LOCUS       sequence-328492-        3162 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Donor DNA for TKIT knockin of SEP to GluA2 in rat.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 3162)
  AUTHORS   Fang H, Bygrave AM, Roth RH, Johnson RC, Huganir RL
  TITLE     An optimized CRISPR/Cas9 approach for precise genome editing in 
            neurons.
  JOURNAL   Elife. 2021 Mar 10;10. pii: 65202. doi: 10.7554/eLife.65202.
  PUBMED    33689678
REFERENCE   2  (bases 1 to 3162)
  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: "Elife."; 
            date: "2021-03-10"; volume: "10. pii: 65202. doi"; pages: " 
            10.7554/eLife.65202"
COMMENT     Sequence Label: pHFS:SEP-rGluA2 TKIT donor
FEATURES             Location/Qualifiers
     source          1..3162
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     606..625
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(844..948)
                     /gene="bla"
                     /label=AmpR promoter
     CDS             1191..1901
                     /codon_start=1
                     /product="pH-sensitive mutant of green fluorescent protein 
                     (Ng et al., 2002)"
                     /label=superecliptic pHluorin
                     /note="excited strongly by blue light at neutral pH but 
                     minimally fluorescent at acidic pH"
                     /translation="SKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTLKF
                     ICTTGKLPVPWPTLVTTLTYGVQCFSRYPDHMKRHDFFKSAMPEGYVQERTIFFKDDGN
                     YKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNDHQVYIMADKQKNGIKAN
                     FKIRHNIEDGGVQLADHYQQNTPIGDGPVLLPDNHYLFTTSTLSKDPNEKRDHMVLLEF
                     VTAAGITHGMDELYK"
     primer_bind     complement(1218..1246)
                     /label=GFP-R
                     /note="GFP, reverse primer. Does NOT anneal to EGFP"
     primer_bind     1841..1861
                     /label=GFP-F
                     /note="GFP, forward primer. Does NOT anneal to EGFP"
     primer_bind     complement(2215..2232)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(2386..2974)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(2466..2485)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(3145..3162,1..774,775..843))
                     /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"
ORIGIN
        1 tgaggcacct atctcagcga tctgtctatt tcgttcatcc atagttgcct gactccccgt
       61 cgtgtagata actacgatac gggagggctt accatctggc cccagtgctg caatgatacc
      121 gcgagaccca cgctcaccgg ctccagattt atcagcaata aaccagccag ccggaagggc
      181 cgagcgcaga agtggtcctg caactttatc cgcctccatc cagtctatta attgttgccg
      241 ggaagctaga gtaagtagtt cgccagttaa tagtttgcgc aacgttgttg ccattgctac
      301 aggcatcgtg gtgtcacgct cgtcgtttgg tatggcttca ttcagctccg gttcccaacg
      361 atcaaggcga gttacatgat cccccatgtt gtgcaaaaaa gcggttagct ccttcggtcc
      421 tccgatcgtt gtcagaagta agttggccgc agtgttatca ctcatggtta tggcagcact
      481 gcataattct cttactgtca tgccatccgt aagatgcttt tctgtgactg gtgagtactc
      541 aaccaagtca ttctgagaat agtgtatgcg gcgaccgagt tgctcttgcc cggcgtcaat
      601 acgggataat accgcgccac atagcagaac tttaaaagtg ctcatcattg gaaaacgttc
      661 ttcggggcga aaactctcaa ggatcttacc gctgttgaga tccagttcga tgtaacccac
      721 tcgtgcaccc aactgatctt cagcatcttt tactttcacc agcgtttctg ggtgagcaaa
      781 aacaggaagg caaaatgccg caaaaaaggg aataagggcg acacggaaat gttgaatact
      841 catactcttc ctttttcaat attattgaag catttatcag ggttattgtc tcatgagcgg
      901 atacatattt gaatgtattt agaaaaataa acaaataggg gttccgcgca catttccccg
      961 actcgagccg ctattgtgtc aaaaggcata cagaggatct aatttgctga ggaaaacagt
     1021 caaagaagga agaggaagaa agggaaacga ggggatattt tgtggatgct ctacttttct
     1081 tggaaatgca aaagattatg catatttctg tcctcctttc tcctgtttta tggggactga
     1141 tttttggtgt ctcttctaac agcatatcta gtggtggcgg tggctctaga agtaaaggag
     1201 aagaactttt cactggagtt gtcccaattc ttgttgaatt agatggtgat gttaatgggc
     1261 acaaattttc tgtcagtgga gagggtgaag gtgatgcaac atacggaaaa cttaccctta
     1321 aatttatttg cactactgga aaactacctg ttccttggcc aacacttgtc actactttaa
     1381 cttatggtgt tcaatgcttt tcaagatacc cagatcatat gaaacggcat gactttttca
     1441 agagtgccat gcccgaaggt tatgtacagg aaagaactat atttttcaaa gatgacggga
     1501 actacaagac acgtgctgaa gtcaagtttg aaggtgatac ccttgttaat agaatcgagt
     1561 taaaaggtat tgattttaaa gaagatggaa acattcttgg acacaaattg gaatacaact
     1621 ataacgatca ccaggtgtac atcatggcag acaaacaaaa gaatggaatc aaagctaact
     1681 tcaaaattag acacaacatt gaagatggag gcgttcaact agcagaccat tatcaacaaa
     1741 atactccaat tggcgatggg cccgtccttt taccagacaa ccattacctg tttacaactt
     1801 ctactctttc gaaagatccc aacgaaaaga gagaccacat ggtccttctt gagtttgtaa
     1861 cagctgctgg gattacacat ggcatggatg aactatacaa gcttggtggc ggtggctcgg
     1921 gcggaggtgg gtcgggtggc ggcggatcag gcggtggcgg atccaacagc atacagatag
     1981 gtaggtacct tttgtcttat gcctttgaat tgtgcataat ttggtgctaa tctttgttca
     2041 cagtgaccaa attaattcat gtcatgtaga cttatgtcat accttgactg cattccagat
     2101 ttaaactacc tagaatatac atgatttttt tttagactga agtaaaaaga catgagtatg
     2161 ccttttgaca caacctctgc attgtgaggt cgatattggg cgctcttccg cttcctcgct
     2221 cactgactcg ctgcgctcgg tcgttcggct gcggcgagcg gtatcagctc actcaaaggc
     2281 ggtaatacgg ttatccacag aatcagggga taacgcagga aagaacatgt gagcaaaagg
     2341 ccagcaaaag gccaggaacc gtaaaaaggc cgcattgctg gcgtttttcc ataggctccg
     2401 cccccctgac gagcatcaca aaaatcgacg ctcaagtcag aggtggcgaa acccgacagg
     2461 actataaaga taccaggcgt ttccccctgg aagctccctc gtgcgctctc ctgttccgac
     2521 cctgccgctt accggatacc tgtccgcctt tctcccttcg ggaagcgtgg cgctttctca
     2581 tagctcacgc tgtaggtatc tcagttcggt gtaggtcgtt cgctccaagc tgggctgtgt
     2641 gcacgaaccc cccgttcagc ccgaccgctg cgccttatcc ggtaactatc gtcttgagtc
     2701 caacccggta agacacgact tatcgccact ggcagcagcc actggtaaca ggattagcag
     2761 agcgaggtat gtaggcggtg ctacagagtt cttgaagtgg tggcctaact acggctacac
     2821 tagaagaaca gtatttggta tctgcgctct gctgaagcca gttaccttcg gaaaaagagt
     2881 tggtagctct tgatccggca aacaaaccac cgctggtagc ggtggttttt ttgtttgcaa
     2941 gcagcagatt acgcgcagaa aaaaaggatc tcaagaagat cctttgatct tttctacggg
     3001 gtctgacgct cagtggaacg aaaactcacg ttaagggatt ttggtcatga gattatcaaa
     3061 aaggatcttc acctagatcc ttttaaatta aaaatgaagt tttaaatcaa tctaaagtat
     3121 atatgagtaa acttggtctg acagttacca atgcttaatc ag
//
