LOCUS       sequence-275233-        6014 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 6014)
  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 6014)
  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..6014
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     polyA_signal    119..200
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(156..175)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     210..229
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     protein_bind    247..280
                     /label=attB
                     /bound_moiety="phage phi-C31 integrase"
                     /note="minimal attB site for the phi-C31 integrase (Groth 
                     et al., 2000)"
     CDS             292..345
                     /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             346..1653
                     /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(1662..1695)
                     /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(1768..1969,1970..2000)
                     /label=P element 3' end
                     /note="P element 3' end"
     primer_bind     complement(2106..2123)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(2277..2865)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(2357..2376)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(3036..3827,3828..3896))
                     /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     3659..3678
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(3897..4001)
                     /gene="bla"
                     /label=AmpR promoter
     primer_bind     complement(4164..4185)
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     misc_feature    join(4401..4431,4432..4986)
                     /label=P element 5' end
     CDS             join(5298..5300,5301..5303,5304..6014,1..3)
                     /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(5343..5364)
                     /label=EGFP-N
                     /note="EGFP, reverse primer"
     primer_bind     complement(5604..5623)
                     /label=EXFP-R
                     /note="For distinguishing EGFP variants, reverse primer"
     primer_bind     5951..5972
                     /label=EGFP-C
                     /note="EGFP, forward primer"
ORIGIN
        1 taaccatggc tagatcataa tcagccatac cacatttgta gaggttttac ttgctttaaa
       61 aaacctccca cacctccccc tgaacctgaa acataaaatg aatgcaattg ttgttgttaa
      121 cttgtttatt gcagcttata atggttacaa ataaagcaat agcatcacaa atttcacaaa
      181 taaagcattt ttttcactgc attctagttg tggtttgtcc aaactcatca atgtatcgaa
      241 ttcgtacgcg cccggggagc ccaagggcac gccctggcac ccggcgcgcc agagggccgc
      301 ggcagcctgc tcacctgcgg cgacgtggaa gagaacccag gaccaatgga ctacaacaag
      361 aggtcttcgg tctcaaccgt gcctaatgca gctcccataa gagtcggatt cgtcggtctc
      421 aacgcagcca aaggatgggc aatcaagaca cattaccccg ccatactgca actatcgtca
      481 caatttcaaa tcactgcctt atacagtcca aaaattgaga cttctattgc caccattcag
      541 cgtctaaaat tgagtaatgc cactgctttt cccactttag agtcatttgc atcatcttcc
      601 actatagata tgatagtgat agctatccaa gtggccagcc attatgaagt tgttatgcct
      661 ctcttggaat tctccaaaaa taatccgaac ctcaagtatc ttttcgtaga atgggccctt
      721 gcatgttcac tagatcaagc cgaatccatt tataaggctg ctgctgaacg tggggttcaa
      781 accatcatct ctttacaagg tcgtaaatca ccatatattt tgagagcaaa agaattaata
      841 tctcaaggct atatcggcga cattaattcg atcgagattg ctggaaatgg cggttggtac
      901 ggctacgaaa ggcctgttaa atcaccaaaa tacatctatg aaatcgggaa cggtgtagac
      961 ctggtaacca caacatttgg tcacacaatc gatattttac aatacatgac aagttcgtac
     1021 ttttccagga taaatgcaat ggttttcaat aatattccag agcaagagct gatagatgag
     1081 cgtggtaacc gattgggcca gcgagtccca aagacagtac cggatcatct tttattccaa
     1141 ggcacattgt taaatggcaa tgttccagtg tcatgcagtt tcaaaggtgg caaacctacc
     1201 aaaaaattta ccaaaaattt ggtcattgac attcacggta ccaagggaga tttgaaactt
     1261 gaaggcgatg ccggcttcgc agaaatttca aatctggtcc tttactacag tggaactaga
     1321 gcaaacgact tcccgctagc caatggacaa caagctcctt tagacccggg gtatgatgca
     1381 ggtaaagaaa tcatggaagt atatcattta cgaaattata atgccattgt gggtaatatt
     1441 catcgactgt atcaatctat ctctgacttc cacttcaata caaagaaaat tcctgaatta
     1501 ccctcacaat ttgtaatgca aggtttcgat ttcgaaggct ttcccacctt gatggatgct
     1561 ctgatattac acaggttaat cgagagcgtt tataaaagta acatgatggg ctccacatta
     1621 aacgttagca atatctcgca ttatagttta taagcggccg cataacttcg tatagcatac
     1681 attatacgaa gttataccgg tggccgcgaa gttcctattc ttcaaatagt ataggaactt
     1741 cgagctcccg gagagatgca gaagctttgc gtactcgcaa attattaaaa ataaaacttt
     1801 aaaaataatt tcgtctaatt aatattatga gttaattcaa accccacgga catgctaagg
     1861 gttaatcaac aatcatatcg ctgtctcact cagactcaat acgacactca gaatactatt
     1921 cctttcactc gcacttattg caagcatacg ttaagtggat gtctcttgcc gacgggacca
     1981 ccttatgtta tttcatcatg gtctggccat tctcatcgtg agcttccggg tgctcgccag
     2041 ctgcattaat gaatcggcca acgcgcgggg agaggcggtt tgcgtattgg gcgctcttcc
     2101 gcttcctcgc tcactgactc gctgcgctcg gtcgttcggc tgcggcgagc ggtatcagct
     2161 cactcaaagg cggtaatacg gttatccaca gaatcagggg ataacgcagg aaagaacatg
     2221 tgagcaaaag gccagcaaaa ggccaggaac cgtaaaaagg ccgcgttgct ggcgtttttc
     2281 cataggctcc gcccccctga cgagcatcac aaaaatcgac gctcaagtca gaggtggcga
     2341 aacccgacag gactataaag ataccaggcg tttccccctg gaagctccct cgtgcgctct
     2401 cctgttccga ccctgccgct taccggatac ctgtccgcct ttctcccttc gggaagcgtg
     2461 gcgctttctc atagctcacg ctgtaggtat ctcagttcgg tgtaggtcgt tcgctccaag
     2521 ctgggctgtg tgcacgaacc ccccgttcag cccgaccgct gcgccttatc cggtaactat
     2581 cgtcttgagt ccaacccggt aagacacgac ttatcgccac tggcagcagc cactggtaac
     2641 aggattagca gagcgaggta tgtaggcggt gctacagagt tcttgaagtg gtggcctaac
     2701 tacggctaca ctagaagaac agtatttggt atctgcgctc tgctgaagcc agttaccttc
     2761 ggaaaaagag ttggtagctc ttgatccggc aaacaaacca ccgctggtag cggtggtttt
     2821 tttgtttgca agcagcagat tacgcgcaga aaaaaaggat ctcaagaaga tcctttgatc
     2881 ttttctacgg ggtctgacgc tcagtggaac gaaaactcac gttaagggat tttggtcatg
     2941 agattatcaa aaaggatctt cacctagatc cttttaaatt aaaaatgaag ttttaaatca
     3001 atctaaagta tatatgagta aacttggtct gacagttacc aatgcttaat cagtgaggca
     3061 cctatctcag cgatctgtct atttcgttca tccatagttg cctgactccc cgtcgtgtag
     3121 ataactacga tacgggaggg cttaccatct ggccccagtg ctgcaatgat accgcgagac
     3181 ccacgctcac cggctccaga tttatcagca ataaaccagc cagccggaag ggccgagcgc
     3241 agaagtggtc ctgcaacttt atccgcctcc atccagtcta ttaattgttg ccgggaagct
     3301 agagtaagta gttcgccagt taatagtttg cgcaacgttg ttgccattgc tacaggcatc
     3361 gtggtgtcac gctcgtcgtt tggtatggct tcattcagct ccggttccca acgatcaagg
     3421 cgagttacat gatcccccat gttgtgcaaa aaagcggtta gctccttcgg tcctccgatc
     3481 gttgtcagaa gtaagttggc cgcagtgtta tcactcatgg ttatggcagc actgcataat
     3541 tctcttactg tcatgccatc cgtaagatgc ttttctgtga ctggtgagta ctcaaccaag
     3601 tcattctgag aatagtgtat gcggcgaccg agttgctctt gcccggcgtc aatacgggat
     3661 aataccgcgc cacatagcag aactttaaaa gtgctcatca ttggaaaacg ttcttcgggg
     3721 cgaaaactct caaggatctt accgctgttg agatccagtt cgatgtaacc cactcgtgca
     3781 cccaactgat cttcagcatc ttttactttc accagcgttt ctgggtgagc aaaaacagga
     3841 aggcaaaatg ccgcaaaaaa gggaataagg gcgacacgga aatgttgaat actcatactc
     3901 ttcctttttc aatattattg aagcatttat cagggttatt gtctcatgag cggatacata
     3961 tttgaatgta tttagaaaaa taaacaaata ggggttccgc gcacatttcc ccgaaaagtg
     4021 ccacctaaat tgtaagcgtt aatattttgt taaaattcgc gttaaatttt tgttaaatca
     4081 gctcattttt taaccaatag gccgaaatcg gcaaaatccc ttataaatca aaagaataga
     4141 ccgagatagg gttgagtgtt gttccagttt ggaacaagag tccactatta aagaacgtgg
     4201 actccaacgt caaagggcga aaaaccgtct atcagggcga tggcccacta cgtgaaccat
     4261 caccctaatc aagttttttg gggtcgaggt gccgtaaagc actaaatcgg aaccctaaag
     4321 ggagcccccg atttagagct tgacggggaa agccggcctt ctccgccgcc gatctgccgc
     4381 tggactacgt gggtctggcc catgatgaaa taacataagg tggtcccgtc gatagccgaa
     4441 gcttaccgaa gtatacactt aaattcagtg cacgtttgct tgttgagagg aaaggttgtg
     4501 tgcggacgaa tttttttttg aaaacattaa cccttacgtg gaataaaaaa aaatgaaata
     4561 ttgcaaattt tgctgcaaag ctgtgactgg agtaaaatta attcacgtgc cgaagtgtgc
     4621 tattaagaga aaattgtggg agcagagcct tgggtgcagc cttggtgaaa actcccaaat
     4681 ttgtgatacc cactttaatg attcgcagtg gaaggctgca cctgcaaaag gtcagacatt
     4741 taaaaggagg cgactcaacg cagatgccgt acctagtaaa gtgatagagc ctgaaccaga
     4801 aaagataaaa gaaggctata ccagtgggag tacacaaaca gagtaagttt gaatagtaaa
     4861 aaaaatcatt tatgtaaaca ataacgtgac tgtgcgttag gtcctgttca ttgtttaatg
     4921 aaaataagag cttgagggaa aaaattcgta ctttggagta cgaaatgcgt cgtttagagc
     4981 agcagcggta ccgggccccc cctcgaggtc gacgaagttc ctattcttca aatagtatag
     5041 gaacttcaag cttcttaagg aattcgagct cgcccgggga tctaattcaa ttagagacta
     5101 attcaattag agctaattca attaggatca gcttatcgat ttcgaaccct cgaccgccgg
     5161 agtataaata gaggcgcttc gtctacggag cgacaattca attcaaacaa gcaaagtgaa
     5221 cacgtcgcta agcgaaagct aagcaaataa acaagcgcag ctgaacaagc taaacaatcg
     5281 ggctagacct aggcaaaatg gtgagcaagg gcgaggagct gttcaccggg gtggtgccca
     5341 tcctggtcga gctggacggc gacgtaaacg gccacaagtt cagcgtgtcc ggcgagggcg
     5401 agggcgatgc cacctacggc aagctgaccc tgaagctcat ctgcaccacc ggcaagctgc
     5461 ccgtgccctg gcccaccctc gtgaccaccc tgggctacgg cctgcagtgc ttcgcccgct
     5521 accccgacca catgaagcag cacgacttct tcaagtccgc catgcccgaa ggctacgtcc
     5581 aggagcgcac catcttcttc aaggacgacg gcaactacaa gacccgcgcc gaggtgaagt
     5641 tcgagggcga caccctggtg aaccgcatcg agctgaaggg catcgacttc aaggaggacg
     5701 gcaacatcct ggggcacaag ctggagtaca actacaacag ccacaacgtc tatatcaccg
     5761 ccgacaagca gaagaacggc atcaaggcca acttcaagat ccgccacaac atcgaggacg
     5821 gcggcgtgca gctcgccgac cactaccagc agaacacccc catcggcgac ggccccgtgc
     5881 tgctgcccga caaccactac ctgagctacc agtccgccct gagcaaagac cccaacgaga
     5941 agcgcgatca catggtcctg ctggagttcg tgaccgccgc cgggatcact ctcggcatgg
     6001 acgagctgta caag
//
