(4374..4385)
                     /codon_start=1
                     /product="Factor Xa recognition and cleavage site"
                     /label=Factor Xa site
                     /translation="IEGR"
     primer_bind     complement(4518..4535)
                     /label=BGH-rev
                     /note="Bovine growth hormone terminator, reverse primer. 
                     Also called BGH reverse"
     polyA_signal    4524..4748
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     rep_origin      4972..5107
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     primer_bind     5293..5313
                     /label=pBABE 3'
                     /note="SV40 enhancer, reverse primer for pBABE vectors"
     rep_origin      complement(5554..6142)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(5634..5653)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(6313..7104,7105..7173))
                     /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     6936..6955
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(7174..7278)
                     /gene="bla"
                     /label=AmpR promoter
     primer_bind     7346..7364
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
ORIGIN
        1 ctcgcgcgtt tcggtgatga cggtgaaaac ctctgacaca tgcagctccc ggagacggtc
       61 acagcttgtc tgtaagcgga tgccgggagc agacaagccc gtcagggcgc gtcagcgggt
      121 gttggcgggt gtcggggctg gcttaactat gcggcatcag agcagattgt actgagagtg
      181 caccattcga cgctctccct tatgcgactc ctgcattagg aagcagccca gtagtaggtt
      241 gaggccgttg agcaccgccg ccgcaaggaa tggtgcatgc aaggagatgg cgcccaacag
      301 tcccccggcc acggggcctg ccaccatacc cacgccgaaa caagcgctca tgagcccgaa
      361 gtggcgagcc cgatcttccc catcggtgat gtcggcgata taggcgccag caaccgcacc
      421 tgtggcgccg gtgatgccgg ccacgatgcg tccggcgtag aggatctggc tagcgatgac
      481 cctgctgatt ggttcgctga ccatttccgg gtgcgggacg gcgttaccag aaactcagaa
      541 ggttcgtcca accaaaccga ctctgacgac agtttacgat agagatgata gggtctgctt
      601 cagtaagcca gatgctacac aattaggctt gtacatattg tcgttagaac gcggctacaa
      661 ttaatacata accttatgta tcatacacat acgatttagg tgacactata gaatacacgg
      721 aattaattct agctgcgcgc tcgctcgctc actgaggccg cccgggcaaa gcccgggcgt
      781 cgggcgacct ttggtcgccc ggcctcagtg agcgagcgag cgcgcagaga gggagtggcc
      841 aactccatca ctaggggttc cttgtagtta atgattaacc cgccatgcta cttatctacc
      901 tcgagggcag aggaagtctt ctaacatgcg gtgacgtgga ggagaatccc ggccctgcac
      961 cgggatccac catggtgagc aagggcgagg agctgttcac cggggtggtg cccatcctgg
     1021 tcgagctgga cggttaatta agccagacgg ccgcagcttt cctttaggaa cagaggcttc
     1081 gagcctttaa ggctgcgtac ttgcttctcc taataccaga gactcaaaaa aaaaaaaaaa
     1141 agttccagat tgctggacaa tgacccgggt ctcatccctt gaccctggga accgggtcca
     1201 cattgaatca ggtgcgaatg ttcgctcgcc ttctctgcct ttcccgcctc ccctcccccg
     1261 gccgcggccc cggttccccc cctgcgctgc accctcagag ttggctgcag ccggcgagct
     1321 gttcccgtca atccctccct cctttacaca ggatgtccat attaggacat ctgcgtcagc
     1381 aggtttccac ggccggtccc tgttgttctg gggggggacc atctccgaaa tcctacacgc
     1441 ggaaggtcta ggagaccccc taagatccca aatgtgaaca ctcataggtg aaagatgtat
     1501 gccaagacgg gggttgaaag cctggggcgt agagttgacg acagagcgcc cgcagagggc
     1561 cttggggcgc gcttcccccc ccttccagtt ccgcccagtg acgtaggaag tccatccatt
     1621 cacagcgctt ctataaaggc gccagctgag gcgcctacta ctccaaccgc gactgcagcg
     1681 agcaactgag aagactggat agagccggcg gttccgcgaa cgagcagtga ccgcgctccc
     1741 acccagctct gctctgcagc tcccaccagt gtctggccgc atcgattcta gaattcgctg
     1801 tctgcgaggg ccagctgttg gggtgagtac tccctctcaa aagcgggcat gacttctgcg
     1861 ctaagattgt cagtttccaa aaacgaggag gatttgatat tcacctggcc cgcggtgatg
     1921 cctttgaggg tggccgcgtc catctggtca gaaaagacaa tctttttgtt gtcaagcttg
     1981 aggtgtggca ggcttgagat ctggccatac acttgagtga caatgacatc cactttgcct
     2041 ttctctccac aggtgtccac tcccaggtcc aactgcagcc caagcggatc ggccgccacc
     2101 atggcttctc gaatcctctg gcatgagatg tggcatgaag gcctggaaga ggcatctcgt
     2161 ttgtactttg gggaaaggaa cgtgaaaggc atgtttgagg tgctggagcc cttgcatgct
     2221 atgatggaac ggggccccca gactctgaag gaaacatcct ttaatcaggc ctatggtcga
     2281 gatttaatgg aggcccaaga gtggtgcagg aagtacatga aatcagggaa tgtcaaggac
     2341 ctcctccaag cctgggacct ctattatcat gtgttccgac gaatctcaaa gactagtgcg
     2401 tacagccgcg cgcgtacgaa aaacaattac gggtctacca tcgagggcct gctcgatctc
     2461 ccggacgacg acgcccccga agaggcgggg ctggcggctc cgcgcctgtc ctttctcccc
     2521 gcgggacaca cgcgcagact atcgacggcc cccccgaccg atgtcagcct gggggacgag
     2581 ctccacttag acggcgagga cgtggcgatg gcgcatgccg acgcgctaga cgatttcgat
     2641 ctggacatgt tgggggacgg ggattccccg ggtccgggat ttacccccca cgactccgcc
     2701 ccctacggcg ctctggatat ggccgacttc gagtttgagc agatgtttac cgatgccctt
     2761 ggaattgacg agtacggtgg gggaagcgga gctactaact tcagcctgct gaagcaggct
     2821 ggagacgtgg aggagaaccc tggacctgaa ttcggggtga gcaagggcga ggagctgttc
     2881 accggggtgg tgcccatcct ggtcgagctg gacggcgacg taaacggcca caagttcagc
     2941 gtgtccggcg agggcgaggg cgatgccacc tacggcaagc tgaccctgaa gttcatctgc
     3001 accaccggca agctgcccgt gccctggccc accctcgtga ccaccctgac ctacggcgtg
     3061 cagtgcttca gccgctaccc cgaccacatg aagcagcacg acttcttcaa gtccgccatg
     3121 cccgaaggct acgtccagga gcgcaccatc ttcttcaagg acgacggcaa ctacaagacc
     3181 cgcgccgagg tgaagttcga gggcgacacc ctggtgaacc gcatcgagct gaagggcatc
     3241 gacttcaagg aggacggcaa catcctgggg cacaagctgg agtacaacta caacagccac
     3301 aacgtctata tcatggccga caagcagaag aacggcatca aggtgaactt caagatccgc
     3361 cacaacatcg aggacggcag cgtgcagctc gccgaccact accagcagaa cacccccatc
     3421 ggcgacggcc ccgtgctgct gcccgacaac cactacctga gcacccagtc cgccctgagc
     3481 aaagacccca acgagaagcg cgatcacatg gtcctgctgg agttcgtgac cgccgccggg
     3541 atcactctcg gcatggacga gctgtacaag tgataaggat ctcgactatc cgtacgacgt
     3601 accagactac gcactcgact aagaattatc catccatcac actggcggcc ggccgctcga
     3661 gcatgcatct agagggccct attctatagt gtcacctaaa tgctagagga tctttgtgaa
     3721 ggaaccttac ttctgtggtg tgacataatt ggacaaacta cctacagaga tttaaagctc
     3781 taaggtaaat ataaaatttt taagtgtata atgtgttaaa ctactgattc taattgtttg
     3841 tgtattttag attccaacct atggaactga tgagcggccg caagctttaa cagcttatcg
     3901 ataatcaacc tctggattac aaaatttgtg aaagattgac tggtattctt aactatgttg
     3961 ctccttttac gctatgtgga tacgctgctt taatgccttt gtatcatgct attgcttccc
     4021 gtatggcttt cattttctcc tccttgtata aatcctggtt gctgtctctt tatgaggagt
     4081 tgtggcccgt tgtcaggcaa cgtggcgtgg tgtgcactgt gtttgctgac gcaaccccca
     4141 ctggttgggg cattgccacc acctgtcagc tcctttccgg gactttcgct ttccccctcc
     4201 ctattgccac ggcggaactc atcgccgcct gccttgcccg ctgctggaca ggggctcggc
     4261 tgttgggcac tgacaattcc gtggtgttgt cggggaaatc atcgtccttt ccttggctgc
     4321 tcgcctgtgt tgccacctgg attctgcgcg ggacgtcctt ctgctacgtc ccttcggccc
     4381 tcaatccagc ggaccttcct tcccgcggcc tgctgccggc tctgcggcct cttccgcgtc
     4441 ttcgccttcg ccctcagacg agtcggatct ccctttgggc cgcctccccg catcgatacc
     4501 gtcgactcgc tgatcagcct cgactgtgcc ttctagttgc cagccatctg ttgtttgccc
     4561 ctcccccgtg ccttccttga ccctggaagg tgccactccc actgtccttt cctaataaaa
     4621 tgaggaaatt gcatcgcatt gtctgagtag gtgtcattct attctggggg gtggggtggg
     4681 gcaggacagc aagggggagg attgggaaga caatagcagg catgctgggg atgcggtggg
     4741 ctctatggct tctgaggcgg aaagaaccag ctggggctcg actagagcat ggctacgtag
     4801 ataagtagca tggcgggtta atcattaact acaaggaacc cctagtgatg gagttggcca
     4861 ctccctctct gcgcgctcgc tcgctcactg aggccgggcg accaaaggtc gcccgacgcc
     4921 cgggcggcct cagtgagcga gcgagcgcgc agagcttttt gcaaaagcct aggcctccaa
     4981 aaaagcctcc tcactacttc tggaatagct cagaggccga ggcggcctcg gcctctgcat
     5041 aaataaaaaa aattagtcag ccatggggcg gagaatgggc ggaactgggc ggagttaggg
     5101 gcgggatggg cggagttagg ggcgggatgg gcggagttag gggcgggact atggttgctg
     5161 actaattgag atgcatgctt tgcatacttc tgcctgctgg ggagcctggg gactttctac
     5221 acctggttgc tgactaattg agatgcatgc tttgcatact tctgcctgct ggggagcctg
     5281 gggactttcc acaccctaac tgacacacat tccacagctg cattaatgaa tcggccaacg
     5341 cgcggggaga ggcggtttgc gtattgggcg ctcttccgct tcctcgctca ctgactcgct
     5401 gcgctcggtc gttcggctgc ggcgagcggt atcagctcac tcaaaggcgg taatacggtt
     5461 atccacagaa tcaggggata acgcaggaaa gaacatgtga gcaaaaggcc agcaaaaggc
     5521 caggaaccgt aaaaaggccg cgttgctggc gtttttccat aggctccgcc cccctgacga
     5581 gcatcacaaa aatcgacgct caagtcagag gtggcgaaac ccgacaggac tataaagata
     5641 ccaggcgttt ccccctggaa gctccctcgt gcgctctcct gttccgaccc tgccgcttac
     5701 cggatacctg tccgcctttc tcccttcggg aagcgtggcg ctttctcata gctcacgctg
     5761 taggtatctc agttcggtgt aggtcgttcg ctccaagctg ggctgtgtgc acgaaccccc
     5821 cgttcagccc gaccgctgcg ccttatccgg taactatcgt cttgagtcca acccggtaag
     5881 acacgactta tcgccactgg cagcagccac tggtaacagg attagcagag cgaggtatgt
     5941 aggcggtgct acagagttct tgaagtggtg gcctaactac ggctacacta gaagaacagt
     6001 atttggtatc tgcgctctgc tgaagccagt taccttcgga aaaagagttg gtagctcttg
     6061 atccggcaaa caaaccaccg ctggtagcgg tggttttttt gtttgcaagc agcagattac
     6121 gcgcagaaaa aaaggatctc aagaagatcc tttgatcttt tctacggggt ctgacgctca
     6181 gtggaacgaa aactcacgtt aagggatttt ggtcatgaga ttatcaaaaa ggatcttcac
     6241 ctagatcctt ttaaattaaa aatgaagttt taaatcaatc taaagtatat atgagtaaac
     6301 ttggtctgac agttaccaat gcttaatcag tgaggcacct atctcagcga tctgtctatt
     6361 tcgttcatcc atagttgcct gactccccgt cgtgtagata actacgatac gggagggctt
     6421 accatctggc cccagtgctg caatgatacc gcgagaccca cgctcaccgg ctccagattt
     6481 atcagcaata aaccagccag ccggaagggc cgagcgcaga agtggtcctg caactttatc
     6541 cgcctccatc cagtctatta attgttgccg ggaagctaga gtaagtagtt cgccagttaa
     6601 tagtttgcgc aacgttgttg ccattgctac aggcatcgtg gtgtcacgct cgtcgtttgg
     6661 tatggcttca ttcagctccg gttcccaacg atcaaggcga gttacatgat cccccatgtt
     6721 gtgcaaaaaa gcggttagct ccttcggtcc tccgatcgtt gtcagaagta agttggccgc
     6781 agtgttatca ctcatggtta tggcagcact gcataattct cttactgtca tgccatccgt
     6841 aagatgcttt tctgtgactg gtgagtactc aaccaagtca ttctgagaat agtgtatgcg
     6901 gcgaccgagt tgctcttgcc cggcgtcaat acgggataat accgcgccac atagcagaac
     6961 tttaaaagtg ctcatcattg gaaaacgttc ttcggggcga aaactctcaa ggatcttacc
     7021 gctgttgaga tccagttcga tgtaacccac tcgtgcaccc aactgatctt cagcatcttt
     7081 tactttcacc agcgtttctg ggtgagcaaa aacaggaagg caaaatgccg caaaaaaggg
     7141 aataagggcg acacggaaat gttgaatact catactcttc ctttttcaat attattgaag
     7201 catttatcag ggttattgtc tcatgagcgg atacatattt gaatgtattt agaaaaataa
     7261 acaaataggg gttccgcgca catttccccg aaaagtgcca cctgacgtct aagaaaacat
     7321 tattatcatg acattaacct ataaaaatag gcgtatcacg aggccctttc gt
//
