LOCUS       sequence-334642-        3619 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Donor DNA for TKIT knockin of GFP to Gephyrin in mouse.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 3619)
  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 3619)
  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: pMiniT:GFP-Gephyrin TKIT donor
FEATURES             Location/Qualifiers
     source          1..3619
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     157..176
                     /label=EXFP-R
                     /note="For distinguishing EGFP variants, reverse primer"
     primer_bind     416..437
                     /label=EGFP-N
                     /note="EGFP, reverse primer"
     promoter        761..865
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(866..934,935..1726)
                     /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(1084..1103)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      1897..2485
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     2386..2405
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     2639..2656
                     /label=L4440
                     /note="L4440 vector, forward primer"
     CDS             complement(join(3385..3619,1..476,477..479))
                     /codon_start=1
                     /product="enhanced GFP"
                     /label=EGFP
                     /translation="VSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTLK
                     FICTTGKLPVPWPTLVTTLTYGVQCFSRYPDHMKQHDFFKSAMPEGYVQERTIFFKDDG
                     NYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIKV
                     NFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSALSKDPNEKRDHMVLLE
                     FVTAAGITLGMDELYK"
     CDS             complement(join(3385..3619,1..476,477..479))
                     /codon_start=1
                     /product="the original enhanced GFP (Yang et al., 1996)"
                     /label=EGFP
                     /note="mammalian codon-optimized"
                     /translation="VSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTLK
                     FICTTGKLPVPWPTLVTTLTYGVQCFSRYPDHMKQHDFFKSAMPEGYVQERTIFFKDDG
                     NYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIKV
                     NFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSALSKDPNEKRDHMVLLE
                     FVTAAGITLGMDELYK"
     primer_bind     complement(3427..3448)
                     /label=EGFP-C
                     /note="EGFP, forward primer"
ORIGIN
        1 ccgttcttct gcttgtcggc catgatatag acgttgtggc tgttgtagtt gtactccagc
       61 ttgtgcccca ggatgttgcc gtcctccttg aagtcgatgc ccttcagctc gatgcggttc
      121 accagggtgt cgccctcgaa cttcacctcg gcgcgggtct tgtagttgcc gtcgtccttg
      181 aagaagatgg tgcgctcctg gacgtagcct tcgggcatgg cggacttgaa gaagtcgtgc
      241 tgcttcatgt ggtcggggta gcggctgaag cactgcacgc cgtaggtcag ggtggtcacg
      301 agggtgggcc agggcacggg cagcttgccg gtggtgcaga tgaacttcag ggtcagcttg
      361 ccgtaggtgg catcgccctc gccctcgccg gacacgctga acttgtggcc gtttacgtcg
      421 ccgtccagct cgaccaggat gggcaccacc ccggtgaaca gctcctcgcc cttgctcact
      481 ctagacatgt ttcccagcgc agtcaccgca ctgacaggag ccaggagaag ccggagcccg
      541 gagcgcgccg gccgggagcg cgcttgcctt ggagagcgcg agagcgaggg cgcccgggga
      601 gcgcgaggcg agcgggaagg agggggccga gcgcccggtg gcctgcgtgg ggtggggggg
      661 agaggccgcg ccaggagcgc gcgggtccgc ggagggggcg gagtcgggcc gcgccgcgac
      721 ttgctggagg tttctcgaat tctcgagtcg gggaaatgtg cgcggaaccc ctatttgttt
      781 atttttctaa atacattcaa atatgtatcc gctcatgaga caataaccct gataaatgct
      841 tcaataatat tgaaaaagga agagtatgag tattcaacat ttccgtgtcg cccttattcc
      901 cttttttgcg gcattttgcc ttcctgtttt tgctcaccca gaaacgctgg tgaaagtaaa
      961 agatgctgaa gatcagttgg gtgcacgagt gggttacatc gaactggatc tcaacagcgg
     1021 taagatcctt gagagttttc gccccgaaga acgttttcca atgatgagca cttttaaagt
     1081 tctgctatgt ggcgcggtat tatcccgtat tgacgccggg caagagcaac tcggtcgccg
     1141 catacactat tctcagaatg acttggttga gtactcacca gtcacagaaa agcatcttac
     1201 ggatggcatg acagtaagag aattatgcag tgctgccata accatgagtg ataacactgc
     1261 ggccaactta cttctgacaa cgatcggagg accgaaggag ctaaccgctt ttttgcacaa
     1321 catgggggat catgtaactc gccttgatcg ttgggaaccg gagctgaatg aagccatacc
     1381 aaacgacgag cgtgacacca cgatgcctgt agcaatggca acaacgttgc gcaaactatt
     1441 aactggcgaa ctacttactc tagcttcccg gcaacaatta atagactgga tggaggcgga
     1501 taaagttgca ggaccacttc tgcgctcggc ccttccggct ggctggttta ttgctgataa
     1561 atctggagcc ggtgagcgtg ggtctcgcgg tatcattgca gcactggggc cagatggtaa
     1621 gccctcccgt atcgtagtta tctacacgac ggggagtcag gcaactatgg atgaacgaaa
     1681 tagacagatc gctgagatag gtgcctcact gattaagcat tggtaactgt cagaccaagt
     1741 ttactcatat atactttaga ttgatttaaa acttcatttt taatttaaaa ggatctaggt
     1801 gaagatcctt tttgataatc tcatgaccaa aatcccttaa cgtgagtttt cgttccactg
     1861 agcgtcagac cccgtagaaa agatcaaagg atcttcttga gatccttttt ttctgcgcgt
     1921 aatctgctgc ttgcaaacaa aaaaaccacc gctaccagcg gtggtttgtt tgccggatca
     1981 agagctacca actctttttc cgaaggtaac tggcttcagc agagcgcaga taccaaatac
     2041 tgttcttcta gtgtagccgt agttaggcca ccacttcaag aactctgtag caccgcctac
     2101 atacctcgct ctgctaatcc tgttaccagt ggctgctgcc agtggcgata agtcgtgtct
     2161 taccgggttg gactcaagac gatagttacc ggataaggcg cagcggtcgg gctgaacggg
     2221 gggttcgtgc acacagccca gcttggagcg aacgacctac accgaactga gatacctaca
     2281 gcgtgagcta tgagaaagcg ccacgcttcc cgaagggaga aaggcggaca ggtatccggt
     2341 aagcggcagg gtcggaacag gagagcgcac gagggagctt ccagggggaa acgcctggta
     2401 tctttatagt cctgtcgggt ttcgccacct ctgacttgag cgtcgatttt tgtgatgctc
     2461 gtcagggggg cggagcctat ggaaaaacgc cagcaatgcg gcctttttac ggttcctggc
     2521 cttttgctgg ccttttgctc acatgttctt tcctgcgtta tcccctgatt ctgtggataa
     2581 ccgtattacc gcctttgagt gagctgatac cgctcgccgc agccgaacga ccgagcgcag
     2641 cgagtcagtg agcgaggaag cggaagagcg cccaatacgc aaaccgcctc tccccgcgcg
     2701 ttggccgatt cattaatgca gccggttcag acaggataaa gaggaacgca gaatgttaga
     2761 caacacccgc ttacgcatag ctattcagaa atcaggccgt ttaagcgatg attcacgaga
     2821 attgctggcc cgctgcggca taaaaattaa tttacacact cagcgctgat gaatccccta
     2881 atgattttgg taaaaatcat taagttaagg tggacacaca tcttgtcata tgattaaatg
     2941 gtttcgcgaa aaatcaataa tcagacaaca agatgtgcga actcgatatt ttacacgact
     3001 ctctttacca attctgcccc gaattacact taaaacgact caacagctta acgttggctt
     3061 gccacgcatt acttgactgt aaaactctca ctcttaccga acttggccgt aacctgccaa
     3121 ccaaagcgag aacaaaacat aacatcaaac gaatcgaccg attgttaggt aatcgtcacc
     3181 tccacaaaga gcgactcgct gtatcgctcg agggatccga attccctccc ggccggactc
     3241 ggggtcgcgg atagtagccg ttgcggctcc cccggacctc ctccccccgt tacctgtgag
     3301 gactccgaca cggatttgat ggtcgtggtt ggtgaggatc attccctcgg tcgctgaccc
     3361 acctccgccc gagccaccgc caagcttgta cagctcgtcc atgccgagag tgatcccggc
     3421 ggcggtcacg aactccagca ggaccatgtg atcgcgcttc tcgttggggt ctttgctcag
     3481 ggcggactgg gtgctcaggt agtggttgtc gggcagcagc acggggccgt cgccgatggg
     3541 ggtgttctgc tggtagtggt cggcgagctg cacgctgccg tcctcgatgt tgtggcggat
     3601 cttgaagttc accttgatg
//
