LOCUS       sequence-423198-        6476 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Expresses the split transcription factors in mouse liver.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 6476)
  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 6476)
  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-CMV-SspB-EGFP-VP16-P2A-FLAG-Gal4DBD-CapN-SsrA-CapC-HA
FEATURES             Location/Qualifiers
     source          1..6476
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     repeat_region   1..130
                     /label=AAV2 ITR (alternate)
                     /note="Functional equivalent of wild-type AAV2 ITR"
     enhancer        201..504
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
     promoter        505..708
                     /label=CMV promoter
                     /note="human cytomegalovirus (CMV) immediate early 
                     promoter"
     primer_bind     659..679
                     /label=CMV-F
                     /note="Human CMV immediate early promoter, forward primer"
     primer_bind     753..772
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        753..771
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     CDS             join(1133..1135,1136..1138,1139..1849)
                     /codon_start=1
                     /product="enhanced GFP"
                     /label=EGFP
                     /translation="MVSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTL
                     KFICTTGKLPVPWPTLVTTLTYGVQCFSRYPDHMKQHDFFKSAMPEGYVQERTIFFKDD
                     GNYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIK
                     VNFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSALSKDPNEKRDHMVLL
                     EFVTAAGITLGMDELYK"
     CDS             join(1133..1135,1136..1138,1139..1849)
                     /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(1178..1199)
                     /label=EGFP-N
                     /note="EGFP, reverse primer"
     primer_bind     complement(1439..1458)
                     /label=EXFP-R
                     /note="For distinguishing EGFP variants, reverse primer"
     primer_bind     1786..1807
                     /label=EGFP-C
                     /note="EGFP, forward primer"
     CDS             1850..1870
                     /codon_start=1
                     /product="nuclear localization signal of SV40 (simian virus
                     40) large T antigen"
                     /label=SV40 NLS
                     /translation="PKKKRKV"
     CDS             1871..2104
                     /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             2114..2170
                     /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             2171..2608
                     /codon_start=1
                     /gene="Saccharomyces cerevisiae GAL4 (truncated)"
                     /product="DNA binding domain of the GAL4 transcriptional 
                     activator"
                     /label=GAL4 DNA binding domain
                     /translation="KLLSSIEQACDICRLKKLKCSKEKPKCAKCLKNNWECRYSPKTKR
                     SPLTRAHLTEVESRLERLEQLFLLIFPREDLDMILKMDSLQDIKALLTGLFVQDNVNKD
                     AVTDRLASVETDMPLTLRQHRISATSSSEESSNKGQRQLTVS"
     CDS             2609..2629
                     /codon_start=1
                     /product="nuclear localization signal of SV40 (simian virus
                     40) large T antigen"
                     /label=SV40 NLS
                     /translation="PKKKRKV"
     CDS             3089..3409
                     /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             3410..3436
                     /codon_start=1
                     /product="HA (human influenza hemagglutinin) epitope tag"
                     /label=HA
                     /translation="YPYDVPDYA"
     CDS             3437..3463
                     /codon_start=1
                     /product="HA (human influenza hemagglutinin) epitope tag"
                     /label=HA
                     /translation="YPYDVPDYA"
     primer_bind     complement(3497..3514)
                     /label=BGH-rev
                     /note="Bovine growth hormone terminator, reverse primer. 
                     Also called BGH reverse"
     polyA_signal    3503..3710
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     repeat_region   3739..3879
                     /label=AAV2 ITR
                     /note="inverted terminal repeat of adeno-associated virus 
                     serotype 2"
     repeat_region   3739..3868
                     /label=AAV2 ITR
     rep_origin      3954..4409
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(4041..4060)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     4251..4272
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     complement(4426..4445)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     4545..4567
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     primer_bind     complement(4605..4623)
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     promoter        4691..4795
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(4796..4864,4865..5656)
                     /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(5014..5033)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      5827..6415
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     6316..6335
                     /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 tagtaatcaa ttacggggtc attagttcat
      181 agcccatata tggagttccg cgttacataa cttacggtaa atggcccgcc tggctgaccg
      241 cccaacgacc cccgcccatt gacgtcaata atgacgtatg ttcccatagt aacgccaata
      301 gggactttcc attgacgtca atgggtggag tatttacggt aaactgccca cttggcagta
      361 catcaagtgt atcatatgcc aagtacgccc cctattgacg tcaatgacgg taaatggccc
      421 gcctggcatt atgcccagta catgacctta tgggactttc ctacttggca gtacatctac
      481 gtattagtca tcgctattac catggtgatg cggttttggc agtacatcaa tgggcgtgga
      541 tagcggtttg actcacgggg atttccaagt ctccacccca ttgacgtcaa tgggagtttg
      601 ttttggcacc aaaatcaacg ggactttcca aaatgtcgta acaactccgc cccattgacg
      661 caaatgggcg gtaggcgtgt acggtgggag gtctatataa gcagagctct ctggctaact
      721 agagaaccca ctgcttactg gcttatcgaa attaatacga ctcactatag ggagacccaa
      781 gcttgccacc atgagccaac tgactcctcg aaggccatat ttgctgaggg ccttttatga
      841 atggctcctc gataatcaac ttaccccaca cttggtggta gatgtaaccc ttcctggcgt
      901 acaggtaccc atggaatacg ccagagacgg tcaaattgta cttaatattg ccccacgcgc
      961 cgttggcaac cttgagctgg caaacgatga ggtccgcttt aacgcacggt ttggaggcat
     1021 tccacgacaa gtatccgtgc ctctggccgc cgtacttgct atatacgccc gcgagaatgg
     1081 agccggcacc atgttcgagc cagaagccgc ctatgatggt agtgggggag gaatggtgag
     1141 caagggcgag gagctgttca ccggggtggt gcccatcctg gtcgagctgg acggcgacgt
     1201 aaacggccac aagttcagcg tgtccggcga gggcgagggc gatgccacct acggcaagct
     1261 gaccctgaag ttcatctgca ccaccggcaa gctgcccgtg ccctggccca ccctcgtgac
     1321 caccctgacc tacggcgtgc agtgcttcag ccgctacccc gaccacatga agcagcacga
     1381 cttcttcaag tccgccatgc ccgaaggcta cgtccaggag cgcaccatct tcttcaagga
     1441 cgacggcaac tacaagaccc gcgccgaggt gaagttcgag ggcgacaccc tggtgaaccg
     1501 catcgagctg aagggcatcg acttcaagga ggacggcaac atcctggggc acaagctgga
     1561 gtacaactac aacagccaca acgtctatat catggccgac aagcagaaga acggcatcaa
     1621 ggtgaacttc aagatccgcc acaacatcga ggacggcagc gtgcagctcg ccgaccacta
     1681 ccagcagaac acccccatcg gcgacggccc cgtgctgctg cccgacaacc actacctgag
     1741 cacccagtcc gccctgagca aagaccccaa cgagaagcgc gatcacatgg tcctgctgga
     1801 gttcgtgacc gccgccggga tcactctcgg catggacgag ctgtacaagc ccaagaagaa
     1861 gaggaaggtg gcccccccga ccgatgtcag cctgggggac gaattgcact tagacggcga
     1921 ggacgtggcg atggcgcatg ccgacgcgct agacgatttc gatctggaca tgttggggga
     1981 cggggattcc ccgggtccgg gatttacccc ccacgactcc gccccctacg gcgctctgga
     2041 tatggccgac ttcgagtttg agcagatgtt taccgatgcc cttggaattg acgagtacgg
     2101 tgggggaagc ggagctacta acttcagcct gctgaagcag gctggagacg tggaggagaa
     2161 ccctggacct aagctactgt cttctatcga acaagcatgc gatatttgcc gacttaaaaa
     2221 gctcaagtgc tccaaagaaa aaccgaagtg cgccaagtgt ctgaagaaca actgggagtg
     2281 tcgctactct cccaaaacca aaaggtctcc gctgactagg gcacatctga cagaagtgga
     2341 atcaaggcta gaaagactgg aacagctatt tctactgatt tttcctcgag aagaccttga
     2401 catgattttg aaaatggatt ctttacagga tataaaagca ttgttaacag gattatttgt
     2461 acaagataat gtgaataaag atgccgtcac agatagattg gcttcagtgg agactgatat
     2521 gcctctaaca ttgagacagc atagaataag tgcgacatca tcatcggaag agagtagtaa
     2581 caaaggtcaa agacagttga ctgtatcgcc caagaagaag aggaaggtgg ctagcggcgt
     2641 ccaagtcgag acaatatctc ccggcgacgg acgaacgttt ccgaagcggg gacagacttg
     2701 cgtcgttcac tacacaggaa tgctggagga tggcaaaaag gtcgattcca gccgggacag
     2761 gaataagccg ttcaaattca tgctgggaaa acaggaggta atacgaggtt gggaggaggg
     2821 tgtggcgcaa atgtctgtcg gtcagcgggc gaaactgacc atttctcctg attatgctta
     2881 cggagcgaca ggccatccgg ggatcattcc gccccacgct accctggtat tcgatgtaga
     2941 actcttgaag ctcgaaacca gaggggttga agaagtagcc agatattccc ccaacctggc
     3001 agcgaatgat gaaaattact tcggtactcc taatttgcgg ccttttggtt caggcggttc
     3061 tggtactgga tctggttctg gaggttctgg agtgcaggtg gaaaccatct ccccaggaga
     3121 cgggcgcacc ttccccaagc gcggccagac ctgcgtggtg cactacaccg ggatgcttga
     3181 agatggaaag aaagttgatt cctcccggga cagaaacaag ccctttaagt ttatgctagg
     3241 caagcaggag gtgatccgag gctgggaaga aggggttgcc cagatgagtg tgggtcagag
     3301 agccaaactg actatatctc cagattatgc ctatggtgcc actgggcacc caggcatcat
     3361 cccaccacat gccactctcg tcttcgatgt ggagcttcta aaactggaat acccatacga
     3421 tgtgccagat tacgcctatc cctatgacgt ccctgattac gcttaagaat tcctcgagct
     3481 agagctcgct gatcagcctc gactgtgcct tctagttgcc agccatctgt tgtttgcccc
     3541 tcccccgtgc cttccttgac cctggaaggt gccactccca ctgtcctttc ctaataaaat
     3601 gaggaaattg catcgcattg tctgagtagg tgtcattcta ttctgggggg tggggtgggg
     3661 caggacagca agggggagga ttgggaagac aatagcaggc atgctgggga ggtaaccacg
     3721 tgcggaccga gcggccgcag gaacccctag tgatggagtt ggccactccc tctctgcgcg
     3781 ctcgctcgct cactgaggcc gggcgaccaa aggtcgcccg acgcccgggc tttgcccggg
     3841 cggcctcagt gagcgagcga gcgcgcagct gcctgcaggg gcgcctgatg cggtattttc
     3901 tccttacgca tctgtgcggt atttcacacc gcatacgtca aagcaaccat agtacgcgcc
     3961 ctgtagcggc gcattaagcg cggcgggtgt ggtggttacg cgcagcgtga ccgctacact
     4021 tgccagcgcc ttagcgcccg ctcctttcgc tttcttccct tcctttctcg ccacgttcgc
     4081 cggctttccc cgtcaagctc taaatcgggg gctcccttta gggttccgat ttagtgcttt
     4141 acggcacctc gaccccaaaa aacttgattt gggtgatggt tcacgtagtg ggccatcgcc
     4201 ctgatagacg gtttttcgcc ctttgacgtt ggagtccacg ttctttaata gtggactctt
     4261 gttccaaact ggaacaacac tcaactctat ctcgggctat tcttttgatt tataagggat
     4321 tttgccgatt tcggtctatt ggttaaaaaa tgagctgatt taacaaaaat ttaacgcgaa
     4381 ttttaacaaa atattaacgt ttacaatttt atggtgcact ctcagtacaa tctgctctga
     4441 tgccgcatag ttaagccagc cccgacaccc gccaacaccc gctgacgcgc cctgacgggc
     4501 ttgtctgctc ccggcatccg cttacagaca agctgtgacc gtctccggga gctgcatgtg
     4561 tcagaggttt tcaccgtcat caccgaaacg cgcgagacga aagggcctcg tgatacgcct
     4621 atttttatag gttaatgtca tgataataat ggtttcttag acgtcaggtg gcacttttcg
     4681 gggaaatgtg cgcggaaccc ctatttgttt atttttctaa atacattcaa atatgtatcc
     4741 gctcatgaga caataaccct gataaatgct tcaataatat tgaaaaagga agagtatgag
     4801 tattcaacat ttccgtgtcg cccttattcc cttttttgcg gcattttgcc ttcctgtttt
     4861 tgctcaccca gaaacgctgg tgaaagtaaa agatgctgaa gatcagttgg gtgcacgagt
     4921 gggttacatc gaactggatc tcaacagcgg taagatcctt gagagttttc gccccgaaga
     4981 acgttttcca atgatgagca cttttaaagt tctgctatgt ggcgcggtat tatcccgtat
     5041 tgacgccggg caagagcaac tcggtcgccg catacactat tctcagaatg acttggttga
     5101 gtactcacca gtcacagaaa agcatcttac ggatggcatg acagtaagag aattatgcag
     5161 tgctgccata accatgagtg ataacactgc ggccaactta cttctgacaa cgatcggagg
     5221 accgaaggag ctaaccgctt ttttgcacaa catgggggat catgtaactc gccttgatcg
     5281 ttgggaaccg gagctgaatg aagccatacc aaacgacgag cgtgacacca cgatgcctgt
     5341 agcaatggca acaacgttgc gcaaactatt aactggcgaa ctacttactc tagcttcccg
     5401 gcaacaatta atagactgga tggaggcgga taaagttgca ggaccacttc tgcgctcggc
     5461 ccttccggct ggctggttta ttgctgataa atctggagcc ggtgagcgtg gaagccgcgg
     5521 tatcattgca gcactggggc cagatggtaa gccctcccgt atcgtagtta tctacacgac
     5581 ggggagtcag gcaactatgg atgaacgaaa tagacagatc gctgagatag gtgcctcact
     5641 gattaagcat tggtaactgt cagaccaagt ttactcatat atactttaga ttgatttaaa
     5701 acttcatttt taatttaaaa ggatctaggt gaagatcctt tttgataatc tcatgaccaa
     5761 aatcccttaa cgtgagtttt cgttccactg agcgtcagac cccgtagaaa agatcaaagg
     5821 atcttcttga gatccttttt ttctgcgcgt aatctgctgc ttgcaaacaa aaaaaccacc
     5881 gctaccagcg gtggtttgtt tgccggatca agagctacca actctttttc cgaaggtaac
     5941 tggcttcagc agagcgcaga taccaaatac tgttcttcta gtgtagccgt agttaggcca
     6001 ccacttcaag aactctgtag caccgcctac atacctcgct ctgctaatcc tgttaccagt
     6061 ggctgctgcc agtggcgata agtcgtgtct taccgggttg gactcaagac gatagttacc
     6121 ggataaggcg cagcggtcgg gctgaacggg gggttcgtgc acacagccca gcttggagcg
     6181 aacgacctac accgaactga gatacctaca gcgtgagcta tgagaaagcg ccacgcttcc
     6241 cgaagggaga aaggcggaca ggtatccggt aagcggcagg gtcggaacag gagagcgcac
     6301 gagggagctt ccagggggaa acgcctggta tctttatagt cctgtcgggt ttcgccacct
     6361 ctgacttgag cgtcgatttt tgtgatgctc gtcagggggg cggagcctat ggaaaaacgc
     6421 cagcaacgcg gcctttttac ggttcctggc cttttgctgg ccttttgctc acatgt
//
