LOCUS       sequence-494260-        7992 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Conditional tagging of endogenous proteins with GFSTF in open 
            reading frame 2, with dual membrane labelling.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 7992)
  TITLE     dFlpTag conditional protein labelling lines
  JOURNAL   Unpublished
REFERENCE   2  (bases 1 to 7992)
  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"; journalName: 
            "Unpublished"
COMMENT     Sequence Label: pFLPtag-SA-SD-2-GFSTF
FEATURES             Location/Qualifiers
     source          1..7992
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     intron          1527..1592
                     /label=small t intron
                     /note="SV40 (simian virus 40) small t antigen intron"
     CDS             1722..1742
                     /codon_start=1
                     /product="nuclear localization signal of SV40 (simian virus
                     40) large T antigen"
                     /label=SV40 NLS
                     /translation="PKKKRKV"
     polyA_signal    2014..2148
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(2051..2070)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     2105..2124
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     CDS             3817..3834
                     /codon_start=1
                     /product="tetracysteine peptide that binds biarsenical 
                     labeling reagents"
                     /label=tetracysteine tag
                     /translation="CCPGCC"
     CDS             3856..3879
                     /codon_start=1
                     /product="peptide that binds Strep-Tactin(R), an engineered
                     form of streptavidin"
                     /label=Strep-Tag II
                     /translation="WSHPQFEK"
     CDS             3880..3900
                     /codon_start=1
                     /product="tobacco etch virus (TEV) protease recognition and
                     cleavage site"
                     /label=TEV site
                     /translation="ENLYFQS"
     CDS             3901..3966
                     /codon_start=1
                     /product="three tandem FLAG(R) epitope tags, followed by an
                     enterokinase cleavage site"
                     /label=3xFLAG
                     /translation="DYKDHDGDYKDHDIDYKDDDDK"
     protein_bind    complement(4161..4194)
                     /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)"
     protein_bind    complement(4259..4306)
                     /label=FRT
                     /bound_moiety="FLP recombinase from the Saccharomyces 
                     cerevisiae 2u plasmid"
                     /note="FLP-mediated recombination occurs in the 8-bp core 
                     sequence TCTAGAAA (Turan and Bode, 2011)."
     promoter        complement(4336..4574)
                     /label=hsp70 promoter
                     /note="Drosophila melanogaster hsp70Bb promoter"
     primer_bind     complement(4368..4389)
                     /label=pCasper-hs
                     /note="Drosophila Hsp70 promoter, forward primer"
     protein_bind    4593..4687
                     /label=5X UAS
                     /bound_moiety="GAL4"
                     /note="five tandem copies of the ""ScaI site"" 17-mer 
                     CGGAGTACTGTCCTCCG, an upstream activating sequence (UAS) 
                     that efficiently binds yeast Gal4 (Webster et al., 1988; 
                     Pfeiffer et al., 2010)"
     protein_bind    4742..4811
                     /label=attB
                     /bound_moiety="phage phi-C31 integrase"
                     /note="attB site for the phi-C31 integrase (Groth et al., 
                     2000)"
     primer_bind     complement(4851..4871)
                     /label=T3
                     /note="T3 promoter, forward primer"
     promoter        complement(4851..4869)
                     /label=T3 promoter
                     /note="promoter for bacteriophage T3 RNA polymerase"
     primer_bind     complement(4890..4906)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(4890..4906)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(4903..4925)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    4914..4930
                     /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(4938..4944,4945..4962,4963..4968))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    4983..5004
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(5121..5138)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(5292..5880)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(5372..5391)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(6051..6842,6843..6911))
                     /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     6674..6693
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(6912..7016)
                     /gene="bla"
                     /label=AmpR promoter
     rep_origin      complement(7042..7497)
                     /direction=LEFT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(7179..7200)
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     7391..7410
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     7624..7646
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     primer_bind     7638..7655
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     7639..7655
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     7665..7684
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        7665..7683
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     protein_bind    7719..7788
                     /label=attB
                     /bound_moiety="phage phi-C31 integrase"
                     /note="attB site for the phi-C31 integrase (Groth et al., 
                     2000)"
     protein_bind    7859..7892
                     /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)"
     protein_bind    7943..7990
                     /label=FRT
                     /bound_moiety="FLP recombinase from the Saccharomyces 
                     cerevisiae 2u plasmid"
                     /note="FLP-mediated recombination occurs in the 8-bp core 
                     sequence TCTAGAAA (Turan and Bode, 2011)."
ORIGIN
        1 ggccgctatg gtgtccaagg gcgaggaagt gatcaaggag ttcatgcgct tcaaagtgcg
       61 catggagggc agcatgaacg gccacgagtt cgagattgag ggcgagggcg agggacgccc
      121 atatgaggga acccagaccg ccaagctgaa agtgaccaag ggcggacccc tgcccttcgc
      181 ctgggatatt ctgagccccc agtttatgta cggcagcaag gcctacgtga agcaccccgc
      241 cgatatcccc gattacaaga agctgagctt ccccgagggc ttcaagtggg agcgcgtgat
      301 gaacttcgag gatggcggcc tcgtgaccgt gacccaggat agcagtctgc aggatggcac
      361 cctgatctac aaagtgaaga tgcgcggcac caacttcccc cccgatggac ccgtgatgca
      421 gaagaagacc atgggctggg aggccagcac cgagcgcctg tatccacgcg acggcgtgct
      481 gaagggcgag attcatcagg ccctgaagct gaaggacggc ggccactacc tggtggagtt
      541 caagaccatc tacatggcca agaagcccgt gcagctgccc ggctactact acgtggacac
      601 caagctggat atcaccagcc acaacgagga ttacaccatc gtggagcagt acgagcgcag
      661 cgagggccgc caccatctgt ttctgggaca cggaaccggc agcaccggaa gcggaagtag
      721 tggaaccgcc agcagcgagg ataacaacat ggccgtgatt aaggagttta tgcgctttaa
      781 agtgcgcatg gagggatcca tgaatggaca tgagtttgag atcgagggcg agggcgaggg
      841 acgcccctac gagggcacgc agacggccaa gctgaaagtg acgaagggcg gaccgctgcc
      901 gtttgcctgg gacatcctgt cgccgcagtt tatgtatggc tcgaaggcct atgtgaagca
      961 cccggccgac attccggact ataagaagct gtcgttccca gagggattca agtgggagcg
     1021 cgtgatgaat tttgaggacg gcggactcgt gaccgtgacg caggacagtt ccctgcagga
     1081 cggaacgctg atctataaag tgaagatgcg cggaacgaat ttcccgccgg acggcccagt
     1141 gatgcagaag aagacgatgg gatgggaggc ctcgaccgag cgcctgtacc cccgcgacgg
     1201 cgtgctgaag ggcgagatcc accaggccct gaagctgaag gatggcggac gctatctggt
     1261 ggagtttaag acgatctata tggccaagaa gccagtgcag ctgccgggat attactatgt
     1321 ggatacgaag ctggacatta cctcgcacaa tgaggactat acgattgtgg agcagtatga
     1381 gcgctcggag ggacgccatc acctgtttct gtacggcatg gatgagctgt ataagtagga
     1441 ggccggcctc gatctttgtg aaggaacctt acttctgtgg tgtgacataa ttggacaaac
     1501 tacctacaga gatttaaagc tctaaggtaa atataaaatt tttaagtgta taatgtgtta
     1561 aactactgat tctaattgtt tgtgtatttt agattccaac ctatggaact gatgaatggg
     1621 agcagtggtg gaatgccttt aatgaggaaa acctgttttg ctcagaagaa atgccatcta
     1681 gtgatgatga ggctactgct gactctcaac attctactcc tccaaaaaag aagagaaagg
     1741 tagaagaccc caaggacttt ccttcagaat tgctaagttt tttgagtcat gctgtgttta
     1801 gtaatagaac tcttgcttgc tttgctattt acaccacaaa ggaaaaagct gcactgctat
     1861 acaagaaaat tatggaaaaa tatttgatgt atagtgcctt gactagagat cataatcagc
     1921 cataccacat ttgtagaggt tttacttgct ttaaaaaacc tcccacacct ccccctgaac
     1981 ctgaaacata aaatgaatgc aattgttgtt gttaacttgt ttattgcagc ttataatggt
     2041 tacaaataaa gcaatagcat cacaaatttc acaaataaag catttttttc actgcattct
     2101 agttgtggtt tgtccaaact catcaatgta tcttatcatg tctggatctg cgtcgcgcca
     2161 cttcaacgct cgatgggagc gtcattggtg ggcggggtaa ccgtcgaaat cagtgtttac
     2221 gcttccaatc gcaacaaaaa attcactgca acactgaaaa gcatacgaaa acgatgaaga
     2281 ttgtacgaga aaccataaag tattttatcc acaaagacac gtatagcaga aaagccaagt
     2341 taactcggcg ataagttgtg tacacaagaa taaaatcggc cagattcagt gttgtcagaa
     2401 ataagaaaac cccactatgt ttttctttgc cttttctttc tcccagcgat cattcatttc
     2461 gtggtgaaag aacggggtca ttgcacggag tttcgactgc gggaaagcag agctgccgtt
     2521 cacttcgtct ataattagcg ctttctattt tccccgattc gggccgctgc tgcgcttttc
     2581 cgcctgctgt ttgtggcaag tgtagcagca ggctgtgcac gcagtgtggc atgcacttgg
     2641 ctttccaccg ttggtatcga ttctctggga cgatgagtca ttcctttcgg gaccacagca
     2701 taatcgttgc cagctcaccg aaatggtgac ttcatttctt aactgccgtc aagcatgcga
     2761 ttgtacatac atacatattt atatatgtac atatttatgt gactatggta ggtcgatata
     2821 atagcaatca acgcaagcaa atgtgtcagt cctgcttaca ggaacgattc tatttagtaa
     2881 ttttcgttgt ataaagtaat tatgtatgta tgtaagcccc ataaatctga aaaccggtag
     2941 tcgatccaac atggcgactt gtcccatccc cggcatgttt aaatatacta attattcttg
     3001 aactaatttt aatcaaccga tttatctctc ttccgcaggt cgggaggttc cggtggaagc
     3061 ggaggtagcg gcggatccgt gtccaagggc gaggagctgt tcaccggcgt ggtgcccatc
     3121 ctggtggagc tggatggcga cgtgaacggc cacaagttca gcgtgcgcgg cgagggcgag
     3181 ggcgacgcca ccaacggcaa gctgaccctg aagttcatct gcaccaccgg caagctgccc
     3241 gtgccctggc ccaccctggt gaccaccctg acctacggcg tgcagtgctt cagccgctac
     3301 cccgatcaca tgaagcagca cgatttcttc aagagcgcca tgcccgaggg ctacgtgcag
     3361 gagcgcacca tcagcttcaa ggatgacggc acctacaaga cccgcgccga ggtgaagttc
     3421 gagggcgata ccctggtgaa ccgcatcgag ctgaagggca tcgatttcaa ggaggatggc
     3481 aacatcctgg gccacaagct ggagtacaac ttcaacagcc acaacgtgta catcaccgcc
     3541 gataagcaga agaacggcat caaggccaac ttcaagatcc gccacaatgt ggaggatggc
     3601 tccgtgcagc tggccgatca ctaccagcag aacaccccca tcggcgacgg cccagtgctg
     3661 ctgcccgata accactacct gagcacccag agcgtgctgt ccaaggaccc caacgagaag
     3721 cgcgatcaca tggtgctgct ggagttcgtg accgccgccg gcatcaccct gggcatggat
     3781 gagctgtaca aggagagcag cggcagcttc ctgaactgct gcccaggatg ctgcatggaa
     3841 ccgggaagcg gcaactggtc ccacccccag ttcgagaagg aaaacctgta cttccagagc
     3901 gattacaagg atcacgacgg cgactacaag gaccacgata ttgattacaa ggacgatgac
     3961 gacaagggcg gatccggagg tagcggtgga agcggaggtt ccggcaggta agttattgaa
     4021 caatggcatc aaatgccttc atcatcacta ccctttagcc cttaagaccc cacaatgacc
     4081 ttacccactc agagaaaaaa gtaaatatga aagcccattt gaacttctaa gcttgtcgac
     4141 ttaattaaat cgatcctagg gaagttccta tactatttga agaataggaa cttcggaata
     4201 ggaacttcta agaggtttca tattgctaat agcagctaca atccagctac cattctgcga
     4261 agttcctata ctttctagag aataggaact tcggaatagg aacttcgctg gtaccctcga
     4321 gcccaattcc ctattcagag ttctcttctt gtattcaata attacttctt ggcagatttc
     4381 agtagttgca gttgatttac ttggttgctg gttactttta attgattcac tttaacttgc
     4441 actttactgc agattgttta gcttgttcag ctgcgcttgt ttatttgctt agctttcgct
     4501 tagcgacgtg ttcactttgc ttgtttgaat tgaattgtcg ctccgtagac gaagcgcctc
     4561 tatttatact ccggcgctcg ctagagtctc cgctcggagg acagtactcc gctcggagga
     4621 cagtactccg ctcggaggac agtactccgc tcggaggaca gtactccgct cggaggacag
     4681 tactccgacc tgcagggaat tcctgcagac tagttctaga gtcgaccatc atgatggacc
     4741 agatgggtga ggtggagtac gcgcccgggg agcccaaggg cacgccctgg cacccgcacc
     4801 gcggcttcga gaccgtgacc tacatcatgc atggtaccca gcttttgttc cctttagtga
     4861 gggttaattg cgcgcttggc gtaatcatgg tcatagctgt ttcctgtgtg aaattgttat
     4921 ccgctcacaa ttccacacaa catacgagcc gggagcataa agtgtaaagc ctggggtgcc
     4981 taatgagtga gctaactcac attaattgcg ttgcgctcac tgcccgcttt ccagtcggga
     5041 aacctgtcgt gccagctgca ttaatgaatc ggccaacgcg cggggagagg cggtttgcgt
     5101 attgggcgct cttccgcttc ctcgctcact gactcgctgc gctcggtcgt tcggctgcgg
     5161 cgagcggtat cagctcactc aaaggcggta atacggttat ccacagaatc aggggataac
     5221 gcaggaaaga acatgtgagc aaaaggccag caaaaggcca ggaaccgtaa aaaggccgcg
     5281 ttgctggcgt ttttccatag gctccgcccc cctgacgagc atcacaaaaa tcgacgctca
     5341 agtcagaggt ggcgaaaccc gacaggacta taaagatacc aggcgtttcc ccctggaagc
     5401 tccctcgtgc gctctcctgt tccgaccctg ccgcttaccg gatacctgtc cgcctttctc
     5461 ccttcgggaa gcgtggcgct ttctcatagc tcacgctgta ggtatctcag ttcggtgtag
     5521 gtcgttcgct ccaagctggg ctgtgtgcac gaaccccccg ttcagcccga ccgctgcgcc
     5581 ttatccggta actatcgtct tgagtccaac ccggtaagac acgacttatc gccactggca
     5641 gcagccactg gtaacaggat tagcagagcg aggtatgtag gcggtgctac agagttcttg
     5701 aagtggtggc ctaactacgg ctacactaga agaacagtat ttggtatctg cgctctgctg
     5761 aagccagtta ccttcggaaa aagagttggt agctcttgat ccggcaaaca aaccaccgct
     5821 ggtagcggtg gtttttttgt ttgcaagcag cagattacgc gcagaaaaaa aggatctcaa
     5881 gaagatcctt tgatcttttc tacggggtct gacgctcagt ggaacgaaaa ctcacgttaa
     5941 gggattttgg tcatgagatt atcaaaaagg atcttcacct agatcctttt aaattaaaaa
     6001 tgaagtttta aatcaatcta aagtatatat gagtaaactt ggtctgacag ttaccaatgc
     6061 ttaatcagtg aggcacctat ctcagcgatc tgtctatttc gttcatccat agttgcctga
     6121 ctccccgtcg tgtagataac tacgatacgg gagggcttac catctggccc cagtgctgca
     6181 atgataccgc gagacccacg ctcaccggct ccagatttat cagcaataaa ccagccagcc
     6241 ggaagggccg agcgcagaag tggtcctgca actttatccg cctccatcca gtctattaat
     6301 tgttgccggg aagctagagt aagtagttcg ccagttaata gtttgcgcaa cgttgttgcc
     6361 attgctacag gcatcgtggt gtcacgctcg tcgtttggta tggcttcatt cagctccggt
     6421 tcccaacgat caaggcgagt tacatgatcc cccatgttgt gcaaaaaagc ggttagctcc
     6481 ttcggtcctc cgatcgttgt cagaagtaag ttggccgcag tgttatcact catggttatg
     6541 gcagcactgc ataattctct tactgtcatg ccatccgtaa gatgcttttc tgtgactggt
     6601 gagtactcaa ccaagtcatt ctgagaatag tgtatgcggc gaccgagttg ctcttgcccg
     6661 gcgtcaatac gggataatac cgcgccacat agcagaactt taaaagtgct catcattgga
     6721 aaacgttctt cggggcgaaa actctcaagg atcttaccgc tgttgagatc cagttcgatg
     6781 taacccactc gtgcacccaa ctgatcttca gcatctttta ctttcaccag cgtttctggg
     6841 tgagcaaaaa caggaaggca aaatgccgca aaaaagggaa taagggcgac acggaaatgt
     6901 tgaatactca tactcttcct ttttcaatat tattgaagca tttatcaggg ttattgtctc
     6961 atgagcggat acatatttga atgtatttag aaaaataaac aaataggggt tccgcgcaca
     7021 tttccccgaa aagtgccacc taaattgtaa gcgttaatat tttgttaaaa ttcgcgttaa
     7081 atttttgtta aatcagctca ttttttaacc aataggccga aatcggcaaa atcccttata
     7141 aatcaaaaga atagaccgag atagggttga gtgttgttcc agtttggaac aagagtccac
     7201 tattaaagaa cgtggactcc aacgtcaaag ggcgaaaaac cgtctatcag ggcgatggcc
     7261 cactacgtga accatcaccc taatcaagtt ttttggggtc gaggtgccgt aaagcactaa
     7321 atcggaaccc taaagggagc ccccgattta gagcttgacg gggaaagccg gcgaacgtgg
     7381 cgagaaagga agggaagaaa gcgaaaggag cgggcgctag ggcgctggca agtgtagcgg
     7441 tcacgctgcg cgtaaccacc acacccgccg cgcttaatgc gccgctacag ggcgcgtccc
     7501 attcgccatt caggctgcgc aactgttggg aagggcgatc ggtgcgggcc tcttcgctat
     7561 tacgccagct ggcgaaaggg ggatgtgctg caaggcgatt aagttgggta acgccagggt
     7621 tttcccagtc acgacgttgt aaaacgacgg ccagtgagcg cgcgtaatac gactcactat
     7681 agggcgaatt ggagctcgct agcgatgtag gtcacggtct cgaagccgcg gtgcgggtgc
     7741 cagggcgtgc ccttgggctc cccgggcgcg tactccacct cacccatctg gtccatcatg
     7801 atggtcgaca agcttggatc cgatgtccca atacgcgtag gcctgaagtt cctattccga
     7861 agttcctatt cttcaaatag tataggaact tctctttgca ccatttcaaa gaataacagt
     7921 gataatttct gggttaaggc aagaagttcc tattccgaag ttcctattct ctagaaagta
     7981 taggaacttc gc
//
