LOCUS       sequence-360118-        4071 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  FlpOFF knock-in for beta3-tubulin GFP (amino acid position: stop 
            codon).
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 4071)
  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 4071)
  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 Tubb3-GFP KI
FEATURES             Location/Qualifiers
     source          1..4071
                     /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    324..357
                     /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    493..526
                     /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             join(580..582,583..1296)
                     /codon_start=1
                     /product="the original enhanced GFP (Yang et al., 1996)"
                     /label=EGFP
                     /note="mammalian codon-optimized"
                     /translation="VSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTLK
                     FICTTGKLPVPWPTLVTTLTYGVQCFSRYPDHMKQHDFFKSAMPEGYVQERTIFFKDDG
                     NYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIKV
                     NFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSALSKDPNEKRDHMVLLE
                     FVTAAGITLGMDELYK"
     CDS             join(580..582,583..1293)
                     /codon_start=1
                     /product="enhanced GFP"
                     /label=EGFP
                     /translation="VSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTLK
                     FICTTGKLPVPWPTLVTTLTYGVQCFSRYPDHMKQHDFFKSAMPEGYVQERTIFFKDDG
                     NYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIKV
                     NFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSALSKDPNEKRDHMVLLE
                     FVTAAGITLGMDELYK"
     primer_bind     complement(622..643)
                     /label=EGFP-N
                     /note="EGFP, reverse primer"
     primer_bind     complement(883..902)
                     /label=EXFP-R
                     /note="For distinguishing EGFP variants, reverse primer"
     primer_bind     1230..1251
                     /label=EGFP-C
                     /note="EGFP, forward primer"
     repeat_region   1372..1512
                     /label=AAV2 ITR
                     /note="inverted terminal repeat of adeno-associated virus 
                     serotype 2"
     repeat_region   1372..1501
                     /label=AAV2 ITR
     rep_origin      1587..2042
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(1674..1693)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     1884..1905
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     complement(2059..2078)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     2178..2200
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     primer_bind     complement(2238..2256)
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     promoter        2324..2428
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(2429..2497,2498..3289)
                     /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(2647..2666)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      3460..4048
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     3949..3968
                     /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 aaacaccgct gcgagcaact
      301 tcacttgttt tagagctatg ctggaagttc ctattctcta gaaagtatag gaacttccag
      361 catagcaagt taaaataagg ctagtccgtt atcaacttga aaaagtggca ccgagtcggt
      421 gcttttttgt tttagagcta gaaatagcaa gttaaaataa ggctagtccg tttttagcgc
      481 gtgcgccaat tcgaagttcc tattctctag aaagtatagg aacttctttt ttttttttct
      541 agaagcttgc tgcgagcaac ttcacttggg ggatcaggcg tgagcaaggg cgaggagctg
      601 ttcaccgggg tggtgcccat cctggtcgag ctggacggcg acgtaaacgg ccacaagttc
      661 agcgtgtccg gcgagggcga gggcgatgcc acctacggca agctgaccct gaagttcatc
      721 tgcaccaccg gcaagctgcc cgtgccctgg cccaccctcg tgaccaccct gacctacggc
      781 gtgcagtgct tcagccgcta ccccgaccac atgaagcagc acgacttctt caagtccgcc
      841 atgcccgaag gctacgtcca ggagcgcacc atcttcttca aggacgacgg caactacaag
      901 acccgcgccg aggtgaagtt cgagggcgac accctggtga accgcatcga gctgaagggc
      961 atcgacttca aggaggacgg caacatcctg gggcacaagc tggagtacaa ctacaacagc
     1021 cacaacgtct atatcatggc cgacaagcag aagaacggca tcaaggtgaa cttcaagatc
     1081 cgccacaaca tcgaggacgg cagcgtgcag ctcgccgacc actaccagca gaacaccccc
     1141 atcggcgacg gccccgtgct gctgcccgac aaccactacc tgagcaccca gtccgccctg
     1201 agcaaagacc ccaacgagaa gcgcgatcac atggtcctgc tggagttcgt gaccgccgcc
     1261 gggatcactc tcggcatgga cgagctgtac aagtaatgtg ctgcgagcaa cttcacttgg
     1321 gctcgaggat cctaggacgc gttaacttac ggtaaatggc ccggcggccg caggaacccc
     1381 tagtgatgga gttggccact ccctctctgc gcgctcgctc gctcactgag gccgggcgac
     1441 caaaggtcgc ccgacgcccg ggctttgccc gggcggcctc agtgagcgag cgagcgcgca
     1501 gctgcctgca ggggcgcctg atgcggtatt ttctccttac gcatctgtgc ggtatttcac
     1561 accgcatacg tcaaagcaac catagtacgc gccctgtagc ggcgcattaa gcgcggcggg
     1621 tgtggtggtt acgcgcagcg tgaccgctac acttgccagc gccttagcgc ccgctccttt
     1681 cgctttcttc ccttcctttc tcgccacgtt cgccggcttt ccccgtcaag ctctaaatcg
     1741 ggggctccct ttagggttcc gatttagtgc tttacggcac ctcgacccca aaaaacttga
     1801 tttgggtgat ggttcacgta gtgggccatc gccctgatag acggtttttc gccctttgac
     1861 gttggagtcc acgttcttta atagtggact cttgttccaa actggaacaa cactcaactc
     1921 tatctcgggc tattcttttg atttataagg gattttgccg atttcggtct attggttaaa
     1981 aaatgagctg atttaacaaa aatttaacgc gaattttaac aaaatattaa cgtttacaat
     2041 tttatggtgc actctcagta caatctgctc tgatgccgca tagttaagcc agccccgaca
     2101 cccgccaaca cccgctgacg cgccctgacg ggcttgtctg ctcccggcat ccgcttacag
     2161 acaagctgtg accgtctccg ggagctgcat gtgtcagagg ttttcaccgt catcaccgaa
     2221 acgcgcgaga cgaaagggcc tcgtgatacg cctattttta taggttaatg tcatgataat
     2281 aatggtttct tagacgtcag gtggcacttt tcggggaaat gtgcgcggaa cccctatttg
     2341 tttatttttc taaatacatt caaatatgta tccgctcatg agacaataac cctgataaat
     2401 gcttcaataa tattgaaaaa ggaagagtat gagtattcaa catttccgtg tcgcccttat
     2461 tccctttttt gcggcatttt gccttcctgt ttttgctcac ccagaaacgc tggtgaaagt
     2521 aaaagatgct gaagatcagt tgggtgcacg agtgggttac atcgaactgg atctcaacag
     2581 cggtaagatc cttgagagtt ttcgccccga agaacgtttt ccaatgatga gcacttttaa
     2641 agttctgcta tgtggcgcgg tattatcccg tattgacgcc gggcaagagc aactcggtcg
     2701 ccgcatacac tattctcaga atgacttggt tgagtactca ccagtcacag aaaagcatct
     2761 tacggatggc atgacagtaa gagaattatg cagtgctgcc ataaccatga gtgataacac
     2821 tgcggccaac ttacttctga caacgatcgg aggaccgaag gagctaaccg cttttttgca
     2881 caacatgggg gatcatgtaa ctcgccttga tcgttgggaa ccggagctga atgaagccat
     2941 accaaacgac gagcgtgaca ccacgatgcc tgtagcaatg gcaacaacgt tgcgcaaact
     3001 attaactggc gaactactta ctctagcttc ccggcaacaa ttaatagact ggatggaggc
     3061 ggataaagtt gcaggaccac ttctgcgctc ggcccttccg gctggctggt ttattgctga
     3121 taaatctgga gccggtgagc gtggaagccg cggtatcatt gcagcactgg ggccagatgg
     3181 taagccctcc cgtatcgtag ttatctacac gacggggagt caggcaacta tggatgaacg
     3241 aaatagacag atcgctgaga taggtgcctc actgattaag cattggtaac tgtcagacca
     3301 agtttactca tatatacttt agattgattt aaaacttcat ttttaattta aaaggatcta
     3361 ggtgaagatc ctttttgata atctcatgac caaaatccct taacgtgagt tttcgttcca
     3421 ctgagcgtca gaccccgtag aaaagatcaa aggatcttct tgagatcctt tttttctgcg
     3481 cgtaatctgc tgcttgcaaa caaaaaaacc accgctacca gcggtggttt gtttgccgga
     3541 tcaagagcta ccaactcttt ttccgaaggt aactggcttc agcagagcgc agataccaaa
     3601 tactgttctt ctagtgtagc cgtagttagg ccaccacttc aagaactctg tagcaccgcc
     3661 tacatacctc gctctgctaa tcctgttacc agtggctgct gccagtggcg ataagtcgtg
     3721 tcttaccggg ttggactcaa gacgatagtt accggataag gcgcagcggt cgggctgaac
     3781 ggggggttcg tgcacacagc ccagcttgga gcgaacgacc tacaccgaac tgagatacct
     3841 acagcgtgag ctatgagaaa gcgccacgct tcccgaaggg agaaaggcgg acaggtatcc
     3901 ggtaagcggc agggtcggaa caggagagcg cacgagggag cttccagggg gaaacgcctg
     3961 gtatctttat agtcctgtcg ggtttcgcca cctctgactt gagcgtcgat ttttgtgatg
     4021 ctcgtcaggg gggcggagcc tatggaaaaa cgccagcaac gcggcctttt t
//
