LOCUS       sequence-275174-        5256 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  TagRFP-T knock-in cassette.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 5256)
  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 5256)
  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: pPRxRF3
FEATURES             Location/Qualifiers
     source          1..5256
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     1..17
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     27..46
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        27..45
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     CDS             130..861
                     /codon_start=1
                     /product="photostable variant of TagRFP (Shaner et al., 
                     2008)"
                     /label=TagRFP-T
                     /translation="MVSKGEELIKENMHMKLYMEGTVNNHHFKCTSEGEGKPYEGTQTM
                     RIKVVEGGPLPFAFDILATSFMYGSRTFINHTQGIPDFFKQSFPEGFTWERVTTYEDGG
                     VLTATQDTSLQDGCLIYNVKIRGVNFPSNGPVMQKKTLGWEANTEMLYPADGGLEGRTD
                     MALKLVGGGHLICNFKTTYRSKKPAKNLKMPGVYYVDHRLERIKEADKETYVEQHEVAV
                     ARYCDLPSKLGHKLNGMDELYK"
     protein_bind    complement(873..906)
                     /label=loxP
                     /bound_moiety="Cre recombinase"
                     /note="Cre-mediated recombination occurs in the 8-bp core 
                     sequence (ATGTATGC) (Shaw et al., 2021)."
     CDS             1444..2121
                     /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(1624..1644)
                     /label=DsRed1-N
                     /note="DsRed1, reverse primer"
     primer_bind     1842..1861
                     /label=mCherry-F
                     /note="mCherry, forward primer"
     primer_bind     2036..2059
                     /label=DsRed1-C
                     /note="DsRed1, forward primer"
     polyA_signal    2237..2318
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(2274..2293)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     2328..2347
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     protein_bind    complement(2362..2395)
                     /label=loxP
                     /bound_moiety="Cre recombinase"
                     /note="Cre-mediated recombination occurs in the 8-bp core 
                     sequence (ATGTATGC) (Shaw et al., 2021)."
     protein_bind    2402..2435
                     /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(2469..2489)
                     /label=T3
                     /note="T3 promoter, forward primer"
     promoter        complement(2469..2487)
                     /label=T3 promoter
                     /note="promoter for bacteriophage T3 RNA polymerase"
     primer_bind     complement(2508..2524)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(2508..2524)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(2521..2543)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    2532..2548
                     /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(2556..2562,2563..2580,2581..2586))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    2601..2622
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(2739..2756)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(2910..3498)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(2990..3009)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(3669..4460,4461..4529))
                     /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     4292..4311
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(4530..4634)
                     /gene="bla"
                     /label=AmpR promoter
     rep_origin      complement(4660..5115)
                     /direction=LEFT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(4797..4818)
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     5009..5028
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     join(5242..5256,1..8)
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     primer_bind     join(5256,1..17)
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
ORIGIN
        1 gtaaaacgac ggccagtgag cgcgcgtaat acgactcact atagggcgaa ttgggtaccc
       61 gtctcaccta cgcccccaac tgagagaact caaaggttac cccagttggg gcactacggc
      121 gcgcccaaaa tggtgtctaa gggcgaagag ctgattaagg agaacatgca catgaagctg
      181 tacatggagg gcaccgtgaa caaccaccac ttcaagtgca catccgaggg cgaaggcaag
      241 ccctacgagg gcacccagac catgagaatc aaggtggtcg agggcggccc tctccccttc
      301 gccttcgaca tcctggctac cagcttcatg tacggcagca gaaccttcat caaccacacc
      361 cagggcatcc ccgatttctt taagcagtcc ttccctgagg gcttcacatg ggagagagtc
      421 accacatacg aagacggggg cgtgctgacc gctacccagg acaccagcct ccaggacggc
      481 tgcctcatct acaacgtcaa gatcagaggg gtgaacttcc catccaacgg ccctgtgatg
      541 cagaagaaaa cactcggctg ggaggccaac accgagatgc tgtaccccgc tgacggcggc
      601 ctggaaggca gaaccgacat ggccctgaag ctcgtgggcg ggggccacct gatctgcaac
      661 ttcaagacca catacagatc caagaaaccc gctaagaacc tcaagatgcc cggcgtctac
      721 tatgtggacc acagactgga aagaatcaag gaggccgaca aagagaccta cgtcgagcag
      781 cacgaggtgg ctgtggccag atactgcgac ctccctagca aactggggca caaacttaat
      841 ggcatggacg agctgtacaa gtaagcggcc gcataacttc gtatagcata cattatacga
      901 agttatattc gaatcggaaa tcaatcgaat tctcgatcaa tgttttgcga aaaaaaatga
      961 aacgagaaca aatgaattca gttgtaaaag atttgttctc ttaacaacgt aatcaataaa
     1021 gtcggatcca attttgttca caagtctaaa caattatggt ttttgttggg gtttcttata
     1081 attaataaga gagatatcaa tcattttacg aggattatga gactgcgttt tgtagtccaa
     1141 gaaagctgat taatgtttag agtgttggta taaatgttaa taggctcatt aatgtagggc
     1201 atgcgacgtc aggcctaagc ttgagctcgc ccggggatct aattcaatta gagactaatt
     1261 caattagagc taattcaatt aggatcagct tatcgatttc gaaccctcga ccgccggagt
     1321 ataaatagag gcgcttcgtc tacggagcga caattcaatt caaacaagca aagtgaacac
     1381 gtcgctaagc gaaagctaag caaataaaca agcgcagctg aacaagctaa acaatcgggc
     1441 aaaatggata gcactgagaa cgtcatcaag cccttcatgc gcttcaaggt gcacatggag
     1501 ggctccgtga acggccacga gttcgagatc gagggcgagg gcgagggcaa gccctacgag
     1561 ggcacccaga ccgccaagct gcaggtgacc aagggcggcc ccctgccctt cgcctgggac
     1621 atcctgtccc cccagttcca gtacggctcc aaggtgtacg tgaagcaccc cgccgacatc
     1681 cccgactaca agaagctgtc cttccccgag ggcttcaagt gggagcgcgt gatgaacttc
     1741 gaggacggcg gcgtggtgac cgtgacccag gactcctccc tgcaggacgg caccttcatc
     1801 taccacgtga agttcatcgg cgtgaacttc ccctccgacg gccccgtaat gcagaagaag
     1861 actctgggct gggagccctc caccgagcgc ctgtaccccc gcgacggcgt gctgaagggc
     1921 gagatccaca aggcgctgaa gctgaagggc ggcggccact acctggtgga gttcaagtca
     1981 atctacatgg ccaagaagcc cgtgaagctg cccggctact actacgtgga ctccaagctg
     2041 gacatcacct cccacaacga ggactacacc gtggtggagc agtacgagcg cgccgaggcc
     2101 cgccaccacc tgttccagta gccatggcta gatcataatc agccatacca catttgtaga
     2161 ggttttactt gctttaaaaa acctcccaca cctccccctg aacctgaaac ataaaatgaa
     2221 tgcaattgtt gttgttaact tgtttattgc agcttataat ggttacaaat aaagcaatag
     2281 catcacaaat ttcacaaata aagcattttt ttcactgcat tctagttgtg gtttgtccaa
     2341 actcatcaat gtatcaagct tataacttcg tatagcatac attatacgaa gttatatgca
     2401 tgaagttcct attcttcaaa tagtatagga acttccgaga cgccgcggtg gagctccagc
     2461 ttttgttccc tttagtgagg gttaattgcg cgcttggcgt aatcatggtc atagctgttt
     2521 cctgtgtgaa attgttatcc gctcacaatt ccacacaaca tacgagccgg aagcataaag
     2581 tgtaaagcct ggggtgccta atgagtgagc taactcacat taattgcgtt gcgctcactg
     2641 cccgctttcc agtcgggaaa cctgtcgtgc cagctgcatt aatgaatcgg ccaacgcgcg
     2701 gggagaggcg gtttgcgtat tgggcgctct tccgcttcct cgctcactga ctcgctgcgc
     2761 tcggtcgttc ggctgcggcg agcggtatca gctcactcaa aggcggtaat acggttatcc
     2821 acagaatcag gggataacgc aggaaagaac atgtgagcaa aaggccagca aaaggccagg
     2881 aaccgtaaaa aggccgcgtt gctggcgttt ttccataggc tccgcccccc tgacgagcat
     2941 cacaaaaatc gacgctcaag tcagaggtgg cgaaacccga caggactata aagataccag
     3001 gcgtttcccc ctggaagctc cctcgtgcgc tctcctgttc cgaccctgcc gcttaccgga
     3061 tacctgtccg cctttctccc ttcgggaagc gtggcgcttt ctcatagctc acgctgtagg
     3121 tatctcagtt cggtgtaggt cgttcgctcc aagctgggct gtgtgcacga accccccgtt
     3181 cagcccgacc gctgcgcctt atccggtaac tatcgtcttg agtccaaccc ggtaagacac
     3241 gacttatcgc cactggcagc agccactggt aacaggatta gcagagcgag gtatgtaggc
     3301 ggtgctacag agttcttgaa gtggtggcct aactacggct acactagaag aacagtattt
     3361 ggtatctgcg ctctgctgaa gccagttacc ttcggaaaaa gagttggtag ctcttgatcc
     3421 ggcaaacaaa ccaccgctgg tagcggtggt ttttttgttt gcaagcagca gattacgcgc
     3481 agaaaaaaag gatctcaaga agatcctttg atcttttcta cggggtctga cgctcagtgg
     3541 aacgaaaact cacgttaagg gattttggtc atgagattat caaaaaggat cttcacctag
     3601 atccttttaa attaaaaatg aagttttaaa tcaatctaaa gtatatatga gtaaacttgg
     3661 tctgacagtt accaatgctt aatcagtgag gcacctatct cagcgatctg tctatttcgt
     3721 tcatccatag ttgcctgact ccccgtcgtg tagataacta cgatacggga gggcttacca
     3781 tctggcccca gtgctgcaat gataccgcga gacccacgct caccggctcc agatttatca
     3841 gcaataaacc agccagccgg aagggccgag cgcagaagtg gtcctgcaac tttatccgcc
     3901 tccatccagt ctattaattg ttgccgggaa gctagagtaa gtagttcgcc agttaatagt
     3961 ttgcgcaacg ttgttgccat tgctacaggc atcgtggtgt cacgctcgtc gtttggtatg
     4021 gcttcattca gctccggttc ccaacgatca aggcgagtta catgatcccc catgttgtgc
     4081 aaaaaagcgg ttagctcctt cggtcctccg atcgttgtca gaagtaagtt ggccgcagtg
     4141 ttatcactca tggttatggc agcactgcat aattctctta ctgtcatgcc atccgtaaga
     4201 tgcttttctg tgactggtga gtactcaacc aagtcattct gagaatagtg tatgcggcga
     4261 ccgagttgct cttgcccggc gtcaatacgg gataataccg cgccacatag cagaacttta
     4321 aaagtgctca tcattggaaa acgttcttcg gggcgaaaac tctcaaggat cttaccgctg
     4381 ttgagatcca gttcgatgta acccactcgt gcacccaact gatcttcagc atcttttact
     4441 ttcaccagcg tttctgggtg agcaaaaaca ggaaggcaaa atgccgcaaa aaagggaata
     4501 agggcgacac ggaaatgttg aatactcata ctcttccttt ttcaatatta ttgaagcatt
     4561 tatcagggtt attgtctcat gagcggatac atatttgaat gtatttagaa aaataaacaa
     4621 ataggggttc cgcgcacatt tccccgaaaa gtgccaccta aattgtaagc gttaatattt
     4681 tgttaaaatt cgcgttaaat ttttgttaaa tcagctcatt ttttaaccaa taggccgaaa
     4741 tcggcaaaat cccttataaa tcaaaagaat agaccgagat agggttgagt gttgttccag
     4801 tttggaacaa gagtccacta ttaaagaacg tggactccaa cgtcaaaggg cgaaaaaccg
     4861 tctatcaggg cgatggccca ctacgtgaac catcacccta atcaagtttt ttggggtcga
     4921 ggtgccgtaa agcactaaat cggaacccta aagggagccc ccgatttaga gcttgacggg
     4981 gaaagccggc gaacgtggcg agaaaggaag ggaagaaagc gaaaggagcg ggcgctaggg
     5041 cgctggcaag tgtagcggtc acgctgcgcg taaccaccac acccgccgcg cttaatgcgc
     5101 cgctacaggg cgcgtcccat tcgccattca ggctgcgcaa ctgttgggaa gggcgatcgg
     5161 tgcgggcctc ttcgctatta cgccagctgg cgaaaggggg atgtgctgca aggcgattaa
     5221 gttgggtaac gccagggttt tcccagtcac gacgtt
//
