LOCUS       sequence-357389-        4435 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  FlpOFF knock-in for smFP-HA-CaV2.3 (amino acid position: G5).
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 4435)
  AUTHORS   Droogers WJ, Willems J, MacGillavry HD, de Jong APH
  TITLE     Duplex Labeling and Manipulation of Neuronal Proteins Using 
            Sequential CRISPR/Cas9 Gene Editing.
  JOURNAL   eNeuro. 2022 Jul 18;9(4):ENEURO.0056-22.2022. doi: 
            10.1523/ENEURO.0056-22.2022.
  PUBMED    35851300
REFERENCE   2  (bases 1 to 4435)
  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: 
            "10.1523/ENEURO.0056-22"; journalName: "eNeuro"; date: "2022-07-18- 
            18"; volume: "9"; issue: "4"; pages: "ENEURO.0056-22.2022"
COMMENT     Sequence Label: pOC5 smFP-HA-Cacna1e KI
FEATURES             Location/Qualifiers
     source          1..4435
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     promoter        39..287
                     /label=U6 promoter
                     /note="RNA polymerase III promoter for human U6 snRNA 
                     (Domitrovich & Kunkel, 2003)"
     primer_bind     39..59
                     /label=hU6-F
                     /note="Human U6 promoter, forward primer"
     primer_bind     210..229
                     /label=LKO.1 5'
                     /note="Human U6 promoter, forward primer"
     protein_bind    326..359
                     /label=FRT (minimal)
                     /bound_moiety="FLP recombinase from the Saccharomyces 
                     cerevisiae 2u plasmid"
                     /note="supports FLP-mediated excision but not integration 
                     (Turan and Bode, 2011)"
     protein_bind    495..528
                     /label=FRT (minimal)
                     /bound_moiety="FLP recombinase from the Saccharomyces 
                     cerevisiae 2u plasmid"
                     /note="supports FLP-mediated excision but not integration 
                     (Turan and Bode, 2011)"
     CDS             595..621
                     /codon_start=1
                     /product="HA (human influenza hemagglutinin) epitope tag"
                     /label=HA
                     /translation="YPYDVPDYA"
     CDS             625..651
                     /codon_start=1
                     /product="HA (human influenza hemagglutinin) epitope tag"
                     /label=HA
                     /translation="YPYDVPDYA"
     CDS             655..681
                     /codon_start=1
                     /product="HA (human influenza hemagglutinin) epitope tag"
                     /label=HA
                     /translation="YPYDVPDYA"
     CDS             1210..1236
                     /codon_start=1
                     /product="HA (human influenza hemagglutinin) epitope tag"
                     /label=HA
                     /translation="YPYDVPDYA"
     CDS             1243..1269
                     /codon_start=1
                     /product="HA (human influenza hemagglutinin) epitope tag"
                     /label=HA
                     /translation="YPYDVPDYA"
     CDS             1282..1308
                     /codon_start=1
                     /product="HA (human influenza hemagglutinin) epitope tag"
                     /label=HA
                     /translation="YPYDVPDYA"
     CDS             1315..1341
                     /codon_start=1
                     /product="HA (human influenza hemagglutinin) epitope tag"
                     /label=HA
                     /translation="YPYDVPDYA"
     CDS             1552..1578
                     /codon_start=1
                     /product="HA (human influenza hemagglutinin) epitope tag"
                     /label=HA
                     /translation="YPYDVPDYA"
     CDS             1582..1608
                     /codon_start=1
                     /product="HA (human influenza hemagglutinin) epitope tag"
                     /label=HA
                     /translation="YPYDVPDYA"
     CDS             1612..1638
                     /codon_start=1
                     /product="HA (human influenza hemagglutinin) epitope tag"
                     /label=HA
                     /translation="YPYDVPDYA"
     repeat_region   1736..1876
                     /label=AAV2 ITR
                     /note="inverted terminal repeat of adeno-associated virus 
                     serotype 2"
     repeat_region   1736..1865
                     /label=AAV2 ITR
     rep_origin      1951..2406
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(2038..2057)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     2248..2269
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     complement(2423..2442)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     2542..2564
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     primer_bind     complement(2602..2620)
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     promoter        2688..2792
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(2793..2861,2862..3653)
                     /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     complement(3011..3030)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      3824..4412
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     4313..4332
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
ORIGIN
        1 acggttcctg gccttttgct ggccttttgc tcacatgtga gggcctattt cccatgattc
       61 cttcatattt gcatatacga tacaaggctg ttagagagat aattggaatt aatttgactg
      121 taaacacaaa gatattagta caaaatacgt gacgtagaaa gtaataattt cttgggtagt
      181 ttgcagtttt aaaattatgt tttaaaatgg actatcatat gcttaccgta acttgaaagt
      241 atttcgattt cttggcttta tatatcttgt ggaaaggacg aaacaccgca ggatggctcg
      301 cttcgggggt tttagagcta tgctggaagt tcctattctc tagaaagtat aggaacttcc
      361 agcatagcaa gttaaaataa ggctagtccg ttatcaactt gaaaaagtgg caccgagtcg
      421 gtgctttttt gttttagagc tagaaatagc aagttaaaat aaggctagtc cgtttttagc
      481 gcgtgcgcca attcgaagtt cctattctct agaaagtata ggaacttctt tttttttttt
      541 ctagaagctt cccccccgaa gcgagccatc ctgatggcta gcggagccac catgtaccct
      601 tatgatgtgc ccgattatgc tggctaccct tatgatgttc ctgattacgc cggatatccg
      661 tatgacgtgc cagactacgc gggaggtgtg agcaagggag aggagttgtt taccggcgtg
      721 gtccccatac tggtggagtt ggatggcgac gttaatggac ataaattctc ggtccgcggc
      781 gagggagagg gagacgccac caatggcaag ctgacgctta agttcatttg cacgactgga
      841 aaattgcctg tcccctggcc gaccctggtc accacactgg gcggaggagt gcagtgcttc
      901 tcccgttacc cagaccacat gaagcagcac gatttcttca agagcgcaat gccggaggga
      961 tacgtgcaag aacggactat ttccttcaag gatgacggaa cctataagac ccgtgcggag
     1021 gtcaaattcg agggtgacac cctggtgaac cgaattgaac tcaaaggaat cgatttcaag
     1081 gaggatggaa atattctggg tcacaagctg gaatacaact tcaacagcca taatgtgtac
     1141 attacggctg ataagcagaa gaacggcatt aaggccaatt tcaagatccg ccacaacgtt
     1201 gagggtggat atccctacga cgtgcccgat tatgcgggcg gatatccgta tgatgttcca
     1261 gattatgctg gtggtggagg ctatccctat gatgtccccg actacgccgg aggttaccca
     1321 tacgacgtgc ccgactacgc tggaggcgat ggcagcgtgc agctggcaga tcattatcaa
     1381 cagaataccc ccataggtga tggccccgtt ctgcttccag ataatcacta cctttccacc
     1441 cagagcgtgc tttcgaaaga cccgaacgaa aagcgtgatc acatggtcct gctggagttt
     1501 gtgaccgcgg ctggaatcac cctgggtatg gacgaactct acaagggtgg ctacccctac
     1561 gatgtgccgg attacgctgg atatccgtat gacgtaccgg actatgccgg ttacccgtac
     1621 gatgtcccgg actacgcttc aaccggtagc gctgtttccc ccccgaagcg agccatcctg
     1681 ctcgactcga ggatcctagg acgcgttaac ttacggtaaa tggcccggcg gccgcaggaa
     1741 cccctagtga tggagttggc cactccctct ctgcgcgctc gctcgctcac tgaggccggg
     1801 cgaccaaagg tcgcccgacg cccgggcttt gcccgggcgg cctcagtgag cgagcgagcg
     1861 cgcagctgcc tgcaggggcg cctgatgcgg tattttctcc ttacgcatct gtgcggtatt
     1921 tcacaccgca tacgtcaaag caaccatagt acgcgccctg tagcggcgca ttaagcgcgg
     1981 cgggtgtggt ggttacgcgc agcgtgaccg ctacacttgc cagcgcctta gcgcccgctc
     2041 ctttcgcttt cttcccttcc tttctcgcca cgttcgccgg ctttccccgt caagctctaa
     2101 atcgggggct ccctttaggg ttccgattta gtgctttacg gcacctcgac cccaaaaaac
     2161 ttgatttggg tgatggttca cgtagtgggc catcgccctg atagacggtt tttcgccctt
     2221 tgacgttgga gtccacgttc tttaatagtg gactcttgtt ccaaactgga acaacactca
     2281 actctatctc gggctattct tttgatttat aagggatttt gccgatttcg gtctattggt
     2341 taaaaaatga gctgatttaa caaaaattta acgcgaattt taacaaaata ttaacgttta
     2401 caattttatg gtgcactctc agtacaatct gctctgatgc cgcatagtta agccagcccc
     2461 gacacccgcc aacacccgct gacgcgccct gacgggcttg tctgctcccg gcatccgctt
     2521 acagacaagc tgtgaccgtc tccgggagct gcatgtgtca gaggttttca ccgtcatcac
     2581 cgaaacgcgc gagacgaaag ggcctcgtga tacgcctatt tttataggtt aatgtcatga
     2641 taataatggt ttcttagacg tcaggtggca cttttcgggg aaatgtgcgc ggaaccccta
     2701 tttgtttatt tttctaaata cattcaaata tgtatccgct catgagacaa taaccctgat
     2761 aaatgcttca ataatattga aaaaggaaga gtatgagtat tcaacatttc cgtgtcgccc
     2821 ttattccctt ttttgcggca ttttgccttc ctgtttttgc tcacccagaa acgctggtga
     2881 aagtaaaaga tgctgaagat cagttgggtg cacgagtggg ttacatcgaa ctggatctca
     2941 acagcggtaa gatccttgag agttttcgcc ccgaagaacg ttttccaatg atgagcactt
     3001 ttaaagttct gctatgtggc gcggtattat cccgtattga cgccgggcaa gagcaactcg
     3061 gtcgccgcat acactattct cagaatgact tggttgagta ctcaccagtc acagaaaagc
     3121 atcttacgga tggcatgaca gtaagagaat tatgcagtgc tgccataacc atgagtgata
     3181 acactgcggc caacttactt ctgacaacga tcggaggacc gaaggagcta accgcttttt
     3241 tgcacaacat gggggatcat gtaactcgcc ttgatcgttg ggaaccggag ctgaatgaag
     3301 ccataccaaa cgacgagcgt gacaccacga tgcctgtagc aatggcaaca acgttgcgca
     3361 aactattaac tggcgaacta cttactctag cttcccggca acaattaata gactggatgg
     3421 aggcggataa agttgcagga ccacttctgc gctcggccct tccggctggc tggtttattg
     3481 ctgataaatc tggagccggt gagcgtggaa gccgcggtat cattgcagca ctggggccag
     3541 atggtaagcc ctcccgtatc gtagttatct acacgacggg gagtcaggca actatggatg
     3601 aacgaaatag acagatcgct gagataggtg cctcactgat taagcattgg taactgtcag
     3661 accaagttta ctcatatata ctttagattg atttaaaact tcatttttaa tttaaaagga
     3721 tctaggtgaa gatccttttt gataatctca tgaccaaaat cccttaacgt gagttttcgt
     3781 tccactgagc gtcagacccc gtagaaaaga tcaaaggatc ttcttgagat cctttttttc
     3841 tgcgcgtaat ctgctgcttg caaacaaaaa aaccaccgct accagcggtg gtttgtttgc
     3901 cggatcaaga gctaccaact ctttttccga aggtaactgg cttcagcaga gcgcagatac
     3961 caaatactgt tcttctagtg tagccgtagt taggccacca cttcaagaac tctgtagcac
     4021 cgcctacata cctcgctctg ctaatcctgt taccagtggc tgctgccagt ggcgataagt
     4081 cgtgtcttac cgggttggac tcaagacgat agttaccgga taaggcgcag cggtcgggct
     4141 gaacgggggg ttcgtgcaca cagcccagct tggagcgaac gacctacacc gaactgagat
     4201 acctacagcg tgagctatga gaaagcgcca cgcttcccga agggagaaag gcggacaggt
     4261 atccggtaag cggcagggtc ggaacaggag agcgcacgag ggagcttcca gggggaaacg
     4321 cctggtatct ttatagtcct gtcgggtttc gccacctctg acttgagcgt cgatttttgt
     4381 gatgctcgtc aggggggcgg agcctatgga aaaacgccag caacgcggcc ttttt
//
