LOCUS       sequence-267869-        5764 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  RMCE vector for generating C-terminal T2A-p65-AD fusion (split 
            GAL4).
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 5764)
  AUTHORS   Kondo S, Takahashi T, Yamagata N, Imanishi Y, Katow H, Hiramatsu S, 
            Lynn K, Abe A, Kumaraswamy A, Tanimoto H
  TITLE     Neurochemical Organization of the Drosophila Brain Visualized by 
            Endogenously Tagged Neurotransmitter Receptors.
  JOURNAL   Cell Rep. 2020 Jan 7;30(1):284-297.e5. doi: 
            10.1016/j.celrep.2019.12.018.
  PUBMED    31914394
REFERENCE   2  (bases 1 to 5764)
  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: 
            "10.1016/j.celrep.2019.12"; journalName: "Cell Rep"; date: 
            "2020-01-7- 7"; volume: "30"; issue: "1"; pages: "284-297.e5"
COMMENT     Sequence Label: pF3BGX-T2A-p65-AD
FEATURES             Location/Qualifiers
     source          1..5764
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     misc_feature    join(44..74,75..628)
                     /label=P element 5' end
     protein_bind    656..689
                     /label=FRT3
                     /bound_moiety="FLP recombinase from the Saccharomyces 
                     cerevisiae 2u plasmid"
                     /note="supports FLP-mediated excision with other FRT3 sites
                     (Bosch et al., 2015)"
     CDS             join(940..942,943..945,946..1659)
                     /codon_start=1
                     /product="yellow fluorescent protein (YFP) with fast and 
                     efficient maturation (Nagai et al., 2002)"
                     /label=Venus
                     /note="mammalian codon-optimized"
                     /translation="MVSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTL
                     KLICTTGKLPVPWPTLVTTLGYGLQCFARYPDHMKQHDFFKSAMPEGYVQERTIFFKDD
                     GNYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYITADKQKNGIK
                     ANFKIRHNIEDGGVQLADHYQQNTPIGDGPVLLPDNHYLSYQSALSKDPNEKRDHMVLL
                     EFVTAAGITLGMDELYK"
     primer_bind     complement(985..1006)
                     /label=EGFP-N
                     /note="EGFP, reverse primer"
     primer_bind     complement(1246..1265)
                     /label=EXFP-R
                     /note="For distinguishing EGFP variants, reverse primer"
     primer_bind     1593..1614
                     /label=EGFP-C
                     /note="EGFP, forward primer"
     polyA_signal    1775..1856
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(1812..1831)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     1866..1885
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     protein_bind    1903..1936
                     /label=attB
                     /bound_moiety="phage phi-C31 integrase"
                     /note="minimal attB site for the phi-C31 integrase (Groth 
                     et al., 2000)"
     CDS             1948..2001
                     /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             2032..2052
                     /codon_start=1
                     /product="nuclear localization signal of SV40 (simian virus
                     40) large T antigen"
                     /label=SV40 NLS
                     /translation="PKKKRKV"
     CDS             2503..2874
                     /codon_start=1
                     /product="transcriptional activation domain of human RelA, 
                     also known as p65 (O'Shea and Perkins, 2008)"
                     /label=RelA (p65) AD
                     /translation="PTQAGEGTLSEALLQLQFDDEDLGALLGNSTDPAVFTDLASVDNS
                     EFQQLLNQGIPVAPHTTEPMLMEYPEAITRLVTGAQRPPDPAPAPLGAPGLPNGLLSGD
                     EDFSSIADMDFSALLSQISS"
     protein_bind    complement(3069..3102)
                     /label=loxP
                     /bound_moiety="Cre recombinase"
                     /note="Cre-mediated recombination occurs in the 8-bp core 
                     sequence (ATGTATGC) (Shaw et al., 2021)."
     protein_bind    3115..3148
                     /label=FRT3
                     /bound_moiety="FLP recombinase from the Saccharomyces 
                     cerevisiae 2u plasmid"
                     /note="supports FLP-mediated excision with other FRT3 sites
                     (Bosch et al., 2015)"
     misc_feature    join(3175..3376,3377..3407)
                     /label=P element 3' end
                     /note="P element 3' end"
     primer_bind     complement(3513..3530)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(3684..4272)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(3764..3783)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(4443..5234,5235..5303))
                     /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     5066..5085
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(5304..5408)
                     /gene="bla"
                     /label=AmpR promoter
     primer_bind     complement(5571..5592)
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
ORIGIN
        1 cttctccgcc gccgatctgc cgctggacta cgtgggtctg gcccatgatg aaataacata
       61 aggtggtccc gtcgatagcc gaagcttacc gaagtataca cttaaattca gtgcacgttt
      121 gcttgttgag aggaaaggtt gtgtgcggac gaattttttt ttgaaaacat taacccttac
      181 gtggaataaa aaaaaatgaa atattgcaaa ttttgctgca aagctgtgac tggagtaaaa
      241 ttaattcacg tgccgaagtg tgctattaag agaaaattgt gggagcagag ccttgggtgc
      301 agccttggtg aaaactccca aatttgtgat acccacttta atgattcgca gtggaaggct
      361 gcacctgcaa aaggtcagac atttaaaagg aggcgactca acgcagatgc cgtacctagt
      421 aaagtgatag agcctgaacc agaaaagata aaagaaggct ataccagtgg gagtacacaa
      481 acagagtaag tttgaatagt aaaaaaaatc atttatgtaa acaataacgt gactgtgcgt
      541 taggtcctgt tcattgttta atgaaaataa gagcttgagg gaaaaattcg tactttggag
      601 tacgaaatgc gtcgtttaga gcagcagcgg taccgggccc cccctcgagg tcgacgaagt
      661 tcctattctt caaatagtat aggaacttca agcttcttaa ggaattcgag ctcgcccggg
      721 gatctaattc aattagagac taattcaatt agagctaatt caattaggat cagcttatcg
      781 atttcgaacc ctcgaccgcc ggagtataaa tagaggcgct tcgtctacgg agcgacaatt
      841 caattcaaac aagcaaagtg aacacgtcgc taagcgaaag ctaagcaaat aaacaagcgc
      901 agctgaacaa gctaaacaat cgggctagac ctaggcaaaa tggtgagcaa gggcgaggag
      961 ctgttcaccg gggtggtgcc catcctggtc gagctggacg gcgacgtaaa cggccacaag
     1021 ttcagcgtgt ccggcgaggg cgagggcgat gccacctacg gcaagctgac cctgaagctc
     1081 atctgcacca ccggcaagct gcccgtgccc tggcccaccc tcgtgaccac cctgggctac
     1141 ggcctgcagt gcttcgcccg ctaccccgac cacatgaagc agcacgactt cttcaagtcc
     1201 gccatgcccg aaggctacgt ccaggagcgc accatcttct tcaaggacga cggcaactac
     1261 aagacccgcg ccgaggtgaa gttcgagggc gacaccctgg tgaaccgcat cgagctgaag
     1321 ggcatcgact tcaaggagga cggcaacatc ctggggcaca agctggagta caactacaac
     1381 agccacaacg tctatatcac cgccgacaag cagaagaacg gcatcaaggc caacttcaag
     1441 atccgccaca acatcgagga cggcggcgtg cagctcgccg accactacca gcagaacacc
     1501 cccatcggcg acggccccgt gctgctgccc gacaaccact acctgagcta ccagtccgcc
     1561 ctgagcaaag accccaacga gaagcgcgat cacatggtcc tgctggagtt cgtgaccgcc
     1621 gccgggatca ctctcggcat ggacgagctg tacaagtaac catggctaga tcataatcag
     1681 ccataccaca tttgtagagg ttttacttgc tttaaaaaac ctcccacacc tccccctgaa
     1741 cctgaaacat aaaatgaatg caattgttgt tgttaacttg tttattgcag cttataatgg
     1801 ttacaaataa agcaatagca tcacaaattt cacaaataaa gcattttttt cactgcattc
     1861 tagttgtggt ttgtccaaac tcatcaatgt atcgaattcg tacgcgcccg gggagcccaa
     1921 gggcacgccc tggcacccgg cgcgccagag ggcagaggca gcctgctcac ctgcggcgac
     1981 gtggaagaga accctgggcc tatggataaa gcggaattaa ttcccgagcc tccaaaaaag
     2041 aagagaaagg tcgaattggg atccacgccg atggagttcc agtacctgcc cgatacggat
     2101 gaccgtcacc gtatcgaaga aaagcggaag cgaacctatg aaaccttcaa gtccatcatg
     2161 aaaaagtccc ccttctcggg ccccacggac ccgcgccccc cgccccgtcg tattgcggtt
     2221 ccttcgcgca gcagtgccag cgtccccaaa cccgcaccgc agccctaccc gttcacttcc
     2281 tcccttagca cgattaacta tgatgagttc cccacgatgg tgttccccag tggacaaatt
     2341 tcccaggcat cggcactggc tccggcccca ccgcaagtcc tcccccaggc gcccgctccg
     2401 gcaccggctc ccgcaatggt gagtgctctg gcccaggccc ccgctccagt ccccgtgctg
     2461 gcgcctggac ccccacaggc agttgcccct cctgctccga aaccaacgca ggcgggcgaa
     2521 ggaaccctga gcgaggccct cttgcagctt cagttcgatg acgaagactt gggagccctg
     2581 ctgggtaaca gcacagaccc tgccgtattc accgatctcg catccgtgga caacagcgag
     2641 tttcagcagc tcttgaatca gggaatcccg gtcgcacctc ataccacaga gcccatgctg
     2701 atggaatacc cggaggctat cacgcgactg gtgaccggcg cacagcgacc acccgatcca
     2761 gcccctgccc cactgggtgc cccgggtttg cccaatggcc tcctcagcgg cgatgaggat
     2821 ttctccagca tcgctgatat ggatttctcc gctttgctga gccagataag ctccgctagc
     2881 ggaggaggtg gtggaggtgg aggtggaggt actagtctgg agatcgaggc cgccttcctg
     2941 gagcgcgaga acaccgccct ggagacccgc gtggccgagc tgcgccagcg cgtgcagcgc
     3001 ctgcgcaacc gcgtgagcca gtaccgcacc cgctacggcc ccctgggcgg cggcaagtaa
     3061 gcggccgcat aacttcgtat agcatacatt atacgaagtt ataccggtgg ccgcgaagtt
     3121 cctattcttc aaatagtata ggaacttcga gctcccggag agatgcagaa gctttgcgta
     3181 ctcgcaaatt attaaaaata aaactttaaa aataatttcg tctaattaat attatgagtt
     3241 aattcaaacc ccacggacat gctaagggtt aatcaacaat catatcgctg tctcactcag
     3301 actcaatacg acactcagaa tactattcct ttcactcgca cttattgcaa gcatacgtta
     3361 agtggatgtc tcttgccgac gggaccacct tatgttattt catcatggtc tggccattct
     3421 catcgtgagc ttccgggtgc tcgccagctg cattaatgaa tcggccaacg cgcggggaga
     3481 ggcggtttgc gtattgggcg ctcttccgct tcctcgctca ctgactcgct gcgctcggtc
     3541 gttcggctgc ggcgagcggt atcagctcac tcaaaggcgg taatacggtt atccacagaa
     3601 tcaggggata acgcaggaaa gaacatgtga gcaaaaggcc agcaaaaggc caggaaccgt
     3661 aaaaaggccg cgttgctggc gtttttccat aggctccgcc cccctgacga gcatcacaaa
     3721 aatcgacgct caagtcagag gtggcgaaac ccgacaggac tataaagata ccaggcgttt
     3781 ccccctggaa gctccctcgt gcgctctcct gttccgaccc tgccgcttac cggatacctg
     3841 tccgcctttc tcccttcggg aagcgtggcg ctttctcata gctcacgctg taggtatctc
     3901 agttcggtgt aggtcgttcg ctccaagctg ggctgtgtgc acgaaccccc cgttcagccc
     3961 gaccgctgcg ccttatccgg taactatcgt cttgagtcca acccggtaag acacgactta
     4021 tcgccactgg cagcagccac tggtaacagg attagcagag cgaggtatgt aggcggtgct
     4081 acagagttct tgaagtggtg gcctaactac ggctacacta gaaggacagt atttggtatc
     4141 tgcgctctgc tgaagccagt taccttcgga aaaagagttg gtagctcttg atccggcaaa
     4201 caaaccaccg ctggtagcgg tggttttttt gtttgcaagc agcagattac gcgcagaaaa
     4261 aaaggatctc aagaagatcc tttgatcttt tctacggggt ctgacgctca gtggaacgaa
     4321 aactcacgtt aagggatttt ggtcatgaga ttatcaaaaa ggatcttcac ctagatcctt
     4381 ttaaattaaa aatgaagttt taaatcaatc taaagtatat atgagtaaac ttggtctgac
     4441 agttaccaat gcttaatcag tgaggcacct atctcagcga tctgtctatt tcgttcatcc
     4501 atagttgcct gactccccgt cgtgtagata actacgatac gggagggctt accatctggc
     4561 cccagtgctg caatgatacc gcgagaccca cgctcaccgg ctccagattt atcagcaata
     4621 aaccagccag ccggaagggc cgagcgcaga agtggtcctg caactttatc cgcctccatc
     4681 cagtctatta attgttgccg ggaagctaga gtaagtagtt cgccagttaa tagtttgcgc
     4741 aacgttgttg ccattgctac aggcatcgtg gtgtcacgct cgtcgtttgg tatggcttca
     4801 ttcagctccg gttcccaacg atcaaggcga gttacatgat cccccatgtt gtgcaaaaaa
     4861 gcggttagct ccttcggtcc tccgatcgtt gtcagaagta agttggccgc agtgttatca
     4921 ctcatggtta tggcagcact gcataattct cttactgtca tgccatccgt aagatgcttt
     4981 tctgtgactg gtgagtactc aaccaagtca ttctgagaat agtgtatgcg gcgaccgagt
     5041 tgctcttgcc cggcgtcaat acgggataat accgcgccac atagcagaac tttaaaagtg
     5101 ctcatcattg gaaaacgttc ttcggggcga aaactctcaa ggatcttacc gctgttgaga
     5161 tccagttcga tgtaacccac tcgtgcaccc aactgatctt cagcatcttt tactttcacc
     5221 agcgtttctg ggtgagcaaa aacaggaagg caaaatgccg caaaaaaggg aataagggcg
     5281 acacggaaat gttgaatact catactcttc ctttttcaat attattgaag catttatcag
     5341 ggttattgtc tcatgagcgg atacatattt gaatgtattt agaaaaataa acaaataggg
     5401 gttccgcgca catttccccg aaaagtgcca cctaaattgt aagcgttaat attttgttaa
     5461 aattcgcgtt aaatttttgt taaatcagct cattttttaa ccaataggcc gaaatcggca
     5521 aaatccctta taaatcaaaa gaatagaccg agatagggtt gagtgttgtt ccagtttgga
     5581 acaagagtcc actattaaag aacgtggact ccaacgtcaa agggcgaaaa accgtctatc
     5641 agggcgatgg cccactacgt gaaccatcac cctaatcaag ttttttgggg tcgaggtgcc
     5701 gtaaagcact aaatcggaac cctaaaggga gcccccgatt tagagcttga cggggaaagc
     5761 cggc
//
