LOCUS       sequence-111286-        5429 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Intron targeting-mediated EGFP knockin at the zebrafish th locus..
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 5429)
  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 5429)
  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: th-P2A-EGFP donor
FEATURES             Location/Qualifiers
     source          1..5429
                     /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             1721..1777
                     /codon_start=1
                     /product="2A peptide from porcine teschovirus-1 
                     polyprotein"
                     /label=P2A
                     /note="Eukaryotic ribosomes fail to insert a peptide bond 
                     between the Gly and Pro residues, yielding separate 
                     polypeptides."
                     /translation="ATNFSLLKQAGDVEENPGP"
     CDS             join(1778..1780,1781..1783,1784..2494)
                     /codon_start=1
                     /product="enhanced GFP"
                     /label=EGFP
                     /translation="MVSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTL
                     KFICTTGKLPVPWPTLVTTLTYGVQCFSRYPDHMKQHDFFKSAMPEGYVQERTIFFKDD
                     GNYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIK
                     VNFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSALSKDPNEKRDHMVLL
                     EFVTAAGITLGMDELYK"
     CDS             join(1778..1780,1781..1783,1784..2494)
                     /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(1823..1844)
                     /label=EGFP-N
                     /note="EGFP, reverse primer"
     primer_bind     complement(2084..2103)
                     /label=EXFP-R
                     /note="For distinguishing EGFP variants, reverse primer"
     primer_bind     2431..2452
                     /label=EGFP-C
                     /note="EGFP, forward primer"
     primer_bind     complement(3208..3224)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(3208..3224)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(3221..3243)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    3232..3248
                     /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(3256..3262,3263..3280,3281..3286))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    3301..3322
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(3439..3456)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(3610..4198)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(3690..3709)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(4369..5160,5161..5229))
                     /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     4992..5011
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(5230..5334)
                     /gene="bla"
                     /label=AmpR promoter
     primer_bind     5402..5420
                     /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 accgcgatta
      421 catggcgtag tacgctattt atattctcct ggatattgta taagtgtggt ggtgcttgct
      481 tctgattttt atataatcca ttaacatatt tatacacata gctaggcctg tttataaact
      541 tttatgttca aaaaagcttg tacacatgga tgtattaaaa gaacttttat tacctgctgc
      601 cttttcttcc gtttacacag tttttttttt actttctccc ctttatgagt gtaacaaccc
      661 ctgggtagtc taggggagtg gggttgcaca cttgaccgaa gattttgttc aaataaacgc
      721 tcaaagaaaa ttgtactcaa agtaatgacc acatacaaga attgctgtgc aaataaagtg
      781 atttattaca ggtggtgata agtgaagcag tgagtgagct atttacaata tttacaattc
      841 acaaccaaaa caaaacataa attagaaaga ctggctatgg gtggcgctaa agcaaagaaa
      901 caatcaaaac caaacagtct tactaatatc aaatccctgc taatctagtc taataaacaa
      961 taatgtgaaa aataaatctt gattgtccaa gatgtcttac ctaaactacc taaaaacgtt
     1021 tggaagtcta taaaagttgt tcagcatttc ttattttgat caatttaaac aattataaag
     1081 aacacaaaac ataaacattt tgatgttatg atagcaattc caatgtagaa gaatcacttt
     1141 ctgccctcca tacacaaata aattcagatg gaggctccat ctgaattttg tgcgtcaaca
     1201 atgacgtcat gtgaaaacaa cctattgcat gcatttggaa atgtacaata acattgcaac
     1261 aaccaacaca acaacaacca caattataat aataatatca gcgataataa caataataaa
     1321 aaatatagat tttattctta ttaaaaatag ttttaaaatc acaaagcaaa aagatgcgat
     1381 aatgagataa aactttctga agttttcaca gtatgtcaac tatcattttt ttgaactagc
     1441 ttttaaaaaa atgcaattaa ctttttttaa agacatatcc cttaaaacag catgaattga
     1501 ctaatcacat ttttatttct taaatgtttt ttatttgtta attattattt acagaacata
     1561 tgtgacccgc atcaagcgac cgttttctgt gcggttcgat ccgtacacgg acagcataga
     1621 agtgctggac aacccactga agatccagaa aggcctggag accataaaag atgaactgaa
     1681 gattctcaca gatgccctga atgtgttggc tggatccgga gctactaatt tctccttgct
     1741 taagcaagct ggtgatgttg aagaaaatcc tggtcctatg gtgagcaagg gcgaggagct
     1801 gttcaccggg gtggtgccca tcctggtcga gctggacggc gacgtaaacg gccacaagtt
     1861 cagcgtgtcc ggcgagggcg agggcgatgc cacctacggc aagctgaccc tgaagttcat
     1921 ctgcaccacc ggcaagctgc ccgtgccctg gcccaccctc gtgaccaccc tgacctacgg
     1981 cgtgcagtgc ttcagccgct accccgacca catgaagcag cacgacttct tcaagtccgc
     2041 catgcccgaa ggctacgtcc aggagcgcac catcttcttc aaggacgacg gcaactacaa
     2101 gacccgcgcc gaggtgaagt tcgagggcga caccctggtg aaccgcatcg agctgaaggg
     2161 catcgacttc aaggaggacg gcaacatcct ggggcacaag ctggagtaca actacaacag
     2221 ccacaacgtc tatatcatgg ccgacaagca gaagaacggc atcaaggtga acttcaagat
     2281 ccgccacaac atcgaggacg gcagcgtgca gctcgccgac cactaccagc agaacacccc
     2341 catcggcgac ggccccgtgc tgctgcccga caaccactac ctgagcaccc agtccgccct
     2401 gagcaaagac cccaacgaga agcgcgatca catggtcctg ctggagttcg tgaccgccgc
     2461 cgggatcact ctcggcatgg acgagctgta caagaccggt taattctgct taatcattcc
     2521 tctatgcttc atgtgtgacg gtctcattgt tatgctgtgc ttctatgtct actgtacatc
     2581 tgctgctata gacattgttg cttctaaaat actgaatgaa aaactttaaa ccttcagcgt
     2641 taaataatgt atccaaaaat gaaagttcac cactcaaact cacattacag tagaatatga
     2701 taatagagat agagaaagag agagacagag agagactttt atttctgaat gaactttttt
     2761 tatttttttt tttaagtcat atatgatcaa aatttaaaat gataacattt tccgctataa
     2821 ttttcatttt ggggtaaatt ataactcgtc taaacagaaa tttctgatat gacatttaat
     2881 taatttatgt acaggattta tttatatttc agatagacgt accctcatta ttaaagcttt
     2941 ttaagtcaaa aggtatatat gaaatattac cagaggtcat attcacaata catattaagc
     3001 ctaaaagcag atcctaagtt tacaatatat atactccaac atacaaataa acatacaaaa
     3061 tctgtcactg aggtggtact ttttcaaaaa atacattttt gtaaaataaa gatggacatt
     3121 agaatcttta gggtacactt ttgtacctta aaggtactag gaggttccca tgtcgacctg
     3181 caggcatgca agcttggcgt aatcatggtc atagctgttt cctgtgtgaa attgttatcc
     3241 gctcacaatt ccacacaaca tacgagccgg aagcataaag tgtaaagcct ggggtgccta
     3301 atgagtgagc taactcacat taattgcgtt gcgctcactg cccgctttcc agtcgggaaa
     3361 cctgtcgtgc cagctgcatt aatgaatcgg ccaacgcgcg gggagaggcg gtttgcgtat
     3421 tgggcgctct tccgcttcct cgctcactga ctcgctgcgc tcggtcgttc ggctgcggcg
     3481 agcggtatca gctcactcaa aggcggtaat acggttatcc acagaatcag gggataacgc
     3541 aggaaagaac atgtgagcaa aaggccagca aaaggccagg aaccgtaaaa aggccgcgtt
     3601 gctggcgttt ttccataggc tccgcccccc tgacgagcat cacaaaaatc gacgctcaag
     3661 tcagaggtgg cgaaacccga caggactata aagataccag gcgtttcccc ctggaagctc
     3721 cctcgtgcgc tctcctgttc cgaccctgcc gcttaccgga tacctgtccg cctttctccc
     3781 ttcgggaagc gtggcgcttt ctcatagctc acgctgtagg tatctcagtt cggtgtaggt
     3841 cgttcgctcc aagctgggct gtgtgcacga accccccgtt cagcccgacc gctgcgcctt
     3901 atccggtaac tatcgtcttg agtccaaccc ggtaagacac gacttatcgc cactggcagc
     3961 agccactggt aacaggatta gcagagcgag gtatgtaggc ggtgctacag agttcttgaa
     4021 gtggtggcct aactacggct acactagaag aacagtattt ggtatctgcg ctctgctgaa
     4081 gccagttacc ttcggaaaaa gagttggtag ctcttgatcc ggcaaacaaa ccaccgctgg
     4141 tagcggtggt ttttttgttt gcaagcagca gattacgcgc agaaaaaaag gatctcaaga
     4201 agatcctttg atcttttcta cggggtctga cgctcagtgg aacgaaaact cacgttaagg
     4261 gattttggtc atgagattat caaaaaggat cttcacctag atccttttaa attaaaaatg
     4321 aagttttaaa tcaatctaaa gtatatatga gtaaacttgg tctgacagtt accaatgctt
     4381 aatcagtgag gcacctatct cagcgatctg tctatttcgt tcatccatag ttgcctgact
     4441 ccccgtcgtg tagataacta cgatacggga gggcttacca tctggcccca gtgctgcaat
     4501 gataccgcga gacccacgct caccggctcc agatttatca gcaataaacc agccagccgg
     4561 aagggccgag cgcagaagtg gtcctgcaac tttatccgcc tccatccagt ctattaattg
     4621 ttgccgggaa gctagagtaa gtagttcgcc agttaatagt ttgcgcaacg ttgttgccat
     4681 tgctacaggc atcgtggtgt cacgctcgtc gtttggtatg gcttcattca gctccggttc
     4741 ccaacgatca aggcgagtta catgatcccc catgttgtgc aaaaaagcgg ttagctcctt
     4801 cggtcctccg atcgttgtca gaagtaagtt ggccgcagtg ttatcactca tggttatggc
     4861 agcactgcat aattctctta ctgtcatgcc atccgtaaga tgcttttctg tgactggtga
     4921 gtactcaacc aagtcattct gagaatagtg tatgcggcga ccgagttgct cttgcccggc
     4981 gtcaatacgg gataataccg cgccacatag cagaacttta aaagtgctca tcattggaaa
     5041 acgttcttcg gggcgaaaac tctcaaggat cttaccgctg ttgagatcca gttcgatgta
     5101 acccactcgt gcacccaact gatcttcagc atcttttact ttcaccagcg tttctgggtg
     5161 agcaaaaaca ggaaggcaaa atgccgcaaa aaagggaata agggcgacac ggaaatgttg
     5221 aatactcata ctcttccttt ttcaatatta ttgaagcatt tatcagggtt attgtctcat
     5281 gagcggatac atatttgaat gtatttagaa aaataaacaa ataggggttc cgcgcacatt
     5341 tccccgaaaa gtgccacctg acgtctaaga aaccattatt atcatgacat taacctataa
     5401 aaataggcgt atcacgaggc cctttcgtc
//
