LOCUS       sequence-275234-        5765 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 5765)
  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 5765)
  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..5765
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     misc_feature    join(1..31,32..586)
                     /label=P element 5' end
     protein_bind    614..647
                     /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(898..900,901..903,904..1617)
                     /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(943..964)
                     /label=EGFP-N
                     /note="EGFP, reverse primer"
     primer_bind     complement(1204..1223)
                     /label=EXFP-R
                     /note="For distinguishing EGFP variants, reverse primer"
     primer_bind     1551..1572
                     /label=EGFP-C
                     /note="EGFP, forward primer"
     polyA_signal    1733..1814
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(1770..1789)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     1824..1843
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     protein_bind    1861..1894
                     /label=attB
                     /bound_moiety="phage phi-C31 integrase"
                     /note="minimal attB site for the phi-C31 integrase (Groth 
                     et al., 2000)"
     CDS             1906..1959
                     /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             1990..2010
                     /codon_start=1
                     /product="nuclear localization signal of SV40 (simian virus
                     40) large T antigen"
                     /label=SV40 NLS
                     /translation="PKKKRKV"
     CDS             2461..2832
                     /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(3027..3060)
                     /label=loxP
                     /bound_moiety="Cre recombinase"
                     /note="Cre-mediated recombination occurs in the 8-bp core 
                     sequence (ATGTATGC) (Shaw et al., 2021)."
     protein_bind    3073..3106
                     /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(3133..3334,3335..3365)
                     /label=P element 3' end
                     /note="P element 3' end"
     primer_bind     complement(3471..3488)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(3642..4230)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(3722..3741)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(4401..5192,5193..5261))
                     /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     5024..5043
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(5262..5366)
                     /gene="bla"
                     /label=AmpR promoter
     primer_bind     complement(5529..5550)
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
ORIGIN
        1 catgatgaaa taacataagg tggtcccgtc gatagccgaa gcttaccgaa gtatacactt
       61 aaattcagtg cacgtttgct tgttgagagg aaaggttgtg tgcggacgaa tttttttttg
      121 aaaacattaa cccttacgtg gaataaaaaa aaatgaaata ttgcaaattt tgctgcaaag
      181 ctgtgactgg agtaaaatta attcacgtgc cgaagtgtgc tattaagaga aaattgtggg
      241 agcagagcct tgggtgcagc cttggtgaaa actcccaaat ttgtgatacc cactttaatg
      301 attcgcagtg gaaggctgca cctgcaaaag gtcagacatt taaaaggagg cgactcaacg
      361 cagatgccgt acctagtaaa gtgatagagc ctgaaccaga aaagataaaa gaaggctata
      421 ccagtgggag tacacaaaca gagtaagttt gaatagtaaa aaaaatcatt tatgtaaaca
      481 ataacgtgac tgtgcgttag gtcctgttca ttgtttaatg aaaataagag cttgagggaa
      541 aaaattcgta ctttggagta cgaaatgcgt cgtttagagc agcagcggta ccgggccccc
      601 cctcgaggtc gacgaagttc ctattcttca aatagtatag gaacttcaag cttcttaagg
      661 aattcgagct cgcccgggga tctaattcaa ttagagacta attcaattag agctaattca
      721 attaggatca gcttatcgat ttcgaaccct cgaccgccgg agtataaata gaggcgcttc
      781 gtctacggag cgacaattca attcaaacaa gcaaagtgaa cacgtcgcta agcgaaagct
      841 aagcaaataa acaagcgcag ctgaacaagc taaacaatcg ggctagacct aggcaaaatg
      901 gtgagcaagg gcgaggagct gttcaccggg gtggtgccca tcctggtcga gctggacggc
      961 gacgtaaacg gccacaagtt cagcgtgtcc ggcgagggcg agggcgatgc cacctacggc
     1021 aagctgaccc tgaagctcat ctgcaccacc ggcaagctgc ccgtgccctg gcccaccctc
     1081 gtgaccaccc tgggctacgg cctgcagtgc ttcgcccgct accccgacca catgaagcag
     1141 cacgacttct tcaagtccgc catgcccgaa ggctacgtcc aggagcgcac catcttcttc
     1201 aaggacgacg gcaactacaa gacccgcgcc gaggtgaagt tcgagggcga caccctggtg
     1261 aaccgcatcg agctgaaggg catcgacttc aaggaggacg gcaacatcct ggggcacaag
     1321 ctggagtaca actacaacag ccacaacgtc tatatcaccg ccgacaagca gaagaacggc
     1381 atcaaggcca acttcaagat ccgccacaac atcgaggacg gcggcgtgca gctcgccgac
     1441 cactaccagc agaacacccc catcggcgac ggccccgtgc tgctgcccga caaccactac
     1501 ctgagctacc agtccgccct gagcaaagac cccaacgaga agcgcgatca catggtcctg
     1561 ctggagttcg tgaccgccgc cgggatcact ctcggcatgg acgagctgta caagtaacca
     1621 tggctagatc ataatcagcc ataccacatt tgtagaggtt ttacttgctt taaaaaacct
     1681 cccacacctc cccctgaacc tgaaacataa aatgaatgca attgttgttg ttaacttgtt
     1741 tattgcagct tataatggtt acaaataaag caatagcatc acaaatttca caaataaagc
     1801 atttttttca ctgcattcta gttgtggttt gtccaaactc atcaatgtat cgaattcgta
     1861 cgcgcccggg gagcccaagg gcacgccctg gcacccggcg cgccagaggg cagaggcagc
     1921 ctgctcacct gcggcgacgt ggaagagaac cctgggccta tggataaagc ggaattaatt
     1981 cccgagcctc caaaaaagaa gagaaaggtc gaattgggat ccacgccgat ggagttccag
     2041 tacctgcccg atacggatga ccgtcaccgt atcgaagaaa agcggaagcg aacctatgaa
     2101 accttcaagt ccatcatgaa aaagtccccc ttctcgggcc ccacggaccc gcgccccccg
     2161 ccccgtcgta ttgcggttcc ttcgcgcagc agtgccagcg tccccaaacc cgcaccgcag
     2221 ccctacccgt tcacttcctc ccttagcacg attaactatg atgagttccc cacgatggtg
     2281 ttccccagtg gacaaatttc ccaggcatcg gcactggctc cggccccacc gcaagtcctc
     2341 ccccaggcgc ccgctccggc accggctccc gcaatggtga gtgctctggc ccaggccccc
     2401 gctccagtcc ccgtgctggc gcctggaccc ccacaggcag ttgcccctcc tgctccgaaa
     2461 ccaacgcagg cgggcgaagg aaccctgagc gaggccctct tgcagcttca gttcgatgac
     2521 gaagacttgg gagccctgct gggtaacagc acagaccctg ccgtattcac cgatctcgca
     2581 tccgtggaca acagcgagtt tcagcagctc ttgaatcagg gaatcccggt cgcacctcat
     2641 accacagagc ccatgctgat ggaatacccg gaggctatca cgcgactggt gaccggcgca
     2701 cagcgaccac ccgatccagc ccctgcccca ctgggtgccc cgggtttgcc caatggcctc
     2761 ctcagcggcg atgaggattt ctccagcatc gctgatatgg atttctccgc tttgctgagc
     2821 cagataagct ccgctagcgg aggaggtggt ggaggtggag gtggaggtac tagtctggag
     2881 atcgaggccg ccttcctgga gcgcgagaac accgccctgg agacccgcgt ggccgagctg
     2941 cgccagcgcg tgcagcgcct gcgcaaccgc gtgagccagt accgcacccg ctacggcccc
     3001 ctgggcggcg gcaagtaagc ggccgcataa cttcgtatag catacattat acgaagttat
     3061 accggtggcc gcgaagttcc tattcttcaa atagtatagg aacttcgagc tcccggagag
     3121 atgcagaagc tttgcgtact cgcaaattat taaaaataaa actttaaaaa taatttcgtc
     3181 taattaatat tatgagttaa ttcaaacccc acggacatgc taagggttaa tcaacaatca
     3241 tatcgctgtc tcactcagac tcaatacgac actcagaata ctattccttt cactcgcact
     3301 tattgcaagc atacgttaag tggatgtctc ttgccgacgg gaccacctta tgttatttca
     3361 tcatggtctg gccattctca tcgtgagctt ccgggtgctc gccagctgca ttaatgaatc
     3421 ggccaacgcg cggggagagg cggtttgcgt attgggcgct cttccgcttc ctcgctcact
     3481 gactcgctgc gctcggtcgt tcggctgcgg cgagcggtat cagctcactc aaaggcggta
     3541 atacggttat ccacagaatc aggggataac gcaggaaaga acatgtgagc aaaaggccag
     3601 caaaaggcca ggaaccgtaa aaaggccgcg ttgctggcgt ttttccatag gctccgcccc
     3661 cctgacgagc atcacaaaaa tcgacgctca agtcagaggt ggcgaaaccc gacaggacta
     3721 taaagatacc aggcgtttcc ccctggaagc tccctcgtgc gctctcctgt tccgaccctg
     3781 ccgcttaccg gatacctgtc cgcctttctc ccttcgggaa gcgtggcgct ttctcatagc
     3841 tcacgctgta ggtatctcag ttcggtgtag gtcgttcgct ccaagctggg ctgtgtgcac
     3901 gaaccccccg ttcagcccga ccgctgcgcc ttatccggta actatcgtct tgagtccaac
     3961 ccggtaagac acgacttatc gccactggca gcagccactg gtaacaggat tagcagagcg
     4021 aggtatgtag gcggtgctac agagttcttg aagtggtggc ctaactacgg ctacactaga
     4081 agaacagtat ttggtatctg cgctctgctg aagccagtta ccttcggaaa aagagttggt
     4141 agctcttgat ccggcaaaca aaccaccgct ggtagcggtg gtttttttgt ttgcaagcag
     4201 cagattacgc gcagaaaaaa aggatctcaa gaagatcctt tgatcttttc tacggggtct
     4261 gacgctcagt ggaacgaaaa ctcacgttaa gggattttgg tcatgagatt atcaaaaagg
     4321 atcttcacct agatcctttt aaattaaaaa tgaagtttta aatcaatcta aagtatatat
     4381 gagtaaactt ggtctgacag ttaccaatgc ttaatcagtg aggcacctat ctcagcgatc
     4441 tgtctatttc gttcatccat agttgcctga ctccccgtcg tgtagataac tacgatacgg
     4501 gagggcttac catctggccc cagtgctgca atgataccgc gagacccacg ctcaccggct
     4561 ccagatttat cagcaataaa ccagccagcc ggaagggccg agcgcagaag tggtcctgca
     4621 actttatccg cctccatcca gtctattaat tgttgccggg aagctagagt aagtagttcg
     4681 ccagttaata gtttgcgcaa cgttgttgcc attgctacag gcatcgtggt gtcacgctcg
     4741 tcgtttggta tggcttcatt cagctccggt tcccaacgat caaggcgagt tacatgatcc
     4801 cccatgttgt gcaaaaaagc ggttagctcc ttcggtcctc cgatcgttgt cagaagtaag
     4861 ttggccgcag tgttatcact catggttatg gcagcactgc ataattctct tactgtcatg
     4921 ccatccgtaa gatgcttttc tgtgactggt gagtactcaa ccaagtcatt ctgagaatag
     4981 tgtatgcggc gaccgagttg ctcttgcccg gcgtcaatac gggataatac cgcgccacat
     5041 agcagaactt taaaagtgct catcattgga aaacgttctt cggggcgaaa actctcaagg
     5101 atcttaccgc tgttgagatc cagttcgatg taacccactc gtgcacccaa ctgatcttca
     5161 gcatctttta ctttcaccag cgtttctggg tgagcaaaaa caggaaggca aaatgccgca
     5221 aaaaagggaa taagggcgac acggaaatgt tgaatactca tactcttcct ttttcaatat
     5281 tattgaagca tttatcaggg ttattgtctc atgagcggat acatatttga atgtatttag
     5341 aaaaataaac aaataggggt tccgcgcaca tttccccgaa aagtgccacc taaattgtaa
     5401 gcgttaatat tttgttaaaa ttcgcgttaa atttttgtta aatcagctca ttttttaacc
     5461 aataggccga aatcggcaaa atcccttata aatcaaaaga atagaccgag atagggttga
     5521 gtgttgttcc agtttggaac aagagtccac tattaaagaa cgtggactcc aacgtcaaag
     5581 ggcgaaaaac cgtctatcag ggcgatggcc cactacgtga accatcaccc taatcaagtt
     5641 ttttggggtc gaggtgccgt aaagcactaa atcggaaccc taaagggagc ccccgattta
     5701 gagcttgacg gggaaagccg gccttctccg ccgccgatct gccgctggac tacgtgggtc
     5761 tggcc
//
