LOCUS Exported 5363 bp ds-DNA circular SYN 12-MAY-2021 DEFINITION Intron targeting-mediated Gal4 knockin at the zebrafish th locus. . ACCESSION . VERSION . KEYWORDS th-P2A-Gal4 donor 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 May 12, 2021 from SnapGene Server 1.1.58 http://www.snapgene.com FEATURES Location/Qualifiers source 1..5363 /organism="synthetic DNA construct" /mol_type="other DNA" 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(3190..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(4303..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 //