LOCUS       sequence-360217-        4413 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 4413)
  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 4413)
  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..4413
                     /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"
     rep_origin      1929..2384
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(2016..2035)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     2226..2247
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     complement(2401..2420)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     2520..2542
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     primer_bind     complement(2580..2598)
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     promoter        2666..2770
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(2771..2839,2840..3631)
                     /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(2989..3008)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      3802..4390
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     4291..4310
                     /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 tcgcccggcc tcagtgagcg agcgagcgcg cagctgcctg caggggcgcc
     1861 tgatgcggta ttttctcctt acgcatctgt gcggtatttc acaccgcata cgtcaaagca
     1921 accatagtac gcgccctgta gcggcgcatt aagcgcggcg ggtgtggtgg ttacgcgcag
     1981 cgtgaccgct acacttgcca gcgccttagc gcccgctcct ttcgctttct tcccttcctt
     2041 tctcgccacg ttcgccggct ttccccgtca agctctaaat cgggggctcc ctttagggtt
     2101 ccgatttagt gctttacggc acctcgaccc caaaaaactt gatttgggtg atggttcacg
     2161 tagtgggcca tcgccctgat agacggtttt tcgccctttg acgttggagt ccacgttctt
     2221 taatagtgga ctcttgttcc aaactggaac aacactcaac tctatctcgg gctattcttt
     2281 tgatttataa gggattttgc cgatttcggt ctattggtta aaaaatgagc tgatttaaca
     2341 aaaatttaac gcgaatttta acaaaatatt aacgtttaca attttatggt gcactctcag
     2401 tacaatctgc tctgatgccg catagttaag ccagccccga cacccgccaa cacccgctga
     2461 cgcgccctga cgggcttgtc tgctcccggc atccgcttac agacaagctg tgaccgtctc
     2521 cgggagctgc atgtgtcaga ggttttcacc gtcatcaccg aaacgcgcga gacgaaaggg
     2581 cctcgtgata cgcctatttt tataggttaa tgtcatgata ataatggttt cttagacgtc
     2641 aggtggcact tttcggggaa atgtgcgcgg aacccctatt tgtttatttt tctaaataca
     2701 ttcaaatatg tatccgctca tgagacaata accctgataa atgcttcaat aatattgaaa
     2761 aaggaagagt atgagtattc aacatttccg tgtcgccctt attccctttt ttgcggcatt
     2821 ttgccttcct gtttttgctc acccagaaac gctggtgaaa gtaaaagatg ctgaagatca
     2881 gttgggtgca cgagtgggtt acatcgaact ggatctcaac agcggtaaga tccttgagag
     2941 ttttcgcccc gaagaacgtt ttccaatgat gagcactttt aaagttctgc tatgtggcgc
     3001 ggtattatcc cgtattgacg ccgggcaaga gcaactcggt cgccgcatac actattctca
     3061 gaatgacttg gttgagtact caccagtcac agaaaagcat cttacggatg gcatgacagt
     3121 aagagaatta tgcagtgctg ccataaccat gagtgataac actgcggcca acttacttct
     3181 gacaacgatc ggaggaccga aggagctaac cgcttttttg cacaacatgg gggatcatgt
     3241 aactcgcctt gatcgttggg aaccggagct gaatgaagcc ataccaaacg acgagcgtga
     3301 caccacgatg cctgtagcaa tggcaacaac gttgcgcaaa ctattaactg gcgaactact
     3361 tactctagct tcccggcaac aattaataga ctggatggag gcggataaag ttgcaggacc
     3421 acttctgcgc tcggcccttc cggctggctg gtttattgct gataaatctg gagccggtga
     3481 gcgtggaagc cgcggtatca ttgcagcact ggggccagat ggtaagccct cccgtatcgt
     3541 agttatctac acgacgggga gtcaggcaac tatggatgaa cgaaatagac agatcgctga
     3601 gataggtgcc tcactgatta agcattggta actgtcagac caagtttact catatatact
     3661 ttagattgat ttaaaacttc atttttaatt taaaaggatc taggtgaaga tcctttttga
     3721 taatctcatg accaaaatcc cttaacgtga gttttcgttc cactgagcgt cagaccccgt
     3781 agaaaagatc aaaggatctt cttgagatcc tttttttctg cgcgtaatct gctgcttgca
     3841 aacaaaaaaa ccaccgctac cagcggtggt ttgtttgccg gatcaagagc taccaactct
     3901 ttttccgaag gtaactggct tcagcagagc gcagatacca aatactgttc ttctagtgta
     3961 gccgtagtta ggccaccact tcaagaactc tgtagcaccg cctacatacc tcgctctgct
     4021 aatcctgtta ccagtggctg ctgccagtgg cgataagtcg tgtcttaccg ggttggactc
     4081 aagacgatag ttaccggata aggcgcagcg gtcgggctga acggggggtt cgtgcacaca
     4141 gcccagcttg gagcgaacga cctacaccga actgagatac ctacagcgtg agctatgaga
     4201 aagcgccacg cttcccgaag ggagaaaggc ggacaggtat ccggtaagcg gcagggtcgg
     4261 aacaggagag cgcacgaggg agcttccagg gggaaacgcc tggtatcttt atagtcctgt
     4321 cgggtttcgc cacctctgac ttgagcgtcg atttttgtga tgctcgtcag gggggcggag
     4381 cctatggaaa aacgccagca acgcggcctt ttt
//
