LOCUS       sequence-334771-        3163 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 3163)
  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 3163)
  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..3163
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     rep_origin      complement(43..631)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(123..142)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(802..1593,1594..1662))
                     /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     1425..1444
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(1663..1767)
                     /gene="bla"
                     /label=AmpR promoter
     CDS             2010..2720
                     /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(2037..2065)
                     /label=GFP-R
                     /note="GFP, reverse primer. Does NOT anneal to EGFP"
     primer_bind     2660..2680
                     /label=GFP-F
                     /note="GFP, forward primer. Does NOT anneal to EGFP"
     primer_bind     complement(3035..3052)
                     /label=L4440
                     /note="L4440 vector, forward primer"
ORIGIN
        1 gcaaaaggcc aggaaccgta aaaaggccgc attgctggcg tttttccata ggctccgccc
       61 ccctgacgag catcacaaaa atcgacgctc aagtcagagg tggcgaaacc cgacaggact
      121 ataaagatac caggcgtttc cccctggaag ctccctcgtg cgctctcctg ttccgaccct
      181 gccgcttacc ggatacctgt ccgcctttct cccttcggga agcgtggcgc tttctcatag
      241 ctcacgctgt aggtatctca gttcggtgta ggtcgttcgc tccaagctgg gctgtgtgca
      301 cgaacccccc gttcagcccg accgctgcgc cttatccggt aactatcgtc ttgagtccaa
      361 cccggtaaga cacgacttat cgccactggc agcagccact ggtaacagga ttagcagagc
      421 gaggtatgta ggcggtgcta cagagttctt gaagtggtgg cctaactacg gctacactag
      481 aagaacagta tttggtatct gcgctctgct gaagccagtt accttcggaa aaagagttgg
      541 tagctcttga tccggcaaac aaaccaccgc tggtagcggt ggtttttttg tttgcaagca
      601 gcagattacg cgcagaaaaa aaggatctca agaagatcct ttgatctttt ctacggggtc
      661 tgacgctcag tggaacgaaa actcacgtta agggattttg gtcatgagat tatcaaaaag
      721 gatcttcacc tagatccttt taaattaaaa atgaagtttt aaatcaatct aaagtatata
      781 tgagtaaact tggtctgaca gttaccaatg cttaatcagt gaggcaccta tctcagcgat
      841 ctgtctattt cgttcatcca tagttgcctg actccccgtc gtgtagataa ctacgatacg
      901 ggagggctta ccatctggcc ccagtgctgc aatgataccg cgagacccac gctcaccggc
      961 tccagattta tcagcaataa accagccagc cggaagggcc gagcgcagaa gtggtcctgc
     1021 aactttatcc gcctccatcc agtctattaa ttgttgccgg gaagctagag taagtagttc
     1081 gccagttaat agtttgcgca acgttgttgc cattgctaca ggcatcgtgg tgtcacgctc
     1141 gtcgtttggt atggcttcat tcagctccgg ttcccaacga tcaaggcgag ttacatgatc
     1201 ccccatgttg tgcaaaaaag cggttagctc cttcggtcct ccgatcgttg tcagaagtaa
     1261 gttggccgca gtgttatcac tcatggttat ggcagcactg cataattctc ttactgtcat
     1321 gccatccgta agatgctttt ctgtgactgg tgagtactca accaagtcat tctgagaata
     1381 gtgtatgcgg cgaccgagtt gctcttgccc ggcgtcaata cgggataata ccgcgccaca
     1441 tagcagaact ttaaaagtgc tcatcattgg aaaacgttct tcggggcgaa aactctcaag
     1501 gatcttaccg ctgttgagat ccagttcgat gtaacccact cgtgcaccca actgatcttc
     1561 agcatctttt actttcacca gcgtttctgg gtgagcaaaa acaggaaggc aaaatgccgc
     1621 aaaaaaggga ataagggcga cacggaaatg ttgaatactc atactcttcc tttttcaata
     1681 ttattgaagc atttatcagg gttattgtct catgagcgga tacatatttg aatgtattta
     1741 gaaaaataaa caaatagggg ttccgcgcac atttccccga ctcgagccgc tattgtgtca
     1801 aaaggcatac agaggatcta atttgctgag gaaaacagtc aaagaaggaa gaggaagaaa
     1861 gggaaacgag gggatatttt gtggatgctc tacttttctt ggaaatgcaa aagattatgc
     1921 atatttctgt cctcctttct cctgttttat ggggactgat ttttggtgtc tcttctaaca
     1981 gcatatctag tggtggcggt ggctctagaa gtaaaggaga agaacttttc actggagttg
     2041 tcccaattct tgttgaatta gatggtgatg ttaatgggca caaattttct gtcagtggag
     2101 agggtgaagg tgatgcaaca tacggaaaac ttacccttaa atttatttgc actactggaa
     2161 aactacctgt tccttggcca acacttgtca ctactttaac ttatggtgtt caatgctttt
     2221 caagataccc agatcatatg aaacggcatg actttttcaa gagtgccatg cccgaaggtt
     2281 atgtacagga aagaactata tttttcaaag atgacgggaa ctacaagaca cgtgctgaag
     2341 tcaagtttga aggtgatacc cttgttaata gaatcgagtt aaaaggtatt gattttaaag
     2401 aagatggaaa cattcttgga cacaaattgg aatacaacta taacgatcac caggtgtaca
     2461 tcatggcaga caaacaaaag aatggaatca aagctaactt caaaattaga cacaacattg
     2521 aagatggagg cgttcaacta gcagaccatt atcaacaaaa tactccaatt ggcgatgggc
     2581 ccgtcctttt accagacaac cattacctgt ttacaacttc tactctttcg aaagatccca
     2641 acgaaaagag agaccacatg gtccttcttg agtttgtaac agctgctggg attacacatg
     2701 gcatggatga actatacaag cttggtggcg gtggctcggg cggaggtggg tcgggtggcg
     2761 gcggatcagg cggtggcgga tccaacagca tacagatagg taggtacctt ttgtcttatg
     2821 cctttgaatt gtgcataatt tggtgctaat ctttgttcac agtgaccaaa ttaattcatg
     2881 tcatgtagac ttatgtcata ccttgactgc attccagatt taaactacct agaatataca
     2941 tgattttttt tttagactga agtaaaaaga catgagtatg ccttttgaca caacctctgc
     3001 attgtgaggt cgatattggg cgctcttccg cttcctcgct cactgactcg ctgcgctcgg
     3061 tcgttcggct gcggcgagcg gtatcagctc actcaaaggc ggtaatacgg ttatccacag
     3121 aatcagggga taacgcagga aagaacatgt gagcaaaagg cca
//
