LOCUS Exported 8294 bp ds-DNA circular SYN 12-MAY-2021 DEFINITION Gene targeting construct for human NANOS3 locus, introducing a P2A-mCherry reporter gene. Contains an frt-PGK-Neo-PA-frt cassette that can be excised.. ACCESSION . VERSION . KEYWORDS Human NANOS3-P2A-mCherry knock-in targeting construct SOURCE synthetic DNA construct ORGANISM synthetic DNA construct REFERENCE 1 (bases 1 to 8294) AUTHORS Naoko Irie, Leehee Weinberger, Walfred W.C. Tang, Toshihiro Kobayashi, Sergey Viukov, Yair S. Manor, Sabine Dietmann, Jacob H. Hanna, M. Azim Surani TITLE SOX17 Is a Critical Specifier of Human Primordial Germ Cell Fate JOURNAL Cell 160, 1–16, January 15, 2015 REFERENCE 2 (bases 1 to 8294) AUTHORS . TITLE Direct Submission JOURNAL Exported May 12, 2021 from SnapGene Server 1.1.58 http://www.snapgene.com FEATURES Location/Qualifiers source 1..8294 /organism="synthetic DNA construct" /mol_type="other DNA" promoter 236..298 /label=Mini-TK promoter /note="minimal herpes simplex virus (HSV) thymidine kinase promoter" CDS 377..1507 /codon_start=1 /product="herpes simplex virus thymidine kinase" /label=HSV TK /note="confers sensitivity to nucleoside analogs such as acyclovir or ganciclovir" /translation="MASYPGHQHASAFDQAARSRGHSNRRTALRPRRQQEATEVRPEQK MPTLLRVYIDGPHGMGKTTTTQLLVALGSRDDIVYVPEPMTYWRVLGASETIANIYTTQ HRLDQGEISAGDAAVVMTSAQITMGMPYTVTDAVLAPHIGGEAGSSHAPPPALTLIFDR HPIAALLCYPAARYLMGSMTPQAVLAFVALIPPTLPGTNIVLGALPEDRHIDRLAKRQR PGERLDLAMLTAIRRVYGLLANTVRYLQGGGSWREDWGQLSGTAVPPQGAEPQSNAGPR PHIGDTLFTLFRAPELLAPNGDLYNVFAWALDVLAKRLRPMHVFILDYDQSPAGCRDAL LQLTSGMIQTHVTTPGSIPTICDLARTFAREMGEAN" primer_bind complement(1505..1524) /label=TK-pA-R /note="Thymidine kinase polyA, reverse primer" polyA_signal 1549..1597 /label=HSV TK poly(A) signal /note="herpes simplex virus thymidine kinase polyadenylation signal (Cole and Stacy, 1985)" primer_bind 2027..2043 /label=M13 fwd /note="common sequencing primer, one of multiple similar variants" protein_bind 2899..2932 /label=loxP /bound_moiety="Cre recombinase" /note="Cre-mediated recombination occurs in the 8-bp core sequence (GCATACAT)." protein_bind 2950..2983 /label=FRT (minimal) /bound_moiety="FLP recombinase from the Saccharomyces cerevisiae 2u plasmid" /note="supports FLP-mediated excision but not integration (Turan and Bode, 2011)" polyA_signal 3026..3250 /label=bGH poly(A) signal /note="bovine growth hormone polyadenylation signal" primer_bind 3239..3256 /label=BGH-rev /note="Bovine growth hormone terminator, reverse primer. Also called BGH reverse" CDS complement(3288..4091) /codon_start=1 /gene="aph(3')-II (or nptII)" /product="aminoglycoside phosphotransferase from Tn5" /label=NeoR/KanR /note="confers resistance to neomycin, kanamycin, and G418 (Geneticin(R))" /translation="MGSAIEQDGLHAGSPAAWVERLFGYDWAQQTIGCSDAAVFRLSAQ GRPVLFVKTDLSGALNELQDEAARLSWLATTGVPCAAVLDVVTEAGRDWLLLGEVPGQD LLSSHLAPAEKVSIMADAMRRLHTLDPATCPFDHQAKHRIERARTRMEAGLVNQDDLDE EHQGLAPAELFARLKARMPDGEDLVVTHGDACLPNIMVENGRFSGFIDCGRLGVADRYQ DIALATRDIAEELGGEWADRFLVLYGIAAPDSQRIAFYRLLDEFF" primer_bind complement(3399..3418) /label=Neo-F /note="Neomycin resistance gene, forward primer" primer_bind 4009..4028 /label=Neo-R /note="Neomycin resistance gene, reverse primer" promoter complement(4105..4605) /label=PGK promoter /note="mouse phosphoglycerate kinase 1 promoter" primer_bind complement(4153..4172) /label=mPGK-F /note="Mouse PGK promoter, forward primer" primer_bind 4547..4570 /label=MSCV-rev /note="Murine stem cell virus, reverse primer" protein_bind 4626..4659 /label=FRT (minimal) /bound_moiety="FLP recombinase from the Saccharomyces cerevisiae 2u plasmid" /note="supports FLP-mediated excision but not integration (Turan and Bode, 2011)" protein_bind 4676..4709 /label=loxP /bound_moiety="Cre recombinase" /note="Cre-mediated recombination occurs in the 8-bp core sequence (GCATACAT)." CDS complement(4733..5443) /codon_start=1 /product="monomeric derivative of DsRed fluorescent protein (Shaner et al., 2004)" /label=mCherry /note="mammalian codon-optimized" /translation="MVSKGEEDNMAIIKEFMRFKVHMEGSVNGHEFEIEGEGEGRPYEG TQTAKLKVTKGGPLPFAWDILSPQFMYGSKAYVKHPADIPDYLKLSFPEGFKWERVMNF EDGGVVTVTQDSSLQDGEFIYKVKLRGTNFPSDGPVMQKKTMGWEASSERMYPEDGALK GEIKQRLKLKDGGHYDAEVKTTYKAKKPVQLPGAYNVNIKLDITSHNEDYTIVEQYERA EGRHSTGGMDELYK" primer_bind complement(5011..5030) /label=mCherry-F /note="mCherry, forward primer" primer_bind 5280..5298 /label=mCherry-R /note="mCherry, reverse primer" regulatory 6475..6484 /regulatory_class="other" /note="vertebrate consensus sequence for strong initiation of translation (Kozak, 1987)" promoter 6528..6632 /gene="bla" /label=AmpR promoter CDS 6633..7493 /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(6851..6870) /label=Amp-R /note="Ampicillin resistance gene, reverse primer" rep_origin 7664..8252 /direction=RIGHT /label=ori /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of replication" primer_bind 8153..8172 /label=pBR322ori-F /note="pBR322 origin, forward primer" ORIGIN 1 cggaccgtag agtcgagcag tgtggttttc aagaggaagc aaaaagcctc tccacccagg 61 cctggaatgt ttccacccaa tgtcgagcag tgtggttttg caagaggaag caaaaagcct 121 ctccacccag gcctggaatg tttccaccca atgtcgagca aaccccgccc agcgtcttgt 181 cattggcgaa ttggaacacg cagatgcagt cggggcggcg cggtcccagg tccacttcgc 241 atattaaggt gacgcgtgtg gcctcgaaca ccgagcgacc ctgcagcgac ccgcttaaca 301 gcgtcaacag cgtgccgcac atcttggtgg cgtgaaactc ccgcacctct tcggccagcg 361 ccttgtagaa gcgcgtatgg cttcgtaccc cggccatcag cacgcgtctg cgttcgacca 421 ggctgcgcgt tctcgcggcc atagcaaccg acgtacggcg ttgcgccctc gccggcagca 481 agaagccacg gaagtccgcc cggagcagaa aatgcccacg ctactgcggg tttatataga 541 cggtccccac gggatgggga aaaccaccac cacgcaactg ctggtggccc tgggttcgcg 601 cgacgatatc gtctacgtac ccgagccgat gacttactgg cgggtgctgg gggcttccga 661 gacaatcgcg aacatctaca ccacacaaca ccgccttgac cagggtgaga tatcggccgg 721 ggacgcggcg gtggtaatga caagcgccca gataacaatg ggcatgcctt ataccgtgac 781 cgacgccgtt ctggctcctc atatcggggg ggaggctggg agctcacatg ccccgccccc 841 ggccctcacc ctcatcttcg accgccatcc catcgccgcc ctcctgtgct acccggccgc 901 gcgatacctt atgggcagca tgacccccca ggccgtgctg gcgttcgtgg ccctcatccc 961 gccgaccttg cccggcacaa acatcgtgtt gggggccctt ccggaggaca gacacatcga 1021 ccgcctggcc aaacgccagc gccccggcga gcggcttgac ctggctatgc tgaccgcgat 1081 tcgccgcgtt tacgggctgc ttgccaatac ggtgcggtat ctgcagggcg gcgggtcgtg 1141 gcgggaggat tggggacagc tttcggggac ggccgtgccg ccccagggtg ccgagcccca 1201 gagcaacgcg ggcccacgac cccatatcgg ggacacgtta tttaccctgt ttcgggcccc 1261 cgagttgctg gcccccaacg gcgacctgta caacgtgttt gcctgggcct tggacgtctt 1321 ggccaaacgc ctccgtccca tgcacgtctt tatcctggat tacgaccaat cgcccgccgg 1381 ctgccgggac gccctgctgc aacttacctc cgggatgatc cagacccacg tcaccacccc 1441 aggctccata ccgacgatct gcgacctggc gcgcacgttt gcacgggaga tgggggaggc 1501 taactgaaac acggaaggag acaataccgg aaggaacccg cgctatgtcg gcaataaaaa 1561 gacagaataa aacgcacggg tgttgggtcg tttgttcata aacgcggggt tcggtcccag 1621 ggctggcact ctgtcgatac cccaccgaga ccccattggg gccaatacgc ccgcgtttct 1681 tccttttccc caccccaccc cccaagttcg ggtgaaggcc cagggctcgc agccaacgtc 1741 gggcggcagg cctgccatag ccacgggccc cgtgggttag ggacggggtc ccccatgggg 1801 aatggtttat ggttcgtggg ggttattatt ttgggcgttg cgtggggtca gtccacgact 1861 ggactgagca gacagaccca tggtttttgg atggcctggg catggaccgc atgtactggc 1921 gcgacacgaa caccgggcgt ctgtggctgc caaacacccc cgacccccaa aaaccaccgc 1981 gcggatttct ggcgccgccg gaccaactaa acctgactac ggaccggtaa aacgacggcc 2041 agtgttaaca agatctggag gtggaggagg caggcagggg tgtggggatt cccccacatg 2101 catttccccc ccgccctgcc agaacagccc catccttctg ctcctgccca agcccaccca 2161 ggacatggtc ccattccctg ccccctaaat gtccctccct tgctctaaac tgcctggctt 2221 gtcactgggg catccatggt gacataaaca ttcaccttcc ttagtgatga ggatgtcggg 2281 ggggggggct ctgtctagtt gtgtgtcaat ttcttcctca aatgaagccc ccacctccag 2341 tacggagctg ggggtgggat gggaggagtc cacccatctt gcatgtcagt gcagggggtg 2401 gctgaaagac caaatgtcca cagaaagggt ctcatgatgt cgatctggct ttgaggtgcc 2461 atgaaccccc ccagctaccc caaggcaccc acttgtgttt ctctgcacaa agcagctcac 2521 cccagcaccc cagggtgccg aaactcaaag aaggagaagg ggagatctag gtacaggact 2581 cccagcaagg ttcacactac accttgtccc aagaccaggg cttgtctgtt gttgatccat 2641 tcataagcat ttattgaggg ctgactggat gcccagcact agggaaacgg cagatggcac 2701 aaagggtggg gtccccaagg acagggcgat ttcagctctt cccttgccaa ccccgggtca 2761 tccccagggc tcaatcccag gctgggggga gggtctggga gtgggggcag tcgtcatagg 2821 gtgggtcttc ctggaaatcc agccgagccc cggtgccctt gcccaggtgt aggcagcctc 2881 gttaactcta ggagatctat aacttcgtat agcatacatt atacgaagtt ataagctttc 2941 tagggtaccg aagttcctat tctctagaaa gtataggaac ttcctcgagg ggcgcgcccc 3001 cagctggttc tttccgcctc agaagccata gagcccaccg catccccagc atgcctgcta 3061 ttgtcttccc aatcctcccc cttgctgtcc tgccccaccc caccccccag aatagaatga 3121 cacctactca gacaatgcga tgcaatttcc tcattttatt aggaaaggac agtgggagtg 3181 gcaccttcca gggtcaagga aggcacgggg gaggggcaaa caacagatgg ctggcaacta 3241 gaaggcacag tcgaggctga tcagcgagct ctagagaatt gatcccctca gaagaactcg 3301 tcaagaaggc gatagaaggc gatgcgctgc gaatcgggag cggcgatacc gtaaagcacg 3361 aggaagcggt cagcccattc gccgccaagc tcttcagcaa tatcacgggt agccaacgct 3421 atgtcctgat agcgatccgc cacacccagc cggccacagt cgatgaatcc agaaaagcgg 3481 ccattttcca ccatgatatt cggcaagcag gcatcgccat gggtcacgac gagatcctcg 3541 ccgtcgggca tgcgcgcctt gagcctggcg aacagttcgg ctggcgcgag cccctgatgc 3601 tcttcgtcca gatcatcctg attgacaaga ccggcttcca tccgagtacg tgctcgctcg 3661 atgcgatgtt tcgcttggtg gtcgaatggg caggtagccg gatcaagcgt atgcagccgc 3721 cgcattgcat cagccatgat ggatactttc tcggcaggag caaggtgaga tgacaggaga 3781 tcctgccccg gcacttcgcc caatagcagc cagtcccttc ccgcttcagt gacaacgtcg 3841 agcacagctg cgcaaggaac gcccgtcgtg gccagccacg atagccgcgc tgcctcgtcc 3901 tgcagttcat tcagggcacc ggacaggtcg gtcttgacaa aaagaaccgg gcgcccctgc 3961 gctgacagcc ggaacacggc ggcatcagag cagccgattg tctgttgtgc ccagtcatag 4021 ccgaatagcc tctccaccca agcggccgga gaacctgcgt gcaatccatc ttgttcaatg 4081 gccgatccca tattggctgc aggtcgaaag gcccggagat gaggaagagg agaacagcgc 4141 ggcagacgtg cgcttttgaa gcgtgcagaa tgccgggctt ccggaggacc ttcgggcgcc 4201 cgccccgccc ctgagcccgc ccctgagccc gcccccggac ccaccccttc ccagcctctg 4261 agcccagaaa gcgaaggagc caaagctgct attggccgct gccccaaagg cctacccgct 4321 tccattgctc agcggtgctg tccatctgca cgagactagt gagacgtgct acttccattt 4381 gtcacgtcct gcacgacgcg agctgcgggg cgggggggaa cttcctgact aggggaggag 4441 tagaaggtgg cgcgaagggg ccaccaaaga acggagccgg ttggcgccta ccggtggatg 4501 tggaatgtgt gcgaggccag aggccacttg tgtagcgcca agtgcccagc ggggctgcta 4561 aagcgcatgc tccagactgc cttgggaaaa gcgcctcccc tacccggtag ggcgcgccgg 4621 gatccgaagt tcctattctc tagaaagtat aggaacttca tcgatcctag aattcataac 4681 ttcgtatagc atacattata cgaagttatc cgcggctaga cccggagcta gcctacttgt 4741 acagctcgtc catgccgccg gtggagtggc ggccctcggc gcgttcgtac tgttccacga 4801 tggtgtagtc ctcgttgtgg gaggtgatgt ccaacttgat gttgacgttg taggcgccgg 4861 gcagctgcac gggcttcttg gccttgtagg tggtcttgac ctcagcgtcg tagtggccgc 4921 cgtccttcag cttcagcctc tgcttgatct cgcccttcag ggcgccgtcc tcggggtaca 4981 tccgctcgga ggaggcctcc cagcccatgg tcttcttctg cattacgggg ccgtcggagg 5041 ggaagttggt gccgcgcagc ttcaccttgt agatgaactc gccgtcctgc agggaggagt 5101 cctgggtcac ggtcaccacg ccgccgtcct cgaagttcat cacgcgctcc cacttgaagc 5161 cctcggggaa ggacagcttc aagtagtcgg ggatgtcggc ggggtgcttc acgtaggcct 5221 tggagccgta catgaactga ggggacagga tgtcccaggc gaagggcagg gggccaccct 5281 tggtcacctt cagcttggcg gtctgggtgc cctcgtaggg gcggccctcg ccctcgccct 5341 cgatctcgaa ctcgtggccg ttcacggagc cctccatgtg caccttgaag cgcatgaact 5401 ccttgatgat ggccatgtta tcctcctcgc ccttgctcac catggtaagc ttcagctgaa 5461 gcttgcatgc gggcccgggg ttggactcga cgtctcccgc aagcttaaga aggtcaaaat 5521 tcaaagtttg cttaccagaa ccgtcgactc tagagtcgcg accgctgtag gtggacatgg 5581 agggagagca ggagggcgaa ggctcagact tcccggcacc tctgaaacct agtggagggg 5641 gagggagaga aatgcttaat cacaggcgat gactcaacgc tccccaagtt gcccacggac 5701 tcggcctccg ggcctgctgg acctctggcg ccacccaggg gctgctccgg agacagcgac 5761 cccgtggcgt ctctggctcc gccgggcggc cccgctggga cccagagccc gagacaaagc 5821 acaggagttc cgattctgcg actgtggtgc cctctcgtgg ccgccgtgct ctggggtccc 5881 cggatctgtt actcccaaaa gttgttcatc agaaaggctg gccgcaggac accatcttcc 5941 ctatgtcctt ttggcctcca gcgtctccca gtgagacagc atcaaacctc acaggaaaca 6001 ctatcttctc ctggaggcag tctttcatgc tttctttttt ttgtgagacg gagtctcact 6061 ctgtcgccca ggctggagtg cagtgacgtg acctcagctc actgcgacct ccgcctcccg 6121 ggttctagcg attctcctgt ctcagccccc cgagtagcag ggattacagg cgcatgccac 6181 catgcccagt taattttttg tattttcagt agagacgggg tttcaccatg ttggccaggc 6241 tggtcttgaa ttcctgacct caggtgatcc acctgcctca gcttccccaa gtactgggat 6301 tacaggcatg agccaccatg accagtgctt tttttttttt ttaagacgag gtcttgctgt 6361 gttgcccagg ctgcagtgca gtggcaccat ctcagctcac tgcagcctct gtcccctgga 6421 cccaagcgat cctccctcgt cagcctccca agtagctggg actacaggca cgcggccgcc 6481 atggtcatag ctgtttgacg tcaggtggca cttttcgggg aaatgtgcgc ggaaccccta 6541 tttgtttatt tttctaaata cattcaaata tgtatccgct cacgagacaa taaccctgat 6601 aaatgcttca ataatattga aaaaggaaga gtatgagtat tcaacatttc cgtgtcgccc 6661 ttattccctt ttttgcggca ttttgccttc ctgtttttgc tcacccagaa acgctggtga 6721 aagtaaaaga tgctgaagat cagttgggtg cacgagtggg ttacatcgaa ctggatctca 6781 acagcggtaa gatccttgag agttttcgcc ccgaagaacg ttttccaatg atgagcactt 6841 ttaaagttct gctatgtggc gcggtattat cccgtattga cgccgggcaa gagcaactcg 6901 gtcgccgcat acactattct cagaatgact tggttgagta ctcaccagtc acagaaaagc 6961 atcttacgga tggcatgaca gtaagagaat tatgcagtgc tgccataacc atgagtgata 7021 acactgcggc caacttactt ctgacaacga tcggaggacc gaaggagcta accgcttttt 7081 tgcacaacat gggggatcat gtaactcgcc ttgatcgttg ggaaccggag ctgaatgaag 7141 ccataccaaa cgacgagcgt gacaccacga tgcctgtagc aatggcaaca acgttgcgca 7201 aactattaac tggcgaacta cttactctag cttcccggca acaattaata gactggatgg 7261 aggcggataa agttgcagga ccacttctgc gctcggccct tccggctggc tggtttattg 7321 ctgataaatc tggagccggt gagcgtgggt ctcgcggtat cattgcagca ctggggccag 7381 atggtaagcc ctcccgtatc gtagttatct acacgacggg gagtcaggca actatggatg 7441 aacgaaatag acagatcgct gagataggtg cctcactgat taagcattgg taactgtcag 7501 accaagttta ctcatatata ctttagattg atttaaaact tcatttttaa tttaaaagga 7561 tctaggtgaa gatccttttt gataatctca tgaccaaaat cccttaacgt gagttttcgt 7621 tccactgagc gtcagacccc gtagaaaaga tcaaaggatc ttcttgagat cctttttttc 7681 tgcgcgtaat ctgctgcttg caaacaaaaa aaccaccgct accagcggtg gtttgtttgc 7741 cggatcaaga gctaccaact ctttttccga aggtaactgg cttcagcaga gcgcagatac 7801 caaatactgt ccttctagtg tagccgtagt taggccacca cttcaagaac tctgtagcac 7861 cgcctacata cctcgctctg ctaatcctgt taccagtggc tgctgccagt ggcgataagt 7921 cgtgtcttac cgggttggac tcaagacgat agttaccgga taaggcgcag cggtcgggct 7981 gaacgggggg ttcgtgcaca cagcccagct tggagcgaac gacctacacc gaactgagat 8041 acctacagcg tgagcattga gaaagcgcca cgcttcccga agggagaaag gcggacaggt 8101 atccggtaag cggcagggtc ggaacaggag agcgcacgag ggagcttcca gggggaaacg 8161 cctggtatct ttatagtcct gtcgggtttc gccacctctg acttgagcgt cgatttttgt 8221 gatgctcgtc aggggggcgg agcctatgga aaaacgccag caacgcggcc tttttacggt 8281 tcctggcctt ttgc //