LOCUS       sequence-320653-        7218 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  T2A-GAL4 knock-in cassette.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 7218)
  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 7218)
  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: pPGxRF3
FEATURES             Location/Qualifiers
     source          1..7218
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     1..20
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        1..19
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     CDS             101..154
                     /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             155..2800
                     /codon_start=1
                     /gene="Saccharomyces cerevisiae GAL4"
                     /product="GAL4 transcriptional activator"
                     /label=GAL4
                     /translation="MKLLSSIEQACDICRLKKLKCSKEKPKCAKCLKNNWECRYSPKTK
                     RSPLTRAHLTEVESRLERLEQLFLLIFPREDLDMILKMDSLQDIKALLTGLFVQDNVNK
                     DAVTDRLASVETDMPLTLRQHRISATSSSEESSNKGQRQLTVSIDSAAHHDNSTIPLDF
                     MPRDALHGFDWSEEDDMSDGLPFLKTDPNNNGFFGDGSLLCILRSIGFKPENYTNSNVN
                     RLPTMITDRYTLASRSTTSRLLQSYLNNFHPYCPIVHSPTLMMLYNNQIEIASKDQWQI
                     LFNCILAIGAWCIEGESTDIDVFYYQNAKSHLTSKVFESGSIILVTALHLLSRYTQWRQ
                     KTNTSYNFHSFSIRMAISLGLNRDLPSSFSDSSILEQRRRIWWSVYSWEIQLSLLYGRS
                     IQLSQNTISFPSSVDDVQRTTTGPTIYHGIIETARLLQVFTKIYELDKTVTAEKSPICA
                     KKCLMICNEIEEVSRQAPKFLQMDISTTALTNLLKEHPWLSFTRFELKWKQLSLIIYVL
                     RDFFTNFTQKKSQLEQDQNDHQSYEVKRCSIMLSDAAQRTVMSVSSYMDNHNVTPYFAW
                     NCSYYLFNAVLVPIKTLLSNSKSNAENNETAQLLQQINTVLMLLKKLATFKIQTCEKYI
                     QVLEEVCAPFLLSQCAIPLPHISYNNSNGSAIKNIVGSATIAQYPTLPEENVNNISVKY
                     VSPGSVGPSPVPLKSGASFSDLVKLLSNRPPSRNSPVTIPRSTPSHRSVTPFLGQQQQL
                     QSLVPLTPSALFGGANFNQSGNIADSSLSFTFTNSSNGPNLITTQTNSQALSQPIASSN
                     VHDNFMNNEITASKIDDGNNSKPLSPGWTDQTAYNAFGITTGMFNTTTMDDVYNYLFDD
                     EDTPPNPKKE"
     protein_bind    complement(2809..2842)
                     /label=loxP
                     /bound_moiety="Cre recombinase"
                     /note="Cre-mediated recombination occurs in the 8-bp core 
                     sequence (ATGTATGC) (Shaw et al., 2021)."
     CDS             3380..4057
                     /codon_start=1
                     /product="noncytotoxic tetrameric variant of DsRed 
                     fluorescent protein (Strack et al., 2008)"
                     /label=DsRed-Express2
                     /note="mammalian codon-optimized"
                     /translation="MDSTENVIKPFMRFKVHMEGSVNGHEFEIEGEGEGKPYEGTQTAK
                     LQVTKGGPLPFAWDILSPQFQYGSKVYVKHPADIPDYKKLSFPEGFKWERVMNFEDGGV
                     VTVTQDSSLQDGTFIYHVKFIGVNFPSDGPVMQKKTLGWEPSTERLYPRDGVLKGEIHK
                     ALKLKGGGHYLVEFKSIYMAKKPVKLPGYYYVDSKLDITSHNEDYTVVEQYERAEARHH
                     LFQ"
     primer_bind     complement(3560..3580)
                     /label=DsRed1-N
                     /note="DsRed1, reverse primer"
     primer_bind     3778..3797
                     /label=mCherry-F
                     /note="mCherry, forward primer"
     primer_bind     3972..3995
                     /label=DsRed1-C
                     /note="DsRed1, forward primer"
     polyA_signal    4173..4254
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(4210..4229)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     4264..4283
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     protein_bind    complement(4298..4331)
                     /label=loxP
                     /bound_moiety="Cre recombinase"
                     /note="Cre-mediated recombination occurs in the 8-bp core 
                     sequence (ATGTATGC) (Shaw et al., 2021)."
     protein_bind    4338..4371
                     /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)"
     primer_bind     complement(4405..4425)
                     /label=T3
                     /note="T3 promoter, forward primer"
     promoter        complement(4405..4423)
                     /label=T3 promoter
                     /note="promoter for bacteriophage T3 RNA polymerase"
     primer_bind     complement(4444..4460)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(4444..4460)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(4457..4479)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    4468..4484
                     /label=lac operator
                     /bound_moiety="lac repressor encoded by lacI"
                     /note="The lac repressor binds to the lac operator to 
                     inhibit transcription in E. coli. This inhibition can be 
                     relieved by adding lactose or 
                     isopropyl-beta-D-thiogalactopyranoside (IPTG)."
     promoter        complement(join(4492..4498,4499..4516,4517..4522))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    4537..4558
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(4675..4692)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(4846..5434)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(4926..4945)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(5605..6396,6397..6465))
                     /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     6228..6247
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(6466..6570)
                     /gene="bla"
                     /label=AmpR promoter
     rep_origin      complement(6596..7051)
                     /direction=LEFT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(6733..6754)
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     6945..6964
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     7178..7200
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     primer_bind     7192..7209
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     7193..7209
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
ORIGIN
        1 taatacgact cactataggg cgaattgggt acccgtctca cctacgcccc caactgagag
       61 aactcaaagg ttaccccagt tggggcacta cggcgcgcca gagggcagag gcagcctgct
      121 cacctgcggc gacgtggaag agaaccctgg gcctatgaag ctactgtctt ctatcgaaca
      181 agcatgcgat atttgccgac ttaaaaagct caagtgctcc aaagaaaaac cgaagtgcgc
      241 caagtgtctg aagaacaact gggagtgtcg ctactctccc aaaaccaaaa ggtctccgct
      301 gactagggca catctgacag aagtggaatc aaggctagaa agactggaac agctatttct
      361 actgattttt cctcgagaag accttgacat gattttgaaa atggattctt tacaggatat
      421 aaaagcattg ttaacaggat tatttgtaca agataatgtg aataaagatg ccgtcacaga
      481 tagattggct tcagtggaga ctgatatgcc tctaacattg agacagcata gaataagtgc
      541 gacatcatca tcggaagaga gtagtaacaa aggtcaaaga cagttgactg tatcgattga
      601 ctcggcagct catcatgata actccacaat tccgttggat tttatgccca gggatgctct
      661 tcatggattt gattggtctg aagaggatga catgtcggat ggcttgccct tcctgaaaac
      721 ggaccccaac aataatgggt tctttggcga cggttctctc ttatgtattc ttcgatctat
      781 tggctttaaa ccggaaaatt acacgaactc taacgttaac aggctcccga ccatgattac
      841 ggatagatac acgttggctt ctagatccac aacatcccgt ttacttcaaa gttatctcaa
      901 taattttcac ccctactgcc ctatcgtgca ctcaccgacg ctaatgatgt tgtataataa
      961 ccagattgaa atcgcgtcga aggatcaatg gcaaatcctt tttaactgca tattagccat
     1021 tggagcctgg tgtatagagg gggaatctac tgatatagat gttttttact atcaaaatgc
     1081 taaatctcat ttgacgagca aggtcttcga gtcaggttcc ataattttgg tgacagccct
     1141 acatcttctg tcgcgatata cacagtggag gcagaaaaca aatactagct ataattttca
     1201 cagcttttcc ataagaatgg ccatatcatt gggcttgaat agggacctcc cctcgtcctt
     1261 cagtgatagc agcattctgg aacaaagacg ccgaatttgg tggtctgtct actcttggga
     1321 gatccaattg tccctgcttt atggtcgatc catccagctt tctcagaata caatctcctt
     1381 cccttcttct gtcgacgatg tgcagcgtac cacaacaggt cccaccatat atcatggcat
     1441 cattgaaaca gcaaggctct tacaagtttt cacaaaaatc tatgaactag acaaaacagt
     1501 aactgcagaa aaaagtccta tatgtgcaaa aaaatgcttg atgatttgta atgagattga
     1561 ggaggtttcg agacaggcac caaagttttt acaaatggat atttccacca ccgctctaac
     1621 caatttgttg aaggaacacc cttggctatc ctttacaaga ttcgaactga agtggaaaca
     1681 gttgtctctt atcatttatg tattaagaga ttttttcact aattttaccc agaaaaagtc
     1741 acaactagaa caggatcaaa atgatcatca aagttatgaa gttaaacgat gctccatcat
     1801 gttaagcgat gcagcacaaa gaactgttat gtctgtaagt agctatatgg acaatcataa
     1861 tgtcacccca tattttgcct ggaattgttc ttattacttg ttcaatgcag tcctagtacc
     1921 cataaagact ctactctcaa actcaaaatc gaatgctgag aataacgaga ccgcacaatt
     1981 attacaacaa attaacactg ttctgatgct attaaaaaaa ctggccactt ttaaaatcca
     2041 gacttgtgaa aaatacattc aagtactgga agaggtatgt gcgccgtttc tgttatcaca
     2101 gtgtgcaatc ccattaccgc atatcagtta taacaatagt aatggtagcg ccattaaaaa
     2161 tattgtcggt tctgcaacta tcgcccaata ccctactctt ccggaggaaa atgtcaacaa
     2221 tatcagtgtt aaatatgttt ctcctggctc agtagggcct tcacctgtgc cattgaaatc
     2281 aggagcaagt ttcagtgatc tagtcaagct gttatctaac cgtccaccct ctcgtaactc
     2341 tccagtgaca ataccaagaa gcacaccttc gcatcgctca gtcacgcctt ttctagggca
     2401 acagcaacag ctgcaatcat tagtgccact gaccccgtct gctttgtttg gtggcgccaa
     2461 ttttaatcaa agtgggaata ttgctgatag ctcattgtcc ttcactttca ctaacagtag
     2521 caacggtccg aacctcataa caactcaaac aaattctcaa gcgctttcac aaccaattgc
     2581 ctcctctaac gttcatgata acttcatgaa taatgaaatc acggctagta aaattgatga
     2641 tggtaataat tcaaaaccac tgtcacctgg ttggacggac caaactgcgt ataacgcgtt
     2701 tggaatcact acagggatgt ttaataccac tacaatggat gatgtatata actatctatt
     2761 cgatgatgaa gataccccac caaacccaaa aaaagagtaa gcggccgcat aacttcgtat
     2821 agcatacatt atacgaagtt atattcgaat cggaaatcaa tcgaattctc gatcaatgtt
     2881 ttgcgaaaaa aaatgaaacg agaacaaatg aattcagttg taaaagattt gttctcttaa
     2941 caacgtaatc aataaagtcg gatccaattt tgttcacaag tctaaacaat tatggttttt
     3001 gttggggttt cttataatta ataagagaga tatcaatcat tttacgagga ttatgagact
     3061 gcgttttgta gtccaagaaa gctgattaat gtttagagtg ttggtataaa tgttaatagg
     3121 ctcattaatg tagggcatgc gacgtcaggc ctaagcttga gctcgcccgg ggatctaatt
     3181 caattagaga ctaattcaat tagagctaat tcaattagga tcagcttatc gatttcgaac
     3241 cctcgaccgc cggagtataa atagaggcgc ttcgtctacg gagcgacaat tcaattcaaa
     3301 caagcaaagt gaacacgtcg ctaagcgaaa gctaagcaaa taaacaagcg cagctgaaca
     3361 agctaaacaa tcgggcaaaa tggatagcac tgagaacgtc atcaagccct tcatgcgctt
     3421 caaggtgcac atggagggct ccgtgaacgg ccacgagttc gagatcgagg gcgagggcga
     3481 gggcaagccc tacgagggca cccagaccgc caagctgcag gtgaccaagg gcggccccct
     3541 gcccttcgcc tgggacatcc tgtcccccca gttccagtac ggctccaagg tgtacgtgaa
     3601 gcaccccgcc gacatccccg actacaagaa gctgtccttc cccgagggct tcaagtggga
     3661 gcgcgtgatg aacttcgagg acggcggcgt ggtgaccgtg acccaggact cctccctgca
     3721 ggacggcacc ttcatctacc acgtgaagtt catcggcgtg aacttcccct ccgacggccc
     3781 cgtaatgcag aagaagactc tgggctggga gccctccacc gagcgcctgt acccccgcga
     3841 cggcgtgctg aagggcgaga tccacaaggc gctgaagctg aagggcggcg gccactacct
     3901 ggtggagttc aagtcaatct acatggccaa gaagcccgtg aagctgcccg gctactacta
     3961 cgtggactcc aagctggaca tcacctccca caacgaggac tacaccgtgg tggagcagta
     4021 cgagcgcgcc gaggcccgcc accacctgtt ccagtagcca tggctagatc ataatcagcc
     4081 ataccacatt tgtagaggtt ttacttgctt taaaaaacct cccacacctc cccctgaacc
     4141 tgaaacataa aatgaatgca attgttgttg ttaacttgtt tattgcagct tataatggtt
     4201 acaaataaag caatagcatc acaaatttca caaataaagc atttttttca ctgcattcta
     4261 gttgtggttt gtccaaactc atcaatgtat caagcttata acttcgtata gcatacatta
     4321 tacgaagtta tatgcatgaa gttcctattc ttcaaatagt ataggaactt ccgagacgcc
     4381 gcggtggagc tccagctttt gttcccttta gtgagggtta attgcgcgct tggcgtaatc
     4441 atggtcatag ctgtttcctg tgtgaaattg ttatccgctc acaattccac acaacatacg
     4501 agccggaagc ataaagtgta aagcctgggg tgcctaatga gtgagctaac tcacattaat
     4561 tgcgttgcgc tcactgcccg ctttccagtc gggaaacctg tcgtgccagc tgcattaatg
     4621 aatcggccaa cgcgcgggga gaggcggttt gcgtattggg cgctcttccg cttcctcgct
     4681 cactgactcg ctgcgctcgg tcgttcggct gcggcgagcg gtatcagctc actcaaaggc
     4741 ggtaatacgg ttatccacag aatcagggga taacgcagga aagaacatgt gagcaaaagg
     4801 ccagcaaaag gccaggaacc gtaaaaaggc cgcgttgctg gcgtttttcc ataggctccg
     4861 cccccctgac gagcatcaca aaaatcgacg ctcaagtcag aggtggcgaa acccgacagg
     4921 actataaaga taccaggcgt ttccccctgg aagctccctc gtgcgctctc ctgttccgac
     4981 cctgccgctt accggatacc tgtccgcctt tctcccttcg ggaagcgtgg cgctttctca
     5041 tagctcacgc tgtaggtatc tcagttcggt gtaggtcgtt cgctccaagc tgggctgtgt
     5101 gcacgaaccc cccgttcagc ccgaccgctg cgccttatcc ggtaactatc gtcttgagtc
     5161 caacccggta agacacgact tatcgccact ggcagcagcc actggtaaca ggattagcag
     5221 agcgaggtat gtaggcggtg ctacagagtt cttgaagtgg tggcctaact acggctacac
     5281 tagaagaaca gtatttggta tctgcgctct gctgaagcca gttaccttcg gaaaaagagt
     5341 tggtagctct tgatccggca aacaaaccac cgctggtagc ggtggttttt ttgtttgcaa
     5401 gcagcagatt acgcgcagaa aaaaaggatc tcaagaagat cctttgatct tttctacggg
     5461 gtctgacgct cagtggaacg aaaactcacg ttaagggatt ttggtcatga gattatcaaa
     5521 aaggatcttc acctagatcc ttttaaatta aaaatgaagt tttaaatcaa tctaaagtat
     5581 atatgagtaa acttggtctg acagttacca atgcttaatc agtgaggcac ctatctcagc
     5641 gatctgtcta tttcgttcat ccatagttgc ctgactcccc gtcgtgtaga taactacgat
     5701 acgggagggc ttaccatctg gccccagtgc tgcaatgata ccgcgagacc cacgctcacc
     5761 ggctccagat ttatcagcaa taaaccagcc agccggaagg gccgagcgca gaagtggtcc
     5821 tgcaacttta tccgcctcca tccagtctat taattgttgc cgggaagcta gagtaagtag
     5881 ttcgccagtt aatagtttgc gcaacgttgt tgccattgct acaggcatcg tggtgtcacg
     5941 ctcgtcgttt ggtatggctt cattcagctc cggttcccaa cgatcaaggc gagttacatg
     6001 atcccccatg ttgtgcaaaa aagcggttag ctccttcggt cctccgatcg ttgtcagaag
     6061 taagttggcc gcagtgttat cactcatggt tatggcagca ctgcataatt ctcttactgt
     6121 catgccatcc gtaagatgct tttctgtgac tggtgagtac tcaaccaagt cattctgaga
     6181 atagtgtatg cggcgaccga gttgctcttg cccggcgtca atacgggata ataccgcgcc
     6241 acatagcaga actttaaaag tgctcatcat tggaaaacgt tcttcggggc gaaaactctc
     6301 aaggatctta ccgctgttga gatccagttc gatgtaaccc actcgtgcac ccaactgatc
     6361 ttcagcatct tttactttca ccagcgtttc tgggtgagca aaaacaggaa ggcaaaatgc
     6421 cgcaaaaaag ggaataaggg cgacacggaa atgttgaata ctcatactct tcctttttca
     6481 atattattga agcatttatc agggttattg tctcatgagc ggatacatat ttgaatgtat
     6541 ttagaaaaat aaacaaatag gggttccgcg cacatttccc cgaaaagtgc cacctaaatt
     6601 gtaagcgtta atattttgtt aaaattcgcg ttaaattttt gttaaatcag ctcatttttt
     6661 aaccaatagg ccgaaatcgg caaaatccct tataaatcaa aagaatagac cgagataggg
     6721 ttgagtgttg ttccagtttg gaacaagagt ccactattaa agaacgtgga ctccaacgtc
     6781 aaagggcgaa aaaccgtcta tcagggcgat ggcccactac gtgaaccatc accctaatca
     6841 agttttttgg ggtcgaggtg ccgtaaagca ctaaatcgga accctaaagg gagcccccga
     6901 tttagagctt gacggggaaa gccggcgaac gtggcgagaa aggaagggaa gaaagcgaaa
     6961 ggagcgggcg ctagggcgct ggcaagtgta gcggtcacgc tgcgcgtaac caccacaccc
     7021 gccgcgctta atgcgccgct acagggcgcg tcccattcgc cattcaggct gcgcaactgt
     7081 tgggaagggc gatcggtgcg ggcctcttcg ctattacgcc agctggcgaa agggggatgt
     7141 gctgcaaggc gattaagttg ggtaacgcca gggttttccc agtcacgacg ttgtaaaacg
     7201 acggccagtg agcgcgcg
//
