LOCUS       sequence-111294-        4975 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Intron targeting-mediated EGFP knockin at the zebrafish gfap locus.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 4975)
  AUTHORS   Li J, Zhang BB, Ren YG, Gu SY, Xiang YH, Huang C, Du JL
  TITLE     Intron targeting-mediated and endogenous gene integrity-maintaining 
            knockin in zebrafish using the CRISPR/Cas9 system.
  JOURNAL   Cell Res. 2015 Apr 7. doi: 10.1038/cr.2015.43.
  PUBMED    25849248
REFERENCE   2  (bases 1 to 4975)
  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: "Cell Res. 
            2015 Apr 7. doi: 10.1038/cr.2015.43."
COMMENT     Sequence Label: gfap-EGFP donor
FEATURES             Location/Qualifiers
     source          1..4975
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     complement(29..51)
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     primer_bind     151..170
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     364..386
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     primer_bind     378..395
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     379..395
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     CDS             join(669..671,672..674,675..1385)
                     /codon_start=1
                     /product="enhanced GFP"
                     /label=EGFP
                     /translation="MVSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTL
                     KFICTTGKLPVPWPTLVTTLTYGVQCFSRYPDHMKQHDFFKSAMPEGYVQERTIFFKDD
                     GNYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIK
                     VNFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSALSKDPNEKRDHMVLL
                     EFVTAAGITLGMDELYK"
     CDS             join(669..671,672..674,675..1385)
                     /codon_start=1
                     /product="the original enhanced GFP (Yang et al., 1996)"
                     /label=EGFP
                     /note="mammalian codon-optimized"
                     /translation="MVSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTL
                     KFICTTGKLPVPWPTLVTTLTYGVQCFSRYPDHMKQHDFFKSAMPEGYVQERTIFFKDD
                     GNYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIK
                     VNFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSALSKDPNEKRDHMVLL
                     EFVTAAGITLGMDELYK"
     primer_bind     complement(714..735)
                     /label=EGFP-N
                     /note="EGFP, reverse primer"
     primer_bind     complement(975..994)
                     /label=EXFP-R
                     /note="For distinguishing EGFP variants, reverse primer"
     primer_bind     1322..1343
                     /label=EGFP-C
                     /note="EGFP, forward primer"
     primer_bind     complement(2754..2770)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(2754..2770)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(2767..2789)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    2778..2794
                     /label=lac operator
                     /bound_moiety="lac repressor encoded by lacI"
                     /note="The lac repressor binds to the lac operator to 
                     inhibit transcription in E. coli. This inhibition can be 
                     relieved by adding lactose or 
                     isopropyl-beta-D-thiogalactopyranoside (IPTG)."
     promoter        complement(join(2802..2808,2809..2826,2827..2832))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    2847..2868
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(2985..3002)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(3156..3744)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(3236..3255)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(3915..4706,4707..4775))
                     /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     4538..4557
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(4776..4880)
                     /gene="bla"
                     /label=AmpR promoter
     primer_bind     4948..4966
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
ORIGIN
        1 tcgcgcgttt cggtgatgac ggtgaaaacc tctgacacat gcagctcccg gagacggtca
       61 cagcttgtct gtaagcggat gccgggagca gacaagcccg tcagggcgcg tcagcgggtg
      121 ttggcgggtg tcggggctgg cttaactatg cggcatcaga gcagattgta ctgagagtgc
      181 accatatgcg gtgtgaaata ccgcacagat gcgtaaggag aaaataccgc atcaggcgcc
      241 attcgccatt caggctgcgc aactgttggg aagggcgatc ggtgcgggcc tcttcgctat
      301 tacgccagct ggcgaaaggg ggatgtgctg caaggcgatt aagttgggta acgccagggt
      361 tttcccagtc acgacgttgt aaaacgacgg ccagtgaatt cgagctcggt acctaagccg
      421 ggaatcgaac tcggatcacc ataagaacca tggtgctatg tgttgcgcac ttaaccacta
      481 ggctataggc gccgaccgat taagttaact tcaattaaag tggattaaac ataaaacaat
      541 taagttgtcc ccaaaaacat ctcaagaatt gtgttgattc tgctcatttt tcattttaca
      601 cattctctct ttcgcagatc attaaagagt ccactacgga gaggaaggat ctgccaggat
      661 cctcttctat ggtgagcaag ggcgaggagc tgttcaccgg ggtggtgccc atcctggtcg
      721 agctggacgg cgacgtaaac ggccacaagt tcagcgtgtc cggcgagggc gagggcgatg
      781 ccacctacgg caagctgacc ctgaagttca tctgcaccac cggcaagctg cccgtgccct
      841 ggcccaccct cgtgaccacc ctgacctacg gcgtgcagtg cttcagccgc taccccgacc
      901 acatgaagca gcacgacttc ttcaagtccg ccatgcccga aggctacgtc caggagcgca
      961 ccatcttctt caaggacgac ggcaactaca agacccgcgc cgaggtgaag ttcgagggcg
     1021 acaccctggt gaaccgcatc gagctgaagg gcatcgactt caaggaggac ggcaacatcc
     1081 tggggcacaa gctggagtac aactacaaca gccacaacgt ctatatcatg gccgacaagc
     1141 agaagaacgg catcaaggtg aacttcaaga tccgccacaa catcgaggac ggcagcgtgc
     1201 agctcgccga ccactaccag cagaacaccc ccatcggcga cggccccgtg ctgctgcccg
     1261 acaaccacta cctgagcacc cagtccgccc tgagcaaaga ccccaacgag aagcgcgatc
     1321 acatggtcct gctggagttc gtgaccgccg ccgggatcac tctcggcatg gacgagctgt
     1381 acaagaccgg ttaatgaggc tcagacactg gcttggctgc agaagaagat ctccttcact
     1441 aatgcttcag gaagctgtgt ttgtcaaacc ttgtattttc tcaccaactt tccctgcttc
     1501 tctcctggta tatatagttc atggaaactg tattcactgt ttttagcaac tacctatatg
     1561 ttttgatctc cttttcttcc tgggcttctc tttttatctg tctgtttctt ctgtggcagc
     1621 gtctttgcaa gatagtagac tagtttatcg cagaatgtgc tataaaaagt catggttgga
     1681 cagatgaagt aaacagtagg acaagatgta gctagtggtc agagcaggtg ggaaatatag
     1741 tggttttttt tttgtcaagc ataaattgta gagaacaagt tcttgagctt tattgtagtc
     1801 tttaagaatg ccagaagaaa aaaagagcaa gtctggagct ggtagttttt aaatctgaac
     1861 agtaggtgtt aaagtaaggc agaagggaat tttaacagca agatgtttgt aagagtttta
     1921 aaggaatgat ggatttggtc agcggtgcta tttgaacatt ttactccaca ttttttttgt
     1981 cctcctccac tgtgtgtgtg ctcagtgttt ttgcacagta gctaactcat ctcttggacc
     2041 taggttaaaa tgctttaaat tgctcgttga cacattactg acctgtagaa gacattaaaa
     2101 tcttaacatt aagcatctta ctgaatgaat tatggtataa atatctgaca cagtgttccc
     2161 catcatgtga ccagatcatt tgattcagat ttatactgat agctttcaat tgtcacgctt
     2221 acatttcctt ggtgaaattg agcaattttt ccatttccat ctgtgtgatc aggcactttt
     2281 gcatgagggt gaaaggttcc tcctgcagtt tatgcttaca tttgagttgt tattctgaat
     2341 tgcacaaata tcatcttggt ttgaaagtga cttaagtgtg agcagtgttt cacacatcac
     2401 tacaatgtta ataatatgca ttaataataa tcaaaactgt gaccatatct tcactgtgag
     2461 ataacattgc ttgatttaca ccgttttcaa ctaagatgga ctaactttca tactgaattg
     2521 gacattactt ctttgtggtg ccttatatta atagaaatta aaaaaaagaa aaatttttat
     2581 atgttagtgt cggttctgtt tgtcaactaa tatctatgtt aagtgtcagc actgcaactg
     2641 atacattatg ataattatga cctaataaag tttagattgt agtcttttaa atactgcatt
     2701 tgcctgtatt tgtgaatgtc gacctgcagg catgcaagct tggcgtaatc atggtcatag
     2761 ctgtttcctg tgtgaaattg ttatccgctc acaattccac acaacatacg agccggaagc
     2821 ataaagtgta aagcctgggg tgcctaatga gtgagctaac tcacattaat tgcgttgcgc
     2881 tcactgcccg ctttccagtc gggaaacctg tcgtgccagc tgcattaatg aatcggccaa
     2941 cgcgcgggga gaggcggttt gcgtattggg cgctcttccg cttcctcgct cactgactcg
     3001 ctgcgctcgg tcgttcggct gcggcgagcg gtatcagctc actcaaaggc ggtaatacgg
     3061 ttatccacag aatcagggga taacgcagga aagaacatgt gagcaaaagg ccagcaaaag
     3121 gccaggaacc gtaaaaaggc cgcgttgctg gcgtttttcc ataggctccg cccccctgac
     3181 gagcatcaca aaaatcgacg ctcaagtcag aggtggcgaa acccgacagg actataaaga
     3241 taccaggcgt ttccccctgg aagctccctc gtgcgctctc ctgttccgac cctgccgctt
     3301 accggatacc tgtccgcctt tctcccttcg ggaagcgtgg cgctttctca tagctcacgc
     3361 tgtaggtatc tcagttcggt gtaggtcgtt cgctccaagc tgggctgtgt gcacgaaccc
     3421 cccgttcagc ccgaccgctg cgccttatcc ggtaactatc gtcttgagtc caacccggta
     3481 agacacgact tatcgccact ggcagcagcc actggtaaca ggattagcag agcgaggtat
     3541 gtaggcggtg ctacagagtt cttgaagtgg tggcctaact acggctacac tagaagaaca
     3601 gtatttggta tctgcgctct gctgaagcca gttaccttcg gaaaaagagt tggtagctct
     3661 tgatccggca aacaaaccac cgctggtagc ggtggttttt ttgtttgcaa gcagcagatt
     3721 acgcgcagaa aaaaaggatc tcaagaagat cctttgatct tttctacggg gtctgacgct
     3781 cagtggaacg aaaactcacg ttaagggatt ttggtcatga gattatcaaa aaggatcttc
     3841 acctagatcc ttttaaatta aaaatgaagt tttaaatcaa tctaaagtat atatgagtaa
     3901 acttggtctg acagttacca atgcttaatc agtgaggcac ctatctcagc gatctgtcta
     3961 tttcgttcat ccatagttgc ctgactcccc gtcgtgtaga taactacgat acgggagggc
     4021 ttaccatctg gccccagtgc tgcaatgata ccgcgagacc cacgctcacc ggctccagat
     4081 ttatcagcaa taaaccagcc agccggaagg gccgagcgca gaagtggtcc tgcaacttta
     4141 tccgcctcca tccagtctat taattgttgc cgggaagcta gagtaagtag ttcgccagtt
     4201 aatagtttgc gcaacgttgt tgccattgct acaggcatcg tggtgtcacg ctcgtcgttt
     4261 ggtatggctt cattcagctc cggttcccaa cgatcaaggc gagttacatg atcccccatg
     4321 ttgtgcaaaa aagcggttag ctccttcggt cctccgatcg ttgtcagaag taagttggcc
     4381 gcagtgttat cactcatggt tatggcagca ctgcataatt ctcttactgt catgccatcc
     4441 gtaagatgct tttctgtgac tggtgagtac tcaaccaagt cattctgaga atagtgtatg
     4501 cggcgaccga gttgctcttg cccggcgtca atacgggata ataccgcgcc acatagcaga
     4561 actttaaaag tgctcatcat tggaaaacgt tcttcggggc gaaaactctc aaggatctta
     4621 ccgctgttga gatccagttc gatgtaaccc actcgtgcac ccaactgatc ttcagcatct
     4681 tttactttca ccagcgtttc tgggtgagca aaaacaggaa ggcaaaatgc cgcaaaaaag
     4741 ggaataaggg cgacacggaa atgttgaata ctcatactct tcctttttca atattattga
     4801 agcatttatc agggttattg tctcatgagc ggatacatat ttgaatgtat ttagaaaaat
     4861 aaacaaatag gggttccgcg cacatttccc cgaaaagtgc cacctgacgt ctaagaaacc
     4921 attattatca tgacattaac ctataaaaat aggcgtatca cgaggccctt tcgtc
//
