LOCUS       sequence-111292-        5363 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Intron targeting-mediated Gal4 knockin at the zebrafish th locus. .
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 5363)
  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 5363)
  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-Gal4 donor
FEATURES             Location/Qualifiers
     source          1..5363
                     /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             1778..2218
                     /codon_start=1
                     /gene="Saccharomyces cerevisiae GAL4 (truncated)"
                     /product="DNA binding domain of the GAL4 transcriptional 
                     activator"
                     /label=GAL4 DNA binding domain
                     /translation="MKLLSSIEQACDICRLKKLKCSKEKPKCAKCLKNNWECRYSPKTK
                     RSPLTRAHLTEVESRLERLEQLFLLIFPREDLDMILKMDSLQDIKALLTGLFVQDNVNK
                     DAVTDRLASVETDMPLTLRQHRISATSSSEESSNKGQRQLTVS"
     primer_bind     complement(3142..3158)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(3142..3158)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(3155..3177)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    3166..3182
                     /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(3190..3196,3197..3214,3215..3220))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    3235..3256
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(3373..3390)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(3544..4132)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(3624..3643)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(4303..5094,5095..5163))
                     /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     4926..4945
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(5164..5268)
                     /gene="bla"
                     /label=AmpR promoter
     primer_bind     5336..5354
                     /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 aagctactgt cttctatcga
     1801 acaagcatgc gatatttgcc gacttaaaaa gctcaagtgc tccaaagaaa aaccgaagtg
     1861 cgccaagtgt ctgaagaaca actgggagtg tcgctactct cccaaaacca aaaggtctcc
     1921 gctgactagg gcacatctga cagaagtgga atcaaggcta gaaagactgg aacagctatt
     1981 tctactgatt tttcctcgag aagaccttga catgattttg aaaatggatt ctttacagga
     2041 tataaaagca ttgttaacag gattatttgt acaagataat gtgaataaag atgccgtcac
     2101 agatagattg gcttcagtgg agactgatat gcctctaaca ttgagacagc atagaataag
     2161 tgcgacatca tcatcggaag agagtagtaa caaaggtcaa agacagttga ctgtatcgtc
     2221 gagatcgacc ccgggaattc agatctctcg agccgccccc ccgaccgatg tcagcctggg
     2281 ggacgagctc cacttagacg gcgaggacgt ggcgatggcg catgccgacg cgctagacga
     2341 tttcgatctg gacatgttgg gggacgggga ttccccgggt ccgggattta ccccccacga
     2401 ctccgccccc tacggcgctc tggatatgac cggttaattc tgcttaatca ttcctctatg
     2461 cttcatgtgt gacggtctca ttgttatgct gtgcttctat gtctactgta catctgctgc
     2521 tatagacatt gttgcttcta aaatactgaa tgaaaaactt taaaccttca gcgttaaata
     2581 atgtatccaa aaatgaaagt tcaccactca aactcacatt acagtagaat atgataatag
     2641 agatagagaa agagagagac agagagagac ttttatttct gaatgaactt tttttatttt
     2701 tttttttaag tcatatatga tcaaaattta aaatgataac attttccgct ataattttca
     2761 ttttggggta aattataact cgtctaaaca gaaatttctg atatgacatt taattaattt
     2821 atgtacagga tttatttata tttcagatag acgtaccctc attattaaag ctttttaagt
     2881 caaaaggtat atatgaaata ttaccagagg tcatattcac aatacatatt aagcctaaaa
     2941 gcagatccta agtttacaat atatatactc caacatacaa ataaacatac aaaatctgtc
     3001 actgaggtgg tactttttca aaaaatacat ttttgtaaaa taaagatgga cattagaatc
     3061 tttagggtac acttttgtac cttaaaggta ctaggaggtt cccatgtcga cctgcaggca
     3121 tgcaagcttg gcgtaatcat ggtcatagct gtttcctgtg tgaaattgtt atccgctcac
     3181 aattccacac aacatacgag ccggaagcat aaagtgtaaa gcctggggtg cctaatgagt
     3241 gagctaactc acattaattg cgttgcgctc actgcccgct ttccagtcgg gaaacctgtc
     3301 gtgccagctg cattaatgaa tcggccaacg cgcggggaga ggcggtttgc gtattgggcg
     3361 ctcttccgct tcctcgctca ctgactcgct gcgctcggtc gttcggctgc ggcgagcggt
     3421 atcagctcac tcaaaggcgg taatacggtt atccacagaa tcaggggata acgcaggaaa
     3481 gaacatgtga gcaaaaggcc agcaaaaggc caggaaccgt aaaaaggccg cgttgctggc
     3541 gtttttccat aggctccgcc cccctgacga gcatcacaaa aatcgacgct caagtcagag
     3601 gtggcgaaac ccgacaggac tataaagata ccaggcgttt ccccctggaa gctccctcgt
     3661 gcgctctcct gttccgaccc tgccgcttac cggatacctg tccgcctttc tcccttcggg
     3721 aagcgtggcg ctttctcata gctcacgctg taggtatctc agttcggtgt aggtcgttcg
     3781 ctccaagctg ggctgtgtgc acgaaccccc cgttcagccc gaccgctgcg ccttatccgg
     3841 taactatcgt cttgagtcca acccggtaag acacgactta tcgccactgg cagcagccac
     3901 tggtaacagg attagcagag cgaggtatgt aggcggtgct acagagttct tgaagtggtg
     3961 gcctaactac ggctacacta gaagaacagt atttggtatc tgcgctctgc tgaagccagt
     4021 taccttcgga aaaagagttg gtagctcttg atccggcaaa caaaccaccg ctggtagcgg
     4081 tggttttttt gtttgcaagc agcagattac gcgcagaaaa aaaggatctc aagaagatcc
     4141 tttgatcttt tctacggggt ctgacgctca gtggaacgaa aactcacgtt aagggatttt
     4201 ggtcatgaga ttatcaaaaa ggatcttcac ctagatcctt ttaaattaaa aatgaagttt
     4261 taaatcaatc taaagtatat atgagtaaac ttggtctgac agttaccaat gcttaatcag
     4321 tgaggcacct atctcagcga tctgtctatt tcgttcatcc atagttgcct gactccccgt
     4381 cgtgtagata actacgatac gggagggctt accatctggc cccagtgctg caatgatacc
     4441 gcgagaccca cgctcaccgg ctccagattt atcagcaata aaccagccag ccggaagggc
     4501 cgagcgcaga agtggtcctg caactttatc cgcctccatc cagtctatta attgttgccg
     4561 ggaagctaga gtaagtagtt cgccagttaa tagtttgcgc aacgttgttg ccattgctac
     4621 aggcatcgtg gtgtcacgct cgtcgtttgg tatggcttca ttcagctccg gttcccaacg
     4681 atcaaggcga gttacatgat cccccatgtt gtgcaaaaaa gcggttagct ccttcggtcc
     4741 tccgatcgtt gtcagaagta agttggccgc agtgttatca ctcatggtta tggcagcact
     4801 gcataattct cttactgtca tgccatccgt aagatgcttt tctgtgactg gtgagtactc
     4861 aaccaagtca ttctgagaat agtgtatgcg gcgaccgagt tgctcttgcc cggcgtcaat
     4921 acgggataat accgcgccac atagcagaac tttaaaagtg ctcatcattg gaaaacgttc
     4981 ttcggggcga aaactctcaa ggatcttacc gctgttgaga tccagttcga tgtaacccac
     5041 tcgtgcaccc aactgatctt cagcatcttt tactttcacc agcgtttctg ggtgagcaaa
     5101 aacaggaagg caaaatgccg caaaaaaggg aataagggcg acacggaaat gttgaatact
     5161 catactcttc ctttttcaat attattgaag catttatcag ggttattgtc tcatgagcgg
     5221 atacatattt gaatgtattt agaaaaataa acaaataggg gttccgcgca catttccccg
     5281 aaaagtgcca cctgacgtct aagaaaccat tattatcatg acattaacct ataaaaatag
     5341 gcgtatcacg aggccctttc gtc
//
