LOCUS       sequence-267870-        6013 bp    DNA     circular SYN 15-APR-2025
DEFINITION  RMCE vector for generating C-terminal T2A-GAL80 fusion.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 6013)
  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 6013)
  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: 
            "10.1016/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-GAL80
FEATURES             Location/Qualifiers
     source          1..6013
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     misc_feature    join(44..74,75..628)
                     /label=P element 5' end
     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             2002..3309
                     /codon_start=1
                     /gene="Saccharomyces cerevisiae GAL80"
                     /product="transcriptional repressor that inhibits GAL4"
                     /label=GAL80
                     /note="GAL80 binds to and inhibits the transcriptional 
                     activation domain of GAL4."
                     /translation="MDYNKRSSVSTVPNAAPIRVGFVGLNAAKGWAIKTHYPAILQLSS
                     QFQITALYSPKIETSIATIQRLKLSNATAFPTLESFASSSTIDMIVIAIQVASHYEVVM
                     PLLEFSKNNPNLKYLFVEWALACSLDQAESIYKAAAERGVQTIISLQGRKSPYILRAKE
                     LISQGYIGDINSIEIAGNGGWYGYERPVKSPKYIYEIGNGVDLVTTTFGHTIDILQYMT
                     SSYFSRINAMVFNNIPEQELIDERGNRLGQRVPKTVPDHLLFQGTLLNGNVPVSCSFKG
                     GKPTKKFTKNLVIDIHGTKGDLKLEGDAGFAEISNLVLYYSGTRANDFPLANGQQAPLD
                     PGYDAGKEIMEVYHLRNYNAIVGNIHRLYQSISDFHFNTKKIPELPSQFVMQGFDFEGF
                     PTLMDALILHRLIESVYKSNMMGSTLNVSNISHYSL"
     protein_bind    complement(3318..3351)
                     /label=loxP
                     /bound_moiety="Cre recombinase"
                     /note="Cre-mediated recombination occurs in the 8-bp core 
                     sequence (ATGTATGC) (Shaw et al., 2021)."
     misc_feature    join(3424..3625,3626..3656)
                     /label=P element 3' end
                     /note="P element 3' end"
     primer_bind     complement(3762..3779)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(3933..4521)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(4013..4032)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(4692..5483,5484..5552))
                     /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     5315..5334
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(5553..5657)
                     /gene="bla"
                     /label=AmpR promoter
     primer_bind     complement(5820..5841)
                     /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 ggccgcggca gcctgctcac ctgcggcgac
     1981 gtggaagaga acccaggacc aatggactac aacaagaggt cttcggtctc aaccgtgcct
     2041 aatgcagctc ccataagagt cggattcgtc ggtctcaacg cagccaaagg atgggcaatc
     2101 aagacacatt accccgccat actgcaacta tcgtcacaat ttcaaatcac tgccttatac
     2161 agtccaaaaa ttgagacttc tattgccacc attcagcgtc taaaattgag taatgccact
     2221 gcttttccca ctttagagtc atttgcatca tcttccacta tagatatgat agtgatagct
     2281 atccaagtgg ccagccatta tgaagttgtt atgcctctct tggaattctc caaaaataat
     2341 ccgaacctca agtatctttt cgtagaatgg gcccttgcat gttcactaga tcaagccgaa
     2401 tccatttata aggctgctgc tgaacgtggg gttcaaacca tcatctcttt acaaggtcgt
     2461 aaatcaccat atattttgag agcaaaagaa ttaatatctc aaggctatat cggcgacatt
     2521 aattcgatcg agattgctgg aaatggcggt tggtacggct acgaaaggcc tgttaaatca
     2581 ccaaaataca tctatgaaat cgggaacggt gtagacctgg taaccacaac atttggtcac
     2641 acaatcgata ttttacaata catgacaagt tcgtactttt ccaggataaa tgcaatggtt
     2701 ttcaataata ttccagagca agagctgata gatgagcgtg gtaaccgatt gggccagcga
     2761 gtcccaaaga cagtaccgga tcatctttta ttccaaggca cattgttaaa tggcaatgtt
     2821 ccagtgtcat gcagtttcaa aggtggcaaa cctaccaaaa aatttaccaa aaatttggtc
     2881 attgacattc acggtaccaa gggagatttg aaacttgaag gcgatgccgg cttcgcagaa
     2941 atttcaaatc tggtccttta ctacagtgga actagagcaa acgacttccc gctagccaat
     3001 ggacaacaag ctcctttaga cccggggtat gatgcaggta aagaaatcat ggaagtatat
     3061 catttacgaa attataatgc cattgtgggt aatattcatc gactgtatca atctatctct
     3121 gacttccact tcaatacaaa gaaaattcct gaattaccct cacaatttgt aatgcaaggt
     3181 ttcgatttcg aaggctttcc caccttgatg gatgctctga tattacacag gttaatcgag
     3241 agcgtttata aaagtaacat gatgggctcc acattaaacg ttagcaatat ctcgcattat
     3301 agtttataag cggccgcata acttcgtata gcatacatta tacgaagtta taccggtggc
     3361 cgcgaagttc ctattcttca aatagtatag gaacttcgag ctcccggaga gatgcagaag
     3421 ctttgcgtac tcgcaaatta ttaaaaataa aactttaaaa ataatttcgt ctaattaata
     3481 ttatgagtta attcaaaccc cacggacatg ctaagggtta atcaacaatc atatcgctgt
     3541 ctcactcaga ctcaatacga cactcagaat actattcctt tcactcgcac ttattgcaag
     3601 catacgttaa gtggatgtct cttgccgacg ggaccacctt atgttatttc atcatggtct
     3661 ggccattctc atcgtgagct tccgggtgct cgccagctgc attaatgaat cggccaacgc
     3721 gcggggagag gcggtttgcg tattgggcgc tcttccgctt cctcgctcac tgactcgctg
     3781 cgctcggtcg ttcggctgcg gcgagcggta tcagctcact caaaggcggt aatacggtta
     3841 tccacagaat caggggataa cgcaggaaag aacatgtgag caaaaggcca gcaaaaggcc
     3901 aggaaccgta aaaaggccgc gttgctggcg tttttccata ggctccgccc ccctgacgag
     3961 catcacaaaa atcgacgctc aagtcagagg tggcgaaacc cgacaggact ataaagatac
     4021 caggcgtttc cccctggaag ctccctcgtg cgctctcctg ttccgaccct gccgcttacc
     4081 ggatacctgt ccgcctttct cccttcggga agcgtggcgc tttctcatag ctcacgctgt
     4141 aggtatctca gttcggtgta ggtcgttcgc tccaagctgg gctgtgtgca cgaacccccc
     4201 gttcagcccg accgctgcgc cttatccggt aactatcgtc ttgagtccaa cccggtaaga
     4261 cacgacttat cgccactggc agcagccact ggtaacagga ttagcagagc gaggtatgta
     4321 ggcggtgcta cagagttctt gaagtggtgg cctaactacg gctacactag aaggacagta
     4381 tttggtatct gcgctctgct gaagccagtt accttcggaa aaagagttgg tagctcttga
     4441 tccggcaaac aaaccaccgc tggtagcggt ggtttttttg tttgcaagca gcagattacg
     4501 cgcagaaaaa aaggatctca agaagatcct ttgatctttt ctacggggtc tgacgctcag
     4561 tggaacgaaa actcacgtta agggattttg gtcatgagat tatcaaaaag gatcttcacc
     4621 tagatccttt taaattaaaa atgaagtttt aaatcaatct aaagtatata tgagtaaact
     4681 tggtctgaca gttaccaatg cttaatcagt gaggcaccta tctcagcgat ctgtctattt
     4741 cgttcatcca tagttgcctg actccccgtc gtgtagataa ctacgatacg ggagggctta
     4801 ccatctggcc ccagtgctgc aatgataccg cgagacccac gctcaccggc tccagattta
     4861 tcagcaataa accagccagc cggaagggcc gagcgcagaa gtggtcctgc aactttatcc
     4921 gcctccatcc agtctattaa ttgttgccgg gaagctagag taagtagttc gccagttaat
     4981 agtttgcgca acgttgttgc cattgctaca ggcatcgtgg tgtcacgctc gtcgtttggt
     5041 atggcttcat tcagctccgg ttcccaacga tcaaggcgag ttacatgatc ccccatgttg
     5101 tgcaaaaaag cggttagctc cttcggtcct ccgatcgttg tcagaagtaa gttggccgca
     5161 gtgttatcac tcatggttat ggcagcactg cataattctc ttactgtcat gccatccgta
     5221 agatgctttt ctgtgactgg tgagtactca accaagtcat tctgagaata gtgtatgcgg
     5281 cgaccgagtt gctcttgccc ggcgtcaata cgggataata ccgcgccaca tagcagaact
     5341 ttaaaagtgc tcatcattgg aaaacgttct tcggggcgaa aactctcaag gatcttaccg
     5401 ctgttgagat ccagttcgat gtaacccact cgtgcaccca actgatcttc agcatctttt
     5461 actttcacca gcgtttctgg gtgagcaaaa acaggaaggc aaaatgccgc aaaaaaggga
     5521 ataagggcga cacggaaatg ttgaatactc atactcttcc tttttcaata ttattgaagc
     5581 atttatcagg gttattgtct catgagcgga tacatatttg aatgtattta gaaaaataaa
     5641 caaatagggg ttccgcgcac atttccccga aaagtgccac ctaaattgta agcgttaata
     5701 ttttgttaaa attcgcgtta aatttttgtt aaatcagctc attttttaac caataggccg
     5761 aaatcggcaa aatcccttat aaatcaaaag aatagaccga gatagggttg agtgttgttc
     5821 cagtttggaa caagagtcca ctattaaaga acgtggactc caacgtcaaa gggcgaaaaa
     5881 ccgtctatca gggcgatggc ccactacgtg aaccatcacc ctaatcaagt tttttggggt
     5941 cgaggtgccg taaagcacta aatcggaacc ctaaagggag cccccgattt agagcttgac
     6001 ggggaaagcc ggc
//
